CA2439503A1 - Recombination super compound interferon used as hepatitis b surface antigen and e antigen inhibitor - Google Patents
Recombination super compound interferon used as hepatitis b surface antigen and e antigen inhibitor Download PDFInfo
- Publication number
- CA2439503A1 CA2439503A1 CA002439503A CA2439503A CA2439503A1 CA 2439503 A1 CA2439503 A1 CA 2439503A1 CA 002439503 A CA002439503 A CA 002439503A CA 2439503 A CA2439503 A CA 2439503A CA 2439503 A1 CA2439503 A1 CA 2439503A1
- Authority
- CA
- Canada
- Prior art keywords
- super
- compound interferon
- interferon
- hepatitis
- compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention relates to the use of recombination super compound interferon (rSIFN-co) as hepatitis B surface antigen and e antigen inhibitor, in which the space structure of said interferon protein has been changed.
Description
CA 02439503 2003-08-27 D~Ct. #792-PCT
RECOMBINATION SUPER COMPOUND INTERFERON USED AS HEPATITIS B
SURFACE ANTIGEN AND E ANTIGEN INHIBITOR
FIELD OF THE INVENTION
This invention is related to a recombinant super-compound interferon (rSIFN-co) with changed dimensional structure.
The characteristic of rSIFN-co in this invention is that it cannot only inhibit DNA (deoxyribonucleic acid) duplication of the hepatitis B virus but also the secretion of HBsAg and HBeAg.
BACKGROUND OF THE INVENTION
rSIFN-co is a new interferon molecule constructed with the most popular conservative amino acid found in natural human a-IFN subtypes using genetic engineering methods. United States Patent Nos. 4,695,623 and 4,897,471 have described it. rSIFN-co had been proved to have broad-spectrum IFN
activity and virus- and tumor-inhibition and natural killer cell activity. United States Patent No. 5,372,808 by Amgen, Inc. addresses treatment rSIFN-co. Chinese Patent No.
97193506.8 by Amgen, Inc. addresses re-treatment of rSIFN
co on hepatitis C. Chinese Patent No. 98114663.5 by Shenzhen Jiusheng Bio-engineering Ltd. addresses treatment of rSIFN-co on hepatitis B and hepatitis C.
The United States Food and Medicine Administration (FDA) authorized Amgen Ltd. to produce rSIFN-co with E. Coli. for clinical hepatitis C treatment at the end of 1997.
Hepatitis B patients can be identified when detecting HBsAg and the HBeAg. a-IFN is commonly used in clinics to treat hepatitis B. IFN binds superficial cell membrane receptors, inhibiting DNA and RNA (ribonucleic acid) duplication, including inducing some enzymes to prevent duplication of the virus in hepatitis-infected cells. All IFNs can inhibit only the DNA duplication of viruses, not the a and s antigen.
RECOMBINATION SUPER COMPOUND INTERFERON USED AS HEPATITIS B
SURFACE ANTIGEN AND E ANTIGEN INHIBITOR
FIELD OF THE INVENTION
This invention is related to a recombinant super-compound interferon (rSIFN-co) with changed dimensional structure.
The characteristic of rSIFN-co in this invention is that it cannot only inhibit DNA (deoxyribonucleic acid) duplication of the hepatitis B virus but also the secretion of HBsAg and HBeAg.
BACKGROUND OF THE INVENTION
rSIFN-co is a new interferon molecule constructed with the most popular conservative amino acid found in natural human a-IFN subtypes using genetic engineering methods. United States Patent Nos. 4,695,623 and 4,897,471 have described it. rSIFN-co had been proved to have broad-spectrum IFN
activity and virus- and tumor-inhibition and natural killer cell activity. United States Patent No. 5,372,808 by Amgen, Inc. addresses treatment rSIFN-co. Chinese Patent No.
97193506.8 by Amgen, Inc. addresses re-treatment of rSIFN
co on hepatitis C. Chinese Patent No. 98114663.5 by Shenzhen Jiusheng Bio-engineering Ltd. addresses treatment of rSIFN-co on hepatitis B and hepatitis C.
The United States Food and Medicine Administration (FDA) authorized Amgen Ltd. to produce rSIFN-co with E. Coli. for clinical hepatitis C treatment at the end of 1997.
Hepatitis B patients can be identified when detecting HBsAg and the HBeAg. a-IFN is commonly used in clinics to treat hepatitis B. IFN binds superficial cell membrane receptors, inhibiting DNA and RNA (ribonucleic acid) duplication, including inducing some enzymes to prevent duplication of the virus in hepatitis-infected cells. All IFNs can inhibit only the DNA duplication of viruses, not the a and s antigen.
DETAILED DESCRIPTION OF THE INVENTION
Invention Component It was surprising to find that rSIFN-co, the dimensional structure of which has been changed, is not only a preparation to inhibit the DNA duplication of hepatitis B, but to inhibit the secretion of HBsAg and HBeAg.
The objective of this invention is to offer a preparation of rSIFN-co to inhibit the DNA duplication of hepatitis B
viruses and the secretion of HBeAg and HBsAg of hepatitis B
and decrease them to normal levels.
Results of this invention: The production of rSIFN-co with recombinant techniques. On the condition of fixed amino acid sequence, the IFN DNA was redesigned according to the E. Coli. codon usage and then the rSIFN-co gene was artificially synthesized.
rSIFN-co cDNA was cloned into the high-expression vector of E. Coli by DNA recombinant techniques, and a high expression of rSIFN-co was gained by using of induce/activate-mechanism of L-arabinose to activate the transcription of PB~ promoter.
Compared with usual thermo-induction, pH induction and IPTG
induction systems of genetic engineering, arabinose induction/activation system has some advantages: (1) Common systems relieve promoter function by creating a "derepression" pattern. Promoters then induce downstream gene expression. So temperature and pH change and the addition of IPTG cannot activate promoters directly. In the system disclosed herein, L-arabinose not only deactivates and represses but also activates the transcription of PB~
promoter which induce a high expression of rSIFN-co.
Therefore, the arabinose induction/activation system is a more effective expression system. (2) The relation between Exogenous and L-arabinose dosage is linearity. This means the concentration of arabinose can be changed to adjust the expression level of the exogenous gene. Therefore, it is easier to control the exogenous gene expression level in E.coli by arabinose than by changing temperature and pH
value. This characteristic is significant for the formation of inclusion bodies. (3) L- arabinose is resourceful cheap and safe, which, on the contrary, are the disadvantages of other inducers such as IPTG.
This invention creates an effective and resistant rSIFN-co-expressing E. Coli. engineering strain with an L-arabinose induction/activation system. The strain is cultivated and fermented under suitable conditions to harvest the bacterial bodies. Inclusion bodies are then purified after destroying bacteria and washing repeatedly. The end result, mass of high-purity, dimensional-structure-changed rSIFN-co protein for this invention and for clinical treatment, was gained from denaturation and renaturation of inclusion bodies and a series of purification steps.
The following are some rSIFN-co preparations: tablets, capsules, oral liquids, pastes, injections, sprays, suppositories, and solutions. Injections are recommended.
It is common to subcutaneously inject or vein-inject the medicine. The medicine carrier could be any acceptance medicine carrier, including carbohydrate, cellulosum, adhesive, collapse, emollient, filling, add-dissolve agent, amortization, preservative, add-thick agent, matching, etc.
DETAILED DESCRIPTION OF THE FIGURE
Figure 1. DNA coding sequence and deduced amino acid sequence of rSIFN-co EXPERIMENTAL DETAILS
Embodiment experience:
The invention disclosed herein also experimentally verifies that the dimensional-structure-changed rSIFN-co can inhibit HBV-DNA duplication and secretion of HBsAg and HBeAg.
Materials Solvent and Dispensing Method: Add lml saline into each vial, dissolve, and mix with MEM culture medium at different concentrations. Mix on the spot.
Control drugs: IFN-a2b (Intron A) as lyophilized, purchased from Schering Plough. 3x106U each, mix to 3x106IU/ml with culture medium; INFERGEN (liquid solution) , purchased from Amgen, 9~g, 0.3m1 each, equal to 9X106IU, and mix with 9X106IU/ml culture medium preserve at 4°C; 2.2.15 cell:
2.2.15 cell line of hepatoma (Hep G2) cloned and transfected by HBV DNA, constructed by Mount Sinai Medical Center.
Reagent: MEM powder, Gibco American Ltd. cattle fetal blood serum, HycloneLab American Ltd. G-418(Geneticin); MEM
dispensing, Gibco American Ltd.; L-Glutamyl, imported and packaged by JING KE Chemical Ltd.; HBsAg and HBeAg solid-phase radioimmunoassay box, Northward Reagent Institute of Chinese Isotope Ltd.; Biograncetina, Northern China Medicine; And Lipofectin, Gibco American Ltd.
Experimental goods and equipment: culture bottle, Denmark TunclonT"'; 24-well and 96-well culture board, Corning American Ltd.; Carbon Dioxide hatching box, Shel-Lab American Ltd.; MEM culture medium 100m1: 10% cattle fetal blood serum, 3% Glutamyll%, 6418 380~g/ml, biograncetina50U/ml.
Method:
2.2.15 cell culture: Added 0.25% pancreatic enzyme into culture box with full of 2.2.15 cell, digest at 37°C for 3 minutes, and add culture medium to stop digest and disturb it to disperse the cells, reproduce with ratio of 1:3. They will reach full growth in 10 days.
Medicine toxicity test: In this test, set groups of different medicine concentrations and a control group in which cell is not acted on with medicine. Digest cell, and dispense to a 100,000 cell/ml solution. Inoculate to 96-well culture board, 200.1 each well, culture at 37°C for 24h with 5o CO2. Test when simple cell layer grows.
Dispense rSIFN-co to 1.8x10'IU/ml solution than prepare a series of solutions diluted at two-fold gradients. Add into 96-well culture board, 3 wells per concentration. Change the solution every 4 days. Test cytopathic effect by microscope after 8 days. Fully destroy as 4, 75% as 3, 50%
as 2, 25% as 1, zero as 0. Calculate average cell lesion and inhibition rate of different concentrations. Calculate TC50 and TCO according to the Reed Muench method.
TC50 = Antilog (B + ~-B x C) A=log >50 ~ medicine concentration, B=log<50 Omedicine concentration, C=log dilution power Inhibition test for HBeAg and HBsAg: Separate into positive and negative HBeAg and HBsAg contrast groups, cell contrast group and medicine concentration groups. Inoculate 700,000 cells/ml of 2.2.15 cell into 6-well culture board, 3 ml each well, culture at 37°C for 24h with 5% COz, then prepare 5 gradiently diluted solutions with 3-fold as the grade (Prepare 5 solutions, each with a different protein concentration. The concentration of Solution 2 is 3 times lower than that of Solution l, the concentration of Solution 3 is 3 times lower than that of Solution 2, etc.) 4.5x106IU/ml, 1.5x106IU/ml, 0.5x106IU/ml, 0.17x1061U/ml, and 0.056x1061U/ml, 1 well per concentration, culture at 37°C
for 24h with 5% CO2. Change solutions every 4 days using the same solution. Collect all culture medium on the 8th day.
Preserve at -20°C Repeat test 3 times to estimate HBsAg and HBeAg with solid-phase radioimmunoassay box (Northward Reagent Institute of Chinese Isotope Ltd.). Estimate cpm value of each well with a 'y- accounting machine.
Medicinal effects calculation: Calculate cpm mean value of contrast groups and different-concentration groups and their standard deviation, P/N value such as inhibition rate, IC50 and SI.
Invention Component It was surprising to find that rSIFN-co, the dimensional structure of which has been changed, is not only a preparation to inhibit the DNA duplication of hepatitis B, but to inhibit the secretion of HBsAg and HBeAg.
The objective of this invention is to offer a preparation of rSIFN-co to inhibit the DNA duplication of hepatitis B
viruses and the secretion of HBeAg and HBsAg of hepatitis B
and decrease them to normal levels.
Results of this invention: The production of rSIFN-co with recombinant techniques. On the condition of fixed amino acid sequence, the IFN DNA was redesigned according to the E. Coli. codon usage and then the rSIFN-co gene was artificially synthesized.
rSIFN-co cDNA was cloned into the high-expression vector of E. Coli by DNA recombinant techniques, and a high expression of rSIFN-co was gained by using of induce/activate-mechanism of L-arabinose to activate the transcription of PB~ promoter.
Compared with usual thermo-induction, pH induction and IPTG
induction systems of genetic engineering, arabinose induction/activation system has some advantages: (1) Common systems relieve promoter function by creating a "derepression" pattern. Promoters then induce downstream gene expression. So temperature and pH change and the addition of IPTG cannot activate promoters directly. In the system disclosed herein, L-arabinose not only deactivates and represses but also activates the transcription of PB~
promoter which induce a high expression of rSIFN-co.
Therefore, the arabinose induction/activation system is a more effective expression system. (2) The relation between Exogenous and L-arabinose dosage is linearity. This means the concentration of arabinose can be changed to adjust the expression level of the exogenous gene. Therefore, it is easier to control the exogenous gene expression level in E.coli by arabinose than by changing temperature and pH
value. This characteristic is significant for the formation of inclusion bodies. (3) L- arabinose is resourceful cheap and safe, which, on the contrary, are the disadvantages of other inducers such as IPTG.
This invention creates an effective and resistant rSIFN-co-expressing E. Coli. engineering strain with an L-arabinose induction/activation system. The strain is cultivated and fermented under suitable conditions to harvest the bacterial bodies. Inclusion bodies are then purified after destroying bacteria and washing repeatedly. The end result, mass of high-purity, dimensional-structure-changed rSIFN-co protein for this invention and for clinical treatment, was gained from denaturation and renaturation of inclusion bodies and a series of purification steps.
The following are some rSIFN-co preparations: tablets, capsules, oral liquids, pastes, injections, sprays, suppositories, and solutions. Injections are recommended.
It is common to subcutaneously inject or vein-inject the medicine. The medicine carrier could be any acceptance medicine carrier, including carbohydrate, cellulosum, adhesive, collapse, emollient, filling, add-dissolve agent, amortization, preservative, add-thick agent, matching, etc.
DETAILED DESCRIPTION OF THE FIGURE
Figure 1. DNA coding sequence and deduced amino acid sequence of rSIFN-co EXPERIMENTAL DETAILS
Embodiment experience:
The invention disclosed herein also experimentally verifies that the dimensional-structure-changed rSIFN-co can inhibit HBV-DNA duplication and secretion of HBsAg and HBeAg.
Materials Solvent and Dispensing Method: Add lml saline into each vial, dissolve, and mix with MEM culture medium at different concentrations. Mix on the spot.
Control drugs: IFN-a2b (Intron A) as lyophilized, purchased from Schering Plough. 3x106U each, mix to 3x106IU/ml with culture medium; INFERGEN (liquid solution) , purchased from Amgen, 9~g, 0.3m1 each, equal to 9X106IU, and mix with 9X106IU/ml culture medium preserve at 4°C; 2.2.15 cell:
2.2.15 cell line of hepatoma (Hep G2) cloned and transfected by HBV DNA, constructed by Mount Sinai Medical Center.
Reagent: MEM powder, Gibco American Ltd. cattle fetal blood serum, HycloneLab American Ltd. G-418(Geneticin); MEM
dispensing, Gibco American Ltd.; L-Glutamyl, imported and packaged by JING KE Chemical Ltd.; HBsAg and HBeAg solid-phase radioimmunoassay box, Northward Reagent Institute of Chinese Isotope Ltd.; Biograncetina, Northern China Medicine; And Lipofectin, Gibco American Ltd.
Experimental goods and equipment: culture bottle, Denmark TunclonT"'; 24-well and 96-well culture board, Corning American Ltd.; Carbon Dioxide hatching box, Shel-Lab American Ltd.; MEM culture medium 100m1: 10% cattle fetal blood serum, 3% Glutamyll%, 6418 380~g/ml, biograncetina50U/ml.
Method:
2.2.15 cell culture: Added 0.25% pancreatic enzyme into culture box with full of 2.2.15 cell, digest at 37°C for 3 minutes, and add culture medium to stop digest and disturb it to disperse the cells, reproduce with ratio of 1:3. They will reach full growth in 10 days.
Medicine toxicity test: In this test, set groups of different medicine concentrations and a control group in which cell is not acted on with medicine. Digest cell, and dispense to a 100,000 cell/ml solution. Inoculate to 96-well culture board, 200.1 each well, culture at 37°C for 24h with 5o CO2. Test when simple cell layer grows.
Dispense rSIFN-co to 1.8x10'IU/ml solution than prepare a series of solutions diluted at two-fold gradients. Add into 96-well culture board, 3 wells per concentration. Change the solution every 4 days. Test cytopathic effect by microscope after 8 days. Fully destroy as 4, 75% as 3, 50%
as 2, 25% as 1, zero as 0. Calculate average cell lesion and inhibition rate of different concentrations. Calculate TC50 and TCO according to the Reed Muench method.
TC50 = Antilog (B + ~-B x C) A=log >50 ~ medicine concentration, B=log<50 Omedicine concentration, C=log dilution power Inhibition test for HBeAg and HBsAg: Separate into positive and negative HBeAg and HBsAg contrast groups, cell contrast group and medicine concentration groups. Inoculate 700,000 cells/ml of 2.2.15 cell into 6-well culture board, 3 ml each well, culture at 37°C for 24h with 5% COz, then prepare 5 gradiently diluted solutions with 3-fold as the grade (Prepare 5 solutions, each with a different protein concentration. The concentration of Solution 2 is 3 times lower than that of Solution l, the concentration of Solution 3 is 3 times lower than that of Solution 2, etc.) 4.5x106IU/ml, 1.5x106IU/ml, 0.5x106IU/ml, 0.17x1061U/ml, and 0.056x1061U/ml, 1 well per concentration, culture at 37°C
for 24h with 5% CO2. Change solutions every 4 days using the same solution. Collect all culture medium on the 8th day.
Preserve at -20°C Repeat test 3 times to estimate HBsAg and HBeAg with solid-phase radioimmunoassay box (Northward Reagent Institute of Chinese Isotope Ltd.). Estimate cpm value of each well with a 'y- accounting machine.
Medicinal effects calculation: Calculate cpm mean value of contrast groups and different-concentration groups and their standard deviation, P/N value such as inhibition rate, IC50 and SI.
A-B
1) Antigen inhibition rate (%) - A x 100 A = cpm of control group; B = cpm of test group;
2) Counting the half-efficiency concentration of the medicine Antigen inhibition IC50 = Antilog (B + A - B x C) A=log>50% medicine concentration, B=log<500medicine concentration, C=log dilution power 3) SI of interspace-conformation changed rSIFN-co effect on HBsAg and HBeAg in 2.2.15 cell culture:
SI -T~sn ll.J U
4) Estimate the differences in cpm of each dilution degree from the control group using student t test Southern blot: (1) HBV-DNA extract in 2.2.15 cell: Culture cell 8 days. Exsuction culture medium (Separate cells from culture medium by means of draining the culture medium.).
Add lysis buffer to break cells, then extract 2 times with a mixture of phenol, chloroform and isoamyl alcohol (1:1:1), 10,0008 centrifuge. Collect the supernatant adding anhydrous alcohol to deposit nucleic acid. Vacuum draw, re-dissolve into 20~,1TE buffer. (2) Electrophoresis: Add 6XDNA
loading buffer, electrophoresis on 1.5% agarose gel, IV/cm, at fixed pressure for 14-18h. (3) Denaturation and hybridization: respectively dip gel into HC1, denaturaion buffer and neutralization buffer. (4) Transmembrane: Make an orderly transfer of DNA to Hybond-N membrane. Bake, hybridize and expose with dot blot hybridization. Scan and analyze relative density with gel-pro software. Calculate inhibition rate and IC50.
Results Results from Tables 1, 2 and 3 show: After maximum innocuous concentration exponent culturing for 8 days with 2.2.15 cell, the maxima is 9.0~Ox106IU/ml and the average - 7 _ inhibition rate of maximum innocuous concentration rSIFN-co to HBeAg is 46.0~5.25a (P<0~001), IC50 is 4.54~1.32X106IU/ml, SI is 3.96; rate to HBsAg is 44.8~ 6.6%, IC50 is 6.49~0.42x106IU/ml, SI is 2.77. This shows that rSIFN-co can significantly inhibit the activity of HBeAg and HBsAg, but that the IFN of the contrast group and INFERGEN cannot. It has also been proved in clinic that rSIFN-co can decrease HBeAg and HBsAg or return them to normal levels.
The following are some examples for the preparation of rSIFN-co:
Example 1: Preparation of lyophilized injection a) rSIFN-co 3 x 106 IU
b) citric acid 0.2 mg c) dibasic sodium phosphate 2.5 mg d) NaCl 4.0 mg e) dextran 20 mg f) Polyoxyethelene anhydrosorbitol monoelaeo-acids ester 0.1 ml g) inject water to a level of 1.0 ml Preparation technique: Weigh materials according to recipe.
Dissolve with sterile and pyrogen-free water. Filter through 0.22~m membrane to de-bacterialize, preserve at 6 10°C. Fill in vials after affirming it is sterile and pyrogen-free. Add 1.0 ml solution to each bottle, and lyophilize in freeze dryer.
Example 2: Preparation of liquid injection a) rSIFN-co 3 x 106 IU
b) citric acid 0.2 mg c) dibasic sodium phosphate 2.5 mg d) NaCl 4.0 mg e) dextran 20 mg f) Polyoxyethelene anhydrosorbitol monoelaeo-acids ester 0 . 1 ml _ g _ g) inject water to a level of 1.0 ml Preparation technique: Weigh materials according to recipe.
Dissolve with sterile and pyrogen-free water. Filter through 0.22~m membrane to de-bacterialize, preserve at 6-10°C. Fill in airtight vial after affirming it is sterile and non-pyrogen at 1.0 ml per vial. Store end product at 2-10°C, and protect from light.
~o O
'[j 1DH M H 'j'J l0tf10100 01 v V'N d0to O N N N n-IO r1 L;, ~
J-~ l!1M ~ d~ ~O 1.1 d~N O ~ ~ t~
O O
f0 M N H M d' (a ~O01h (~to 10 -r1 -r1 ~-1 0101l~M d~ r-1 O V~L~10a0 M
J-1 J.-) -ri ~DM 101D V~ ~ O M 01I~H t~
-r1 E d~o ao.~ ~ E ioM m av r ,c7 .y -r-i H O ~ O ~ 0100r1LON
v -ri v U o M o o N U n N r-to 0 ,~ ~
~ ~
~
~ 0 0 0 0 0 ~ ~ o 0 0 0 o -r- W -.-i -I
? ~
-~
G s~
O O
.,.~ .,.
1J 1~
(fS H d'00H r~ ~ 01d' N
ri N N CO0101 r-1 M N 10M d' 0101O O O ~ h L~Lf1M h E N d~01~Hd E ('~O h O f~ O
01Lf1t!1L!1Lh O ~ N h l~O h U Lf1N O O O Lf1 U ~OM N N O
U . U ~ H
U
c-I O H N M d' H H O riN M d~ O
!~, Q,' >:." s~
O O
1J Q1 01 .1.1 N 01 ((j 0101M \D (~ 00l~M I~00 r1 O N M O~ r1 U10100n-i00 01OD0001 ~ N 0101ODl!1 E tf1ODO d' E N O1N 41N
M O O ~ N d~01O1N
U 01Lf1N O U O1tf1N H H
U . M U
Ff,' 0 0 0 0 o O ~ O O o o O M
>~ C
O O O
o~r iot~ G -~ rtd~u~ m >~
v r a~M rn O v ~ r-mo M oo O
~ ~
Ol O 01OJ01 -ri al N O n-iN In -r-i -r1 -r1 (IS I~I~lfld' J~ (IS N l~M h N J-1 .~ .~
a,~-I O M 01O ~ S-I I~lf101I~N
-rl -rl v v O 11 d~M r-IO ri v M N O O ri r1 ..C
x ~ o 0 0 0 o A ~ o 0 0 0 o A
-~ -~
~ ~
O1O~ M M 01H M
u~~ o w dmo r ~ U 10N u1 ~0O rit0n-i I ''OU1Q1O ''CSN r-I00l~t~
P7 v ?-IV~l0O ~-I0101ri01L
r-~ r1 x ~ -H M M N -r1M H H O O
r1 r1 ~
'~ ~ ~ C 0 0 0 0 o O ~ ~ H 0 0 0 0 o O
-~ 3 I
O ~ W
r. pq o 00~ H o~
., ~ x M ; ~ a ~u o a o~~,o 0 o a ~ ~o~ H
w ~ -~o M ~ o ,~-~o ~oM ~o ~-I ~
x -'1-I-~U d'N O 1~U M M O O N
~ ~
O H .~a 0 0 0 0 0 0 ~ ~ ~ 0 0 0 0 0 0 v G W
O w w o U H l~Il1a0n-I ~ H l!1H l~M
I N l~O 01 U10001O Ll1 ,"'~, N M tf101 ~ y -I01W 0001 0 (x, J-11001~OV' .y.'O l~ N M ~DN N
H N M 0100~-i ~'a-~ U1 d~V'L~ODCO f,' r1 r-1 U1 1-id~M M O (ISy~ ?.1M N O O O (d r1 r-I
.,1 . . . ,.-I-~ .,1. . . . . ,--I
~ ~
c~ 0 0 0 0 o pp ~ t~ o 0 0 0 o pp 4., 3 ., g O
b ~ 00o w d' G 'd o 0 0 N ~-I l~01O N M H S-I d~~DM LW-i H ml J.-) -ri V~O ODOD01 -ri ril~M LhO7 r1 r-1 ~'-, O O N ~O00 ~'-, H V'O O O
v v E- ~ H ~ H ~I a H r ov~IH H
o .d -'1 C N N 10O O (..,-' O
O l0ODO O I~ H O O a0N r-IN H
.-I ~-1 -~ U L~O O M N O U doh h H Lf1 r1 ri ~ v 01N l0T N ~D N N l~O H M c-I
N v -~ cn 00~ ,-I~ N ,-I ~n t~r .-aH o~ ,-r ~ O
-'1 U J~ O N J-1 ODd'N O
W l0~ON (~l~ r1 Ul l010r1d'IW -1 4-1 ~ H ' ri r ~-I N riN ( r1 r~ 1-I O InCOI~~D n-i O Ci-i ri ~ r-i -ri O ~O00Lf101 v -r1 l~00O O O N
H v ovo~o~,-i~ U v ~ m ,-i~ ri U
f~ f~
,-G
u~ o 0 v .~ -~ -.~
a o x a, it rt E E
y M H S-1 M H
W
H .R J.~ W '~1ri r1 .!-1 M r1 ri , ,"]
f-i M r1 O Ci M H O
H H
v ~ v M H s~ v M ,--I
~ p r1 u7 U M H 1J ~ U M H 1~
O O
?d !~ o O O '4.,' (-'., o o O
'"~ H
c~ -.~ O o O o M ~ O O o 0 o M r-IO
x x H w a o,~,,~M ~ a i a o,M ~ M ,-~a o a O
N u~ ~ ~ b r t m n ao00 L'-V'O1 00M d~ v d' d'~i'M N lD
Ci a ~ N M M h ao +~ ~ ~ ~ m tmn O O
fd r1M W M O h t~ Lf1O d~N l~01 -rl -ri r-1 N l0r1 M ODtn r1 M r1M M M r-i 1J 1~
a 111U1O O~d'M a 00 N 00~ON O~
-rl -rl E h l~N M ~-iO~ ~ V~ N u1r-If-1O
.R ~
a M V'01 ~OM a M U7d'O O
-.1 -.-1 v v U h d~H o o u7 U O N o o O r--~
.~ .~
J.-) 1W
~p U
~
~
Q,' o 0 0 0 o M F(,' o O o o O l0 -ri -r1 ~.I ~-I
C
O O
-.1 -r1 1~ 1~
b N ~OM 01 BLS h h N N M
r-1 O101N I~H r1 M tnr1n-iN
01V'c~ h OD ~ 01 O~10l0LO
h o vo voao E M r-~ ~ ~ O
a ao~o~ d~N o a ra o ,--i~ 0 0 U d~O OD h 10V7 U u1 N .-~Ir-ir1u1 U U ~ U
U
ri O n-1r1 N M H H O r1N M crH
A,' A,' G
O O
-ri J-1 r1N Lf1 J-1 00 r-I
la H M N V'ri (~ h M V~O101 r1 ODl~Lf1O Lf7 r1 I'~n-IM ODOD
a H ~-'~~ ~ ~ a d' d'V'O O
o~,~ r~r ~ M r m un a cmn M ao~ a a~ 0 0 0 0 U M a0d' r-1H U OD W r-~O o U O U O
r-io 0 0 o M ~ o o O o o M
O O O
m r N M ~ G -~ M m ~ G
N o ovm unO v ~ oo~ aoO
~ a O a1-~i O d'O l001-ri a1-r1 O 01M O -ri f~ N I'-d~ 01L~-1.J ((S l0 tf1l0 t171J
.f.~ ,Sa S-I r1N d~ 10n-ia S-1 0~ O 01 O
-r1 -r1 0l N
l~ U1V'N O n--Ir~ N d' r'~1O O ri .~
J..!
C ~ q ~ p ~ ~
~ ~
-~ 0 0 0 0 o - 0 0 0 0 o t V'd'N 1I l -i N L~
O
L(1OJC~ v-11D I~ LC1M ~D
O
O d0O O~W tf1V~M N
'O f~t~V~ N n-i '~ l~ 00V~ U1 S-1l0l~V~ I~N 1-1O1 I~ W -i O r-I r1 p -rid~V~N (W-I -rld' N r1 O
ri r1 H '~
p v 0 0 0 0 0 ~ ~ v ( 0 0 0 0 o D
-~
ro ~ m N O~ N Gq~ M u1N
x lf1N O d'01 C;~ M L~V~
d'd'01 h N O 0 .(".nN 01I~
r-I'-I01a0 O N y~-~O l0 U1N
r1 H U toM r-~O N ~ U d' N O
r-I r1 ~ v ~ t a c 0 0 0 0 0 0 ~ . c 0 0 0 0 0 0 n n v -G
h H 00l0d' 0110 O H tf1~-id~
l~O1M tf1O l0 L~01 M M r1 N O ~. N L~N O
d~o ov d~ovx 0 ~ m o,h x UI U1H 01 N O ~i.~ W 01 L~d' 'Sr' r1 ri S-1Ind'N n-iO (ISy~ ~-1d' M r1 c0 r1 r1 O . . . . . ,--~-~ .,1 O
g o 0 0 0 o W ~ ~, 0 0 0 0 o pq .r-, 'b N 0010 ro ~
~-I O O 1D OJO H ri N 00H Lf7 ri -ri Lf1lDN r1d' -ri 01 r-1N d~M
r1 r1 ,i; M H M l0Ll1 ~,' L~ M O M H
v v E-~ 0~~ ~ .-1~ O (-~ m ao~ ~ ~ D
3 g .(", 00CO d~N N ('., N N V~
O 1ON N H M l0 O 00 V'r11DM
r1 r1 U r1l0N d~l0O1 U 41 M N M 10 ri ri v U7O d~ h M ~O v r1 Lf1H N r-1 N v cn ao~ ~ ~ ~ ~ u1 ~ o0 O
U
+m r ~ ~r~ O ~ N aoO
m aod~om o ao~ m ~ o o d~ri~
~ ~
?-I riM N M M r-1 S-1 OD InM 01d~r1 ri r~
H -ri 00O N tnl~v -.-I t~ r1N M N N
N N
C~ t~~ ,-ir-i~ U C.-~ u~ r o~,--i,-iU
O O
v ~ ~
s s ~ M H 1 M ri 4i M r-iO C M H O
H H
C; N M H 1-I N M ~-iSJ
~' O U M r1L1 U M r11~
O O
U ~ 0 0 0 G C 0 0 0 ~ ~
O O O O O M ri0 O O O O M r1O
X x tn U o~M ~ M ~ U U a~ M .--iM ,~U
D O
O
M r M M O
-r o rn t~
N ~ N N 111M M
C
1.~ Lf1r-1~0 v-1lfl01 J-1 N O
O O
(d 01 01 r1 01 lf1l0 (~ 00 O
-rl -r1 r r-~ O1 Lf1di H r 01 rI M 01 l~ 1J
N
-rl !I1OD 10 Lf1a0 d' ~ 00 10 -ri O
~ O ~ r N
p N O ~' r 00 ~ r r d~ r1 o O N -L', W , . n U
U 0o . d ~' U
~ ~
rtf -ri O O o o O M F~ o o S-1 -ri ~.I
O o o l0 1~ 1.1 td 61 M N o-Ir r1 l0 O M M r-1 r1 d~ r r r 10 M l0 O ~ M W O~
00 tf1O
~ CO M O ~ M l0 l0 l0 N M 00 O ~ ri M M M O
lI1O 01 ' O r 1D In U in N N N (f1 F, ri O H r~ N M H r1 O 1 O
FC A
-rl ' -O
, r N d'H
O O
--i -ri IlS M N l0 Ln In tO (O M
ri CO U1 CO r1 r1 r-I N r E ~O tn N lD If? E M t0 W --iu~ o m n ~ ~ r U M c0 d' N O r N
U O U
~C ~ o 0 o O M ~ o o 0 o G ~
O M
r-IlD W -1 r-aj"" -r1 c--I
N M 1D r O ~ O N t(1r O
J~ 11 ~ -ri ~ (d ri M l0 O U1 1J ((S (p .Q ,L~ ~
~ 1 0 N di V~ r1 O r1 N V~ N
.4' ~
~1 S-1 .
~ 0 0 0 0 o A ~-~ o 0 0 q p 0 o U
N
w r io ~ o p o r w O O (f7lp r O N
b H
~ ~
~ N r r ~-1N ~D
ri '" -r1~ d' ri N O -ril!1M
r1 r1 H ~
v O O O O O D O [ O O O
N 1D r N H -~ ~ N
O p 1J1.1 1~
r-1 -rlfI$
~-I ,S]S-I d~ t!1r M y..~
~ m N
-rl O l0 r d' O~
" ~ ~"''~ O d' ,"O F. M 00 H d~ ~ tnO C: N O
H -riO M O Lf1d' 01 "',l,'-rlO ~ M
i-i ri a. V' N N O 1-1U ~N N
r1 -.-IN
QJ
.~ ~n 0 0 0 0 0 0 ~ q ~ a o 0 , 0 0 0 C: M U ~ V~
H l0 N H cH v H ~l't!1 O
-I r N !n w t0 N
M
4 w M o~ D w au r D a0 u~
l~ c0 er a0 O ~ x Ul N r M r-~ ("" UI O1 M N
r1 ri N
S-1V~ ~i O ri Il3~" S-IWit'N ~ M
ri ri -ri . . . ,-1O -r1 . ~ r-1 Cu 0 0 0 o Cq-~ f=.,0 0 0 0 o W
~ ~ O N
w ~ M N ~ ~ r1 ~D 1D 0 r V ~A
1 f ' -iI O - r1 W 01 M H M
I , r~
H3 ~ ~ m ~ ~ H M
m o -r O N3 ~ ~ ~ ~ - O
o r r 'b '~ ~r f; N 00 d' l0 d~ ~ O N 1DOpM
-I ' d' O
r d M OD H SO d' O CO O lDN M N ~
r1 0 O vo m M u~ U N ov M m M u~N
ri o ii m Q ~ ~ ~ ~ ~ N n ~ ~ ~
v t o ~ a -r' o 1 O o -, ~ ,-i d~
O ~ N O CO
O Ul N cr H d0 N r1 U1 l0 N N U1V'r1 r1 ri S-1 O ~i M O 01 r( ~-1 r1 d~ d~l0H -i r1 r-1 w ~ 1 ~ ~ N U ~ ~ N r 3 ~
w a f. N ~ , ,-~ u~
o ~ -ii U ..
H
~n H
N
G ~
N
-.-i O y~
b1 U .L~
~
a ~
M -1-IF~, v n M H ~.1 N
\
M ..j U M H yJ U , ~
~C
Gi O O O H M r11Jr.C
c ~i O O O O M r-iO O O O
O O
O v1r-i H U 01 M r-iM H U U 01 t r1 H
M r-1M r1a x x b d~ '~ tD
N N N M
G G
~ ~ a ~ m o o ro ~ ro ~ o -~ -.~
r o ~ 00 -.1 E m w E u,o w ,c~ .d ~ ~ ~ ~ ~
-~ -~
a~ a~
U~a o a U.~i~ ~ o a U ~ U
~ G
ro ro A,' O O O O O Cx, F(,' O O O O O fi-, -r-I -rl S.-I S-1 L"
O O
-ri -ri 1~ i~
ro M M M M M ro InIn lf1Lf1 ri U7Lf1Lf1tf11f1 r-I O O O O
N N N N N ~ N t~h l~L
r-W-1r1 H n-I~ ~ 01O O O O
M M M M M O ~ N H r1 n-1H O
U 0101O1 0101u1 U 0000a0 00ODu1 U U ~ U
U
H O ~-1N M d~H H O H N M V~H
~[', A', G
O O
-rl l~ l~ h ro ~ ro ~ ~r r-i d' r-~ 0101 f~ ~ N V' W
~D ~ DOv-f U O U ~ o U O U
FI,' O O O O O M IS O O O O O M
N
x O 0 O
-r-I h Li -.-i Lll O N d~ O N o~ O
~ t~
1J ro O ~0 1J
.~
.J-1~-I l0 ~ S-I h v-I
-r1 -ri ~ QJ
PO U N o r-I N --Io r-I
f'.. ~
1~ 1W
x a~~ -,~ ~
a ~
ro ro w r.~ 0 0 0 0 o to FC 0 0 0 0 0 q -~ -~
~ ~
w N
ro 01 V'N
Gi N 111(~
b l0 '~ O d~
U1 1-1 S-il~ S-1N l0 r1 r1 N Cp -r1 -rlH -r1Lf1M
r1 r1 H ue 0 0 0 0 o O ~ ~ C 0 0 0 0 o O
-i ~ ~ ~ m H r1 f~ 1010 -a x ~ o m ~7 p d r N
O N ~ ~ O V~lf1 r1 ri U O -,.iU N O
r1 r1 w ~3 U ~3 a :~c o o o 0 0 o ~ o o 0 0 0 0 n w w H H
N O
V~
W ~.iO N LO
ri ri ~ ro-~ ~ ~ ro r-, f-~
F: -.1 ri~ -ri r-1 N N
H fi,0 0 0 0 o pq~ G~~0 0 0 0 o W
~u -0 d N O DDM 's''L~ ODV' O ~O
~-I O 00O l0OD ~"L-I 00N M O OD
r1 r-i r1 L(1H d' riO H -rl Ll10001 O OD
ri ri ,5.," 01tt7~O l0N ,L", ~DO M N O
N N
ro H 0~,--i~ ~ ~ O E a~
~7 O
~ d'O 10 N M d~ G O O N ~OlD
O N 01H d'00d' O ~OV~OD d'OD40 r1 ri U h ODI~ O O U1 U 01M 01 M OD00 r-I r1 N n-I10r-1tl7N l~ N r1O N N O O
N N
cn .-a~ N ,~~ ~ cn ao~ '--i,-i~ ,--i a ao00o w M ~.m o o ~r~ o V7 r110~O lf100r1 Ul lDW If1M 00r-i r1 r1 W ~-I 0100h 00O r1 S-I N 01N l~ODr-i ri r1 O -.-1 d'd'~O O N N -~ N O N N O N
N N
G4 r-I,-~r-~N r-iU Cr., a~r-I~-Ir-ir-~U
G
~n O O
N -~ -~
O O
~
~
. M n-Id~ ~1 M r1d' \
N l.~ M H O r-i .U t'~1w-~iO r1 '-~- ",~' ("-, M ril~O S~ M r1C~O
H H
N N M n-IM ~-I N M w1M S-1 d' d' r-i U M r1O 1~ U M r-iO 1~
~ ~
f~''-i O O C~~ C.'H O O f~
ro O O O M H O O O O M H O
X X
H U M n-1t'-WIM U U M ~--IM ~-1M U
~ O
~ro~ao N r 21 .-iM m o ao N r ~ r a~r N ~ ~ u~,~M
~ G
1-1 N ~fl\D tJlM 11 r~O 01d~00 O O
(d O d~l0 ~OH Ib L~N l0d'h -ri --I
r-i r1N H O O r-I d~N O O \0 r1 h O 01 M 00O ~ ~Od~ lDf~H W
-ri ,G~ d'N Q1 L~Lf1W E V'O M N N
,.Q
~ tf1O N O O U~ ~ N r1 O O O
-ri -r1 N N
U N ~ 0 0 o U 0 0 0 0 0 .y..~ .W
..~
U . . ~ U
C ~
r~ ~6 r.~ O o O O O fz, ~ O o 0 0 o O
-.-i -ri S-1 ~.-1 C
O O
-,-r -t~
ro u,~ N N M ra r-I N ~OM M V'O r1 U1cr d~d~M
COI~N N QO ~ l~l0 1ptDOD
tf1I~r1 H M E toU1 N tf1crO
Q~I~N N O~O ~ 01N 0000L~
a aor ~ r ~oin U o~m o~~ o~U
i . . . . U i H
U U
H O r1N M d~H o-i O v-iN M d~
~[,' F~, O O
1~ l~ l~ 1~ 111 r~
(~ h N H 01 t0 M N N a000 r1 U1ODN 0001 r1 lpH O O O
'-i01O~ M M ~ ri01 00W 00 E ~ ,-iM r r E u~0 0 0 0 O O OD N N ~ N N H ~-Ir-i U M N o 0 o U o 0 0 0 o U U
F(,' O O O O O M ~C, O O O O O M
~, ~ O O
' - Lf1N ri .('., d' 00C', ' O N r ~ M o~O N n o o O
~ W
,NO1-ri r1O lf1 M -r1 ~71-ri N H OD-rl 1~ d~07~O L~.l-1 f13 V~O O 1~
.~ .~
y~?-1 O v-iIn N ~ S-I O r1 ri -ri -ri N N
U v H H O O r-i N o o O r~
,t," .fi 1~ 1.) q ~ q ~
~
w w 0 0 0 0 o -. 0 0 0 0 o -a W n o~ r 01 ~ d W 0 W
O N n-1 N
'b O N 'b aD
S-1 O~ OD 7-r N
ri r1 M x ~ -rl O O -rl O
ri r-I
H '~
b ~ ~ 0 0 0 0 o O ~ v E 0 0 0 0 o O
-~
a~
H ~-I 0101 ~ ~'-~ M
~' ~ x f~' ~
, b 00d j~ l M
" -' O .( O N O O ~.,N O
., ''1O N r1 ,N''1O v-1M
r1 r1 U N N ~ U 0 0 ri r1 ~ a O .~3 0 0 0 0 0 o U .s7r 0 0 0 0 0 0 n ~ G
r , ~, O H Lf71D01 ~ H (~
IflL~d' M
y o r M
G ~ o .~ .-i S~ o ,-io ~ ~ ~ o it ~ r-i ,1 _,~ .
H v f~ 0 0 0 0 o W ~ t=,0 0 0 0 o p4 O
'b ~0N d' O O .~.,''Cj d~O ~OO O~
O S-I O l!101 d~N H S-I M Lf1a0N O
ri r-I
-r~ M N H M M -ri N M O O tf1 ri ri ~i I~~Od' ~Od~ .Li N N tf1M n-i N N
~ a H ,~,~ ~ ,~,~o G
o b rd -r1 W O 00d' d'N N (.,'' N V' ~O00V~M
J-1 O lf1ODM M InO O d1CO 01N 00V~
rl r1 -r1 U H N a0 O 10~O U l0d' 100101OD
r1 r1 N N N d0 10I~Lf1 N n-Iri d'N ~-iri N N
u~ ~ ,~~ ~ ~ ,~ cn .-~,-a~
~ G
N o ~r N w o ~ o o w N d~a 0~ m r o~~ N o ~ m aow o~M o~
~ ~--i ~ M M m n ~ ~ o m m o ~
~
O N -r~ ~ M d' Lf1h N -ri N N N t1W-1UJ
O N
w ~-I~-ir~ r~r-~U , C~ r-~r-ir~r-Ir-iU
m ~
r~ ~..
C-~' G
u~ O O
-r1 'ri ~
R: U J-1 1~
r~ .-1 a to ~
E E
,4 M r1 LI M n-I
\ \
M .O J.-I M rir1 1~ W ~1'-iri ,."7 '~
Ci M r1O Li M n-IO
H H
N 11 O M ri~.1 N M r-I.~1 a' d' r-I UI U M r-iJJ U r'W--~1~
O O
,L? ~-1 C." O o o ~ ~ O O O
~"~ H
(a -ri O O O O M r1O O O O O M n-fO
x X
E-a C~, U ovM ~ M ~ U U o~M ~ M ~ U
O O
b umn ~ ~ o~
v O O lf7 N M 'd~O
.(-', Ci, y m O m -i r w o M
O O
rtf o ~ d~ ~o r m o o -~ -~
~ ov~ ov .-~ ~ ~ d~m +~ ~
N ~ r O ~ ~n O w r O
-~
M 10 U1 Cra ~ r 01H M (c~
.L~
-ri N d' O V7 ~ N C N c-i U7 v -ri v N o o ~ '-~0 0 0 v ~ ro ~ v ~
r.~ 0 0 0 0 o t~, ~ o 0 0 0 o Cv -~ -~
?.~ ?a G C
O O
(~ M Lf1LllLll fa l0 01d~m m r-i M 111~Ol0l0a ri 01 o M M M
10N M M M ~ o~ r m m m 01n-I1nUlLfl E m M M Ol01 O d' N N N O ~ Lf1N H Lf7(f1O
U m r r r r U7 U O1 O141m m t(1 i U i U
U U
r-i O v-IN M V~H o-I O r1N M d~H
~, F[,' O O
-ri -rl 1~ m .L W -1 lf1 (~ Lf1r t11 ftS N m H tD
M to m m o u~r o~
~DN m ~ r1 r1m 01 tf1 ~ O 01M M
r m ~ ~ a o~m n U N O o U r1 O o O
U U
FI,' O O O O O M A~ O O O O O M
Ci i.~' O O
-ri r M Lf1 ~ -r1 d' r d~l0 Ca' v o m m O v O m r o~ O
W -a dl-ri M M m -ri (TI-rl O N m 01 -rl (a O m In yJ (a r1 t1101M 1~
,.Q ,!a o,~ ~ ~ 1a d~ M o u~
-~
v v ~ o o .1 v 0 0 0 0 ~ ,W
~r .-~
-r., ~
v b -.~ 0 0 0 0 o q r.C 0 0 0 0 o A
S~ -~
~, i' io r m U
m r m M
~ N r1 01 M t11 '~ M 01 ?-Im m Sd d~ N
ri r1 -r1r W -riO O
ri -1 n-I
H '~
v 0 0 0 0 o O v t 0 0 0 0 o O
o -~
ro ~
. ~
u~ ~ W n u~ o~
~0 to x to N N
'b 1f1 1D ~ '~ l0 M Lf1 ~ O
H
U H O U O O r-i r-i r1 a ~
~
c 0 0 0 0 0 0 ~ .s13 0 0 0 0 0 0 n W
r " o H r1r ~ H r-Ir M 10 Lf1r r O N N
O m ~ ri Ldl0U7 ~
U1 O C'..~ UI M 01N c-1 L;
r1 r~
S-~N O (!fy~ 1-IO O O O fO
ri r1 -~.~ -~
~
t=,o 0 0 0 o p7,1Z f~ 0 0 0 0 o a1 g _ 3 Tf ,S'.,'C3 d' ~ON d'm ~-1 m d~ O N m H ~-I r rim d~N
r1 r1 -rl O 01 O1m V~ -ri N r 01d'm r1 r1 ,Tr' d'M r1~Om ,~ N M M N N
v v H ~ ~ m ~ r O H ~ ~ ~ ~ ~ O
b C m m m vom m O ~DN d''d'm m O ~O O l0d'N N
r-~ m1 U r o~ r a d~d~ U N r ~ M m m ~ .-i v M O d'H m m v N N M r1N N
v N
u) voo~ r m r r cn r-i.-i,~
v tr a-i O ~ ~r o m vom O
U1 m N O O m r1 U1 ~O 01d~r1N ri r1 ri v S-I r 01 10M V'r1 ~-1 M lfld'10m r1 ri r1 -ri N l0 d1Lnm N -ri N H N N N v N N
C=, vor m m r U fj.~ ~ ~ ~ ~ ~ U
H
O O
U -~ -~
O O
1J t~ 1W
r-~ 1 r~ td ~
~I M r1 ~1 M r1 \ \
1J M n-Iri JJ M r1r1 "~] ,"',]
'[f ~.,' ', M H O C,' M r1O
H H
~i N I'. M H S.1 v M r11-I
d' d' O U Ii M r-IJ.! U r1r-1J.-!
O O
U ~ o I o . ~ ~ 0 0 0 p ~ o ~ I
v O o 'I o M ~ o O o 0 o I ~
X o x M
m U ovII,r-iM r-IU U o~ M ~ M ~ U
D M O
N M ~O r1 r v U1 Ci --fa J-1 r U1 O l0 1.J r ftS r1 ~D M ri (IS l0 -r-1 -r1 .-a ~ r ~o ~r ~
~ D
E H N t11r1 W ~ tf1 .~ .~
-r1 O W ri O Cn ~ r1 V1 v -r1 Ol O O O O
U N ~ U ~C
~ p N (IS
b 0 O o o O C7.n ~ 0 0 0 o O fia -ri 111d' r ri '~ l0 ~-1 -.~i O
O
1J 1~
f~ 01 M r1 H O (fS M M M M M
r1 d~ ~D 00 O O ri OD OD0000 CO
p O1 ~p l0 10 ~ O O O O O
M ~O r N N ~ d' d'd'd' l0 N OD OD COO ~ OD 00N 00 4DO
U 00 00 r r r u1 U T 0101O\ O\tI1 i . . . . U 1 . . . . . U
U U
-I O r1 N M d'H ri O H N M V'H
L~ ~
('-., .('-, .,1 -ri IJ 01 CAD 1~
01 r oo ~ r~ r 01 ~ In m M d~ M O
r O d' t11 ~ U1 r1 00 d~ O ~ H
U N o 0 o U O
U U
(,'' O O O O O M F.~ O O O O O M
~
O
ri ~D N C,' ' r f.,' u1 01 r m O W ~ O
-~
(J) 01 O N O -r1 t31-rl O7 -fi -ri v N
~o ~, .a ~
1-I M M M O ~ ~-1 H
-ri -'1 (C IlS
v r~ O O O '~ ~-1 ~
~.i h .i ,5 -'1 ., -!-'a 0 0 0 0 o Ca ~-~ 0 0 0 0 0 ~1 ~
U
Q 01 ~O O
M N N
~ O O N ~
r1 H
-ri N r1 O
r-i N ,i.,'' N
O
H o o 0 0 0 ~ ~ E-~0 0 0 0 o O
3 M a1 01 3 u1 ~
-I ~ ~ ~ ~ 0 p .-i O P ~
G y~U ~ 0 U
r-I ~-i -~v x .,~N
v v H W o 0 0 0 0 0 ~ cn 0 0 0 0 0 0 g io 3 H 01 d~ d' y~H lfl u1 u1 N Q r M ~, x v ~ r x (A l0 01 r1 C.,''y.~ N V' .f", r1 r-i S1 O O O cd~ ~1 O (a r1 r-i -r-I. . . ,.-~~ -r-~ r-i N v fl-,0 0 0 0 o pp C.,0 0 0 0 o W
O
.d 1J'~ N ODl0O N
S-1 aD N N ~0 O ''1S-1 O 01d'l0 O
r-i r1 ri tn O t11r d' ~ -rl 01 r I!1M 10 r1 ri ri 01 1f1vD r ''1~ rl '-1N N r1 N
H ~ ~ r oo r D ~ H ~ ~ ~ ~ ~ O o H O
~ A
~. ~0 l0O OO N N
O N l0 10 l0 O O O t!101~Dtf1O O A
ri r1 U d' OD N l0 d~d' U OD a0rid' tovO
r-I r-I
v o r r m r r v ~ N M N
W v cn ~ ao 01 m r r ~n t~ N a ~ ao d woo N ~
W O r l0 N O r1 U7 W t11d'a0 O r1p r1 r1 Y-1 d' O ~-IN V~r1 S-1 O d'00l0 ~Or-i r1 r1 -r1 N v-1O ~L1r v -ri v-IM N N r1~ ..
v N
r ~ r r r U C~ ~ ~ ~ ~ ~ U o U
o O 'n H
w -1 ~-f CJ
H X X ~
H
N
- (d U 1-i 1, ' 1~
~ ~
~
~ M ri j ~ M r1 r yJ M rir1~.J-1 M rir1 ,~
M ~ O ~ M .--i0 s ~ ~, O
' ''t~ N M r1?-1',N M r1~-I~
~ J7 al S-a U M r1J-1~~~U M .-I-1.-!FC
~ ~ " FC
-ri C; O O O Fi' Ci O O O .,N
H ~ H ~ cn O O O M r1O I O O O O M riO (Yj a W
H U o1 M ~ M ~ U ~ U 01 M ~ M ~ a x p p x
1) Antigen inhibition rate (%) - A x 100 A = cpm of control group; B = cpm of test group;
2) Counting the half-efficiency concentration of the medicine Antigen inhibition IC50 = Antilog (B + A - B x C) A=log>50% medicine concentration, B=log<500medicine concentration, C=log dilution power 3) SI of interspace-conformation changed rSIFN-co effect on HBsAg and HBeAg in 2.2.15 cell culture:
SI -T~sn ll.J U
4) Estimate the differences in cpm of each dilution degree from the control group using student t test Southern blot: (1) HBV-DNA extract in 2.2.15 cell: Culture cell 8 days. Exsuction culture medium (Separate cells from culture medium by means of draining the culture medium.).
Add lysis buffer to break cells, then extract 2 times with a mixture of phenol, chloroform and isoamyl alcohol (1:1:1), 10,0008 centrifuge. Collect the supernatant adding anhydrous alcohol to deposit nucleic acid. Vacuum draw, re-dissolve into 20~,1TE buffer. (2) Electrophoresis: Add 6XDNA
loading buffer, electrophoresis on 1.5% agarose gel, IV/cm, at fixed pressure for 14-18h. (3) Denaturation and hybridization: respectively dip gel into HC1, denaturaion buffer and neutralization buffer. (4) Transmembrane: Make an orderly transfer of DNA to Hybond-N membrane. Bake, hybridize and expose with dot blot hybridization. Scan and analyze relative density with gel-pro software. Calculate inhibition rate and IC50.
Results Results from Tables 1, 2 and 3 show: After maximum innocuous concentration exponent culturing for 8 days with 2.2.15 cell, the maxima is 9.0~Ox106IU/ml and the average - 7 _ inhibition rate of maximum innocuous concentration rSIFN-co to HBeAg is 46.0~5.25a (P<0~001), IC50 is 4.54~1.32X106IU/ml, SI is 3.96; rate to HBsAg is 44.8~ 6.6%, IC50 is 6.49~0.42x106IU/ml, SI is 2.77. This shows that rSIFN-co can significantly inhibit the activity of HBeAg and HBsAg, but that the IFN of the contrast group and INFERGEN cannot. It has also been proved in clinic that rSIFN-co can decrease HBeAg and HBsAg or return them to normal levels.
The following are some examples for the preparation of rSIFN-co:
Example 1: Preparation of lyophilized injection a) rSIFN-co 3 x 106 IU
b) citric acid 0.2 mg c) dibasic sodium phosphate 2.5 mg d) NaCl 4.0 mg e) dextran 20 mg f) Polyoxyethelene anhydrosorbitol monoelaeo-acids ester 0.1 ml g) inject water to a level of 1.0 ml Preparation technique: Weigh materials according to recipe.
Dissolve with sterile and pyrogen-free water. Filter through 0.22~m membrane to de-bacterialize, preserve at 6 10°C. Fill in vials after affirming it is sterile and pyrogen-free. Add 1.0 ml solution to each bottle, and lyophilize in freeze dryer.
Example 2: Preparation of liquid injection a) rSIFN-co 3 x 106 IU
b) citric acid 0.2 mg c) dibasic sodium phosphate 2.5 mg d) NaCl 4.0 mg e) dextran 20 mg f) Polyoxyethelene anhydrosorbitol monoelaeo-acids ester 0 . 1 ml _ g _ g) inject water to a level of 1.0 ml Preparation technique: Weigh materials according to recipe.
Dissolve with sterile and pyrogen-free water. Filter through 0.22~m membrane to de-bacterialize, preserve at 6-10°C. Fill in airtight vial after affirming it is sterile and non-pyrogen at 1.0 ml per vial. Store end product at 2-10°C, and protect from light.
~o O
'[j 1DH M H 'j'J l0tf10100 01 v V'N d0to O N N N n-IO r1 L;, ~
J-~ l!1M ~ d~ ~O 1.1 d~N O ~ ~ t~
O O
f0 M N H M d' (a ~O01h (~to 10 -r1 -r1 ~-1 0101l~M d~ r-1 O V~L~10a0 M
J-1 J.-) -ri ~DM 101D V~ ~ O M 01I~H t~
-r1 E d~o ao.~ ~ E ioM m av r ,c7 .y -r-i H O ~ O ~ 0100r1LON
v -ri v U o M o o N U n N r-to 0 ,~ ~
~ ~
~
~ 0 0 0 0 0 ~ ~ o 0 0 0 o -r- W -.-i -I
? ~
-~
G s~
O O
.,.~ .,.
1J 1~
(fS H d'00H r~ ~ 01d' N
ri N N CO0101 r-1 M N 10M d' 0101O O O ~ h L~Lf1M h E N d~01~Hd E ('~O h O f~ O
01Lf1t!1L!1Lh O ~ N h l~O h U Lf1N O O O Lf1 U ~OM N N O
U . U ~ H
U
c-I O H N M d' H H O riN M d~ O
!~, Q,' >:." s~
O O
1J Q1 01 .1.1 N 01 ((j 0101M \D (~ 00l~M I~00 r1 O N M O~ r1 U10100n-i00 01OD0001 ~ N 0101ODl!1 E tf1ODO d' E N O1N 41N
M O O ~ N d~01O1N
U 01Lf1N O U O1tf1N H H
U . M U
Ff,' 0 0 0 0 o O ~ O O o o O M
>~ C
O O O
o~r iot~ G -~ rtd~u~ m >~
v r a~M rn O v ~ r-mo M oo O
~ ~
Ol O 01OJ01 -ri al N O n-iN In -r-i -r1 -r1 (IS I~I~lfld' J~ (IS N l~M h N J-1 .~ .~
a,~-I O M 01O ~ S-I I~lf101I~N
-rl -rl v v O 11 d~M r-IO ri v M N O O ri r1 ..C
x ~ o 0 0 0 o A ~ o 0 0 0 o A
-~ -~
~ ~
O1O~ M M 01H M
u~~ o w dmo r ~ U 10N u1 ~0O rit0n-i I ''OU1Q1O ''CSN r-I00l~t~
P7 v ?-IV~l0O ~-I0101ri01L
r-~ r1 x ~ -H M M N -r1M H H O O
r1 r1 ~
'~ ~ ~ C 0 0 0 0 o O ~ ~ H 0 0 0 0 o O
-~ 3 I
O ~ W
r. pq o 00~ H o~
., ~ x M ; ~ a ~u o a o~~,o 0 o a ~ ~o~ H
w ~ -~o M ~ o ,~-~o ~oM ~o ~-I ~
x -'1-I-~U d'N O 1~U M M O O N
~ ~
O H .~a 0 0 0 0 0 0 ~ ~ ~ 0 0 0 0 0 0 v G W
O w w o U H l~Il1a0n-I ~ H l!1H l~M
I N l~O 01 U10001O Ll1 ,"'~, N M tf101 ~ y -I01W 0001 0 (x, J-11001~OV' .y.'O l~ N M ~DN N
H N M 0100~-i ~'a-~ U1 d~V'L~ODCO f,' r1 r-1 U1 1-id~M M O (ISy~ ?.1M N O O O (d r1 r-I
.,1 . . . ,.-I-~ .,1. . . . . ,--I
~ ~
c~ 0 0 0 0 o pp ~ t~ o 0 0 0 o pp 4., 3 ., g O
b ~ 00o w d' G 'd o 0 0 N ~-I l~01O N M H S-I d~~DM LW-i H ml J.-) -ri V~O ODOD01 -ri ril~M LhO7 r1 r-1 ~'-, O O N ~O00 ~'-, H V'O O O
v v E- ~ H ~ H ~I a H r ov~IH H
o .d -'1 C N N 10O O (..,-' O
O l0ODO O I~ H O O a0N r-IN H
.-I ~-1 -~ U L~O O M N O U doh h H Lf1 r1 ri ~ v 01N l0T N ~D N N l~O H M c-I
N v -~ cn 00~ ,-I~ N ,-I ~n t~r .-aH o~ ,-r ~ O
-'1 U J~ O N J-1 ODd'N O
W l0~ON (~l~ r1 Ul l010r1d'IW -1 4-1 ~ H ' ri r ~-I N riN ( r1 r~ 1-I O InCOI~~D n-i O Ci-i ri ~ r-i -ri O ~O00Lf101 v -r1 l~00O O O N
H v ovo~o~,-i~ U v ~ m ,-i~ ri U
f~ f~
,-G
u~ o 0 v .~ -~ -.~
a o x a, it rt E E
y M H S-1 M H
W
H .R J.~ W '~1ri r1 .!-1 M r1 ri , ,"]
f-i M r1 O Ci M H O
H H
v ~ v M H s~ v M ,--I
~ p r1 u7 U M H 1J ~ U M H 1~
O O
?d !~ o O O '4.,' (-'., o o O
'"~ H
c~ -.~ O o O o M ~ O O o 0 o M r-IO
x x H w a o,~,,~M ~ a i a o,M ~ M ,-~a o a O
N u~ ~ ~ b r t m n ao00 L'-V'O1 00M d~ v d' d'~i'M N lD
Ci a ~ N M M h ao +~ ~ ~ ~ m tmn O O
fd r1M W M O h t~ Lf1O d~N l~01 -rl -ri r-1 N l0r1 M ODtn r1 M r1M M M r-i 1J 1~
a 111U1O O~d'M a 00 N 00~ON O~
-rl -rl E h l~N M ~-iO~ ~ V~ N u1r-If-1O
.R ~
a M V'01 ~OM a M U7d'O O
-.1 -.-1 v v U h d~H o o u7 U O N o o O r--~
.~ .~
J.-) 1W
~p U
~
~
Q,' o 0 0 0 o M F(,' o O o o O l0 -ri -r1 ~.I ~-I
C
O O
-.1 -r1 1~ 1~
b N ~OM 01 BLS h h N N M
r-1 O101N I~H r1 M tnr1n-iN
01V'c~ h OD ~ 01 O~10l0LO
h o vo voao E M r-~ ~ ~ O
a ao~o~ d~N o a ra o ,--i~ 0 0 U d~O OD h 10V7 U u1 N .-~Ir-ir1u1 U U ~ U
U
ri O n-1r1 N M H H O r1N M crH
A,' A,' G
O O
-ri J-1 r1N Lf1 J-1 00 r-I
la H M N V'ri (~ h M V~O101 r1 ODl~Lf1O Lf7 r1 I'~n-IM ODOD
a H ~-'~~ ~ ~ a d' d'V'O O
o~,~ r~r ~ M r m un a cmn M ao~ a a~ 0 0 0 0 U M a0d' r-1H U OD W r-~O o U O U O
r-io 0 0 o M ~ o o O o o M
O O O
m r N M ~ G -~ M m ~ G
N o ovm unO v ~ oo~ aoO
~ a O a1-~i O d'O l001-ri a1-r1 O 01M O -ri f~ N I'-d~ 01L~-1.J ((S l0 tf1l0 t171J
.f.~ ,Sa S-I r1N d~ 10n-ia S-1 0~ O 01 O
-r1 -r1 0l N
l~ U1V'N O n--Ir~ N d' r'~1O O ri .~
J..!
C ~ q ~ p ~ ~
~ ~
-~ 0 0 0 0 o - 0 0 0 0 o t V'd'N 1I l -i N L~
O
L(1OJC~ v-11D I~ LC1M ~D
O
O d0O O~W tf1V~M N
'O f~t~V~ N n-i '~ l~ 00V~ U1 S-1l0l~V~ I~N 1-1O1 I~ W -i O r-I r1 p -rid~V~N (W-I -rld' N r1 O
ri r1 H '~
p v 0 0 0 0 0 ~ ~ v ( 0 0 0 0 o D
-~
ro ~ m N O~ N Gq~ M u1N
x lf1N O d'01 C;~ M L~V~
d'd'01 h N O 0 .(".nN 01I~
r-I'-I01a0 O N y~-~O l0 U1N
r1 H U toM r-~O N ~ U d' N O
r-I r1 ~ v ~ t a c 0 0 0 0 0 0 ~ . c 0 0 0 0 0 0 n n v -G
h H 00l0d' 0110 O H tf1~-id~
l~O1M tf1O l0 L~01 M M r1 N O ~. N L~N O
d~o ov d~ovx 0 ~ m o,h x UI U1H 01 N O ~i.~ W 01 L~d' 'Sr' r1 ri S-1Ind'N n-iO (ISy~ ~-1d' M r1 c0 r1 r1 O . . . . . ,--~-~ .,1 O
g o 0 0 0 o W ~ ~, 0 0 0 0 o pq .r-, 'b N 0010 ro ~
~-I O O 1D OJO H ri N 00H Lf7 ri -ri Lf1lDN r1d' -ri 01 r-1N d~M
r1 r1 ,i; M H M l0Ll1 ~,' L~ M O M H
v v E-~ 0~~ ~ .-1~ O (-~ m ao~ ~ ~ D
3 g .(", 00CO d~N N ('., N N V~
O 1ON N H M l0 O 00 V'r11DM
r1 r1 U r1l0N d~l0O1 U 41 M N M 10 ri ri v U7O d~ h M ~O v r1 Lf1H N r-1 N v cn ao~ ~ ~ ~ ~ u1 ~ o0 O
U
+m r ~ ~r~ O ~ N aoO
m aod~om o ao~ m ~ o o d~ri~
~ ~
?-I riM N M M r-1 S-1 OD InM 01d~r1 ri r~
H -ri 00O N tnl~v -.-I t~ r1N M N N
N N
C~ t~~ ,-ir-i~ U C.-~ u~ r o~,--i,-iU
O O
v ~ ~
s s ~ M H 1 M ri 4i M r-iO C M H O
H H
C; N M H 1-I N M ~-iSJ
~' O U M r1L1 U M r11~
O O
U ~ 0 0 0 G C 0 0 0 ~ ~
O O O O O M ri0 O O O O M r1O
X x tn U o~M ~ M ~ U U a~ M .--iM ,~U
D O
O
M r M M O
-r o rn t~
N ~ N N 111M M
C
1.~ Lf1r-1~0 v-1lfl01 J-1 N O
O O
(d 01 01 r1 01 lf1l0 (~ 00 O
-rl -r1 r r-~ O1 Lf1di H r 01 rI M 01 l~ 1J
N
-rl !I1OD 10 Lf1a0 d' ~ 00 10 -ri O
~ O ~ r N
p N O ~' r 00 ~ r r d~ r1 o O N -L', W , . n U
U 0o . d ~' U
~ ~
rtf -ri O O o o O M F~ o o S-1 -ri ~.I
O o o l0 1~ 1.1 td 61 M N o-Ir r1 l0 O M M r-1 r1 d~ r r r 10 M l0 O ~ M W O~
00 tf1O
~ CO M O ~ M l0 l0 l0 N M 00 O ~ ri M M M O
lI1O 01 ' O r 1D In U in N N N (f1 F, ri O H r~ N M H r1 O 1 O
FC A
-rl ' -O
, r N d'H
O O
--i -ri IlS M N l0 Ln In tO (O M
ri CO U1 CO r1 r1 r-I N r E ~O tn N lD If? E M t0 W --iu~ o m n ~ ~ r U M c0 d' N O r N
U O U
~C ~ o 0 o O M ~ o o 0 o G ~
O M
r-IlD W -1 r-aj"" -r1 c--I
N M 1D r O ~ O N t(1r O
J~ 11 ~ -ri ~ (d ri M l0 O U1 1J ((S (p .Q ,L~ ~
~ 1 0 N di V~ r1 O r1 N V~ N
.4' ~
~1 S-1 .
~ 0 0 0 0 o A ~-~ o 0 0 q p 0 o U
N
w r io ~ o p o r w O O (f7lp r O N
b H
~ ~
~ N r r ~-1N ~D
ri '" -r1~ d' ri N O -ril!1M
r1 r1 H ~
v O O O O O D O [ O O O
N 1D r N H -~ ~ N
O p 1J1.1 1~
r-1 -rlfI$
~-I ,S]S-I d~ t!1r M y..~
~ m N
-rl O l0 r d' O~
" ~ ~"''~ O d' ,"O F. M 00 H d~ ~ tnO C: N O
H -riO M O Lf1d' 01 "',l,'-rlO ~ M
i-i ri a. V' N N O 1-1U ~N N
r1 -.-IN
QJ
.~ ~n 0 0 0 0 0 0 ~ q ~ a o 0 , 0 0 0 C: M U ~ V~
H l0 N H cH v H ~l't!1 O
-I r N !n w t0 N
M
4 w M o~ D w au r D a0 u~
l~ c0 er a0 O ~ x Ul N r M r-~ ("" UI O1 M N
r1 ri N
S-1V~ ~i O ri Il3~" S-IWit'N ~ M
ri ri -ri . . . ,-1O -r1 . ~ r-1 Cu 0 0 0 o Cq-~ f=.,0 0 0 0 o W
~ ~ O N
w ~ M N ~ ~ r1 ~D 1D 0 r V ~A
1 f ' -iI O - r1 W 01 M H M
I , r~
H3 ~ ~ m ~ ~ H M
m o -r O N3 ~ ~ ~ ~ - O
o r r 'b '~ ~r f; N 00 d' l0 d~ ~ O N 1DOpM
-I ' d' O
r d M OD H SO d' O CO O lDN M N ~
r1 0 O vo m M u~ U N ov M m M u~N
ri o ii m Q ~ ~ ~ ~ ~ N n ~ ~ ~
v t o ~ a -r' o 1 O o -, ~ ,-i d~
O ~ N O CO
O Ul N cr H d0 N r1 U1 l0 N N U1V'r1 r1 ri S-1 O ~i M O 01 r( ~-1 r1 d~ d~l0H -i r1 r-1 w ~ 1 ~ ~ N U ~ ~ N r 3 ~
w a f. N ~ , ,-~ u~
o ~ -ii U ..
H
~n H
N
G ~
N
-.-i O y~
b1 U .L~
~
a ~
M -1-IF~, v n M H ~.1 N
\
M ..j U M H yJ U , ~
~C
Gi O O O H M r11Jr.C
c ~i O O O O M r-iO O O O
O O
O v1r-i H U 01 M r-iM H U U 01 t r1 H
M r-1M r1a x x b d~ '~ tD
N N N M
G G
~ ~ a ~ m o o ro ~ ro ~ o -~ -.~
r o ~ 00 -.1 E m w E u,o w ,c~ .d ~ ~ ~ ~ ~
-~ -~
a~ a~
U~a o a U.~i~ ~ o a U ~ U
~ G
ro ro A,' O O O O O Cx, F(,' O O O O O fi-, -r-I -rl S.-I S-1 L"
O O
-ri -ri 1~ i~
ro M M M M M ro InIn lf1Lf1 ri U7Lf1Lf1tf11f1 r-I O O O O
N N N N N ~ N t~h l~L
r-W-1r1 H n-I~ ~ 01O O O O
M M M M M O ~ N H r1 n-1H O
U 0101O1 0101u1 U 0000a0 00ODu1 U U ~ U
U
H O ~-1N M d~H H O H N M V~H
~[', A', G
O O
-rl l~ l~ h ro ~ ro ~ ~r r-i d' r-~ 0101 f~ ~ N V' W
~D ~ DOv-f U O U ~ o U O U
FI,' O O O O O M IS O O O O O M
N
x O 0 O
-r-I h Li -.-i Lll O N d~ O N o~ O
~ t~
1J ro O ~0 1J
.~
.J-1~-I l0 ~ S-I h v-I
-r1 -ri ~ QJ
PO U N o r-I N --Io r-I
f'.. ~
1~ 1W
x a~~ -,~ ~
a ~
ro ro w r.~ 0 0 0 0 o to FC 0 0 0 0 0 q -~ -~
~ ~
w N
ro 01 V'N
Gi N 111(~
b l0 '~ O d~
U1 1-1 S-il~ S-1N l0 r1 r1 N Cp -r1 -rlH -r1Lf1M
r1 r1 H ue 0 0 0 0 o O ~ ~ C 0 0 0 0 o O
-i ~ ~ ~ m H r1 f~ 1010 -a x ~ o m ~7 p d r N
O N ~ ~ O V~lf1 r1 ri U O -,.iU N O
r1 r1 w ~3 U ~3 a :~c o o o 0 0 o ~ o o 0 0 0 0 n w w H H
N O
V~
W ~.iO N LO
ri ri ~ ro-~ ~ ~ ro r-, f-~
F: -.1 ri~ -ri r-1 N N
H fi,0 0 0 0 o pq~ G~~0 0 0 0 o W
~u -0 d N O DDM 's''L~ ODV' O ~O
~-I O 00O l0OD ~"L-I 00N M O OD
r1 r-i r1 L(1H d' riO H -rl Ll10001 O OD
ri ri ,5.," 01tt7~O l0N ,L", ~DO M N O
N N
ro H 0~,--i~ ~ ~ O E a~
~7 O
~ d'O 10 N M d~ G O O N ~OlD
O N 01H d'00d' O ~OV~OD d'OD40 r1 ri U h ODI~ O O U1 U 01M 01 M OD00 r-I r1 N n-I10r-1tl7N l~ N r1O N N O O
N N
cn .-a~ N ,~~ ~ cn ao~ '--i,-i~ ,--i a ao00o w M ~.m o o ~r~ o V7 r110~O lf100r1 Ul lDW If1M 00r-i r1 r1 W ~-I 0100h 00O r1 S-I N 01N l~ODr-i ri r1 O -.-1 d'd'~O O N N -~ N O N N O N
N N
G4 r-I,-~r-~N r-iU Cr., a~r-I~-Ir-ir-~U
G
~n O O
N -~ -~
O O
~
~
. M n-Id~ ~1 M r1d' \
N l.~ M H O r-i .U t'~1w-~iO r1 '-~- ",~' ("-, M ril~O S~ M r1C~O
H H
N N M n-IM ~-I N M w1M S-1 d' d' r-i U M r1O 1~ U M r-iO 1~
~ ~
f~''-i O O C~~ C.'H O O f~
ro O O O M H O O O O M H O
X X
H U M n-1t'-WIM U U M ~--IM ~-1M U
~ O
~ro~ao N r 21 .-iM m o ao N r ~ r a~r N ~ ~ u~,~M
~ G
1-1 N ~fl\D tJlM 11 r~O 01d~00 O O
(d O d~l0 ~OH Ib L~N l0d'h -ri --I
r-i r1N H O O r-I d~N O O \0 r1 h O 01 M 00O ~ ~Od~ lDf~H W
-ri ,G~ d'N Q1 L~Lf1W E V'O M N N
,.Q
~ tf1O N O O U~ ~ N r1 O O O
-ri -r1 N N
U N ~ 0 0 o U 0 0 0 0 0 .y..~ .W
..~
U . . ~ U
C ~
r~ ~6 r.~ O o O O O fz, ~ O o 0 0 o O
-.-i -ri S-1 ~.-1 C
O O
-,-r -t~
ro u,~ N N M ra r-I N ~OM M V'O r1 U1cr d~d~M
COI~N N QO ~ l~l0 1ptDOD
tf1I~r1 H M E toU1 N tf1crO
Q~I~N N O~O ~ 01N 0000L~
a aor ~ r ~oin U o~m o~~ o~U
i . . . . U i H
U U
H O r1N M d~H o-i O v-iN M d~
~[,' F~, O O
1~ l~ l~ 1~ 111 r~
(~ h N H 01 t0 M N N a000 r1 U1ODN 0001 r1 lpH O O O
'-i01O~ M M ~ ri01 00W 00 E ~ ,-iM r r E u~0 0 0 0 O O OD N N ~ N N H ~-Ir-i U M N o 0 o U o 0 0 0 o U U
F(,' O O O O O M ~C, O O O O O M
~, ~ O O
' - Lf1N ri .('., d' 00C', ' O N r ~ M o~O N n o o O
~ W
,NO1-ri r1O lf1 M -r1 ~71-ri N H OD-rl 1~ d~07~O L~.l-1 f13 V~O O 1~
.~ .~
y~?-1 O v-iIn N ~ S-I O r1 ri -ri -ri N N
U v H H O O r-i N o o O r~
,t," .fi 1~ 1.) q ~ q ~
~
w w 0 0 0 0 o -. 0 0 0 0 o -a W n o~ r 01 ~ d W 0 W
O N n-1 N
'b O N 'b aD
S-1 O~ OD 7-r N
ri r1 M x ~ -rl O O -rl O
ri r-I
H '~
b ~ ~ 0 0 0 0 o O ~ v E 0 0 0 0 o O
-~
a~
H ~-I 0101 ~ ~'-~ M
~' ~ x f~' ~
, b 00d j~ l M
" -' O .( O N O O ~.,N O
., ''1O N r1 ,N''1O v-1M
r1 r1 U N N ~ U 0 0 ri r1 ~ a O .~3 0 0 0 0 0 o U .s7r 0 0 0 0 0 0 n ~ G
r , ~, O H Lf71D01 ~ H (~
IflL~d' M
y o r M
G ~ o .~ .-i S~ o ,-io ~ ~ ~ o it ~ r-i ,1 _,~ .
H v f~ 0 0 0 0 o W ~ t=,0 0 0 0 o p4 O
'b ~0N d' O O .~.,''Cj d~O ~OO O~
O S-I O l!101 d~N H S-I M Lf1a0N O
ri r-I
-r~ M N H M M -ri N M O O tf1 ri ri ~i I~~Od' ~Od~ .Li N N tf1M n-i N N
~ a H ,~,~ ~ ,~,~o G
o b rd -r1 W O 00d' d'N N (.,'' N V' ~O00V~M
J-1 O lf1ODM M InO O d1CO 01N 00V~
rl r1 -r1 U H N a0 O 10~O U l0d' 100101OD
r1 r1 N N N d0 10I~Lf1 N n-Iri d'N ~-iri N N
u~ ~ ,~~ ~ ~ ,~ cn .-~,-a~
~ G
N o ~r N w o ~ o o w N d~a 0~ m r o~~ N o ~ m aow o~M o~
~ ~--i ~ M M m n ~ ~ o m m o ~
~
O N -r~ ~ M d' Lf1h N -ri N N N t1W-1UJ
O N
w ~-I~-ir~ r~r-~U , C~ r-~r-ir~r-Ir-iU
m ~
r~ ~..
C-~' G
u~ O O
-r1 'ri ~
R: U J-1 1~
r~ .-1 a to ~
E E
,4 M r1 LI M n-I
\ \
M .O J.-I M rir1 1~ W ~1'-iri ,."7 '~
Ci M r1O Li M n-IO
H H
N 11 O M ri~.1 N M r-I.~1 a' d' r-I UI U M r-iJJ U r'W--~1~
O O
,L? ~-1 C." O o o ~ ~ O O O
~"~ H
(a -ri O O O O M r1O O O O O M n-fO
x X
E-a C~, U ovM ~ M ~ U U o~M ~ M ~ U
O O
b umn ~ ~ o~
v O O lf7 N M 'd~O
.(-', Ci, y m O m -i r w o M
O O
rtf o ~ d~ ~o r m o o -~ -~
~ ov~ ov .-~ ~ ~ d~m +~ ~
N ~ r O ~ ~n O w r O
-~
M 10 U1 Cra ~ r 01H M (c~
.L~
-ri N d' O V7 ~ N C N c-i U7 v -ri v N o o ~ '-~0 0 0 v ~ ro ~ v ~
r.~ 0 0 0 0 o t~, ~ o 0 0 0 o Cv -~ -~
?.~ ?a G C
O O
(~ M Lf1LllLll fa l0 01d~m m r-i M 111~Ol0l0a ri 01 o M M M
10N M M M ~ o~ r m m m 01n-I1nUlLfl E m M M Ol01 O d' N N N O ~ Lf1N H Lf7(f1O
U m r r r r U7 U O1 O141m m t(1 i U i U
U U
r-i O v-IN M V~H o-I O r1N M d~H
~, F[,' O O
-ri -rl 1~ m .L W -1 lf1 (~ Lf1r t11 ftS N m H tD
M to m m o u~r o~
~DN m ~ r1 r1m 01 tf1 ~ O 01M M
r m ~ ~ a o~m n U N O o U r1 O o O
U U
FI,' O O O O O M A~ O O O O O M
Ci i.~' O O
-ri r M Lf1 ~ -r1 d' r d~l0 Ca' v o m m O v O m r o~ O
W -a dl-ri M M m -ri (TI-rl O N m 01 -rl (a O m In yJ (a r1 t1101M 1~
,.Q ,!a o,~ ~ ~ 1a d~ M o u~
-~
v v ~ o o .1 v 0 0 0 0 ~ ,W
~r .-~
-r., ~
v b -.~ 0 0 0 0 o q r.C 0 0 0 0 o A
S~ -~
~, i' io r m U
m r m M
~ N r1 01 M t11 '~ M 01 ?-Im m Sd d~ N
ri r1 -r1r W -riO O
ri -1 n-I
H '~
v 0 0 0 0 o O v t 0 0 0 0 o O
o -~
ro ~
. ~
u~ ~ W n u~ o~
~0 to x to N N
'b 1f1 1D ~ '~ l0 M Lf1 ~ O
H
U H O U O O r-i r-i r1 a ~
~
c 0 0 0 0 0 0 ~ .s13 0 0 0 0 0 0 n W
r " o H r1r ~ H r-Ir M 10 Lf1r r O N N
O m ~ ri Ldl0U7 ~
U1 O C'..~ UI M 01N c-1 L;
r1 r~
S-~N O (!fy~ 1-IO O O O fO
ri r1 -~.~ -~
~
t=,o 0 0 0 o p7,1Z f~ 0 0 0 0 o a1 g _ 3 Tf ,S'.,'C3 d' ~ON d'm ~-1 m d~ O N m H ~-I r rim d~N
r1 r1 -rl O 01 O1m V~ -ri N r 01d'm r1 r1 ,Tr' d'M r1~Om ,~ N M M N N
v v H ~ ~ m ~ r O H ~ ~ ~ ~ ~ O
b C m m m vom m O ~DN d''d'm m O ~O O l0d'N N
r-~ m1 U r o~ r a d~d~ U N r ~ M m m ~ .-i v M O d'H m m v N N M r1N N
v N
u) voo~ r m r r cn r-i.-i,~
v tr a-i O ~ ~r o m vom O
U1 m N O O m r1 U1 ~O 01d~r1N ri r1 ri v S-I r 01 10M V'r1 ~-1 M lfld'10m r1 ri r1 -ri N l0 d1Lnm N -ri N H N N N v N N
C=, vor m m r U fj.~ ~ ~ ~ ~ ~ U
H
O O
U -~ -~
O O
1J t~ 1W
r-~ 1 r~ td ~
~I M r1 ~1 M r1 \ \
1J M n-Iri JJ M r1r1 "~] ,"',]
'[f ~.,' ', M H O C,' M r1O
H H
~i N I'. M H S.1 v M r11-I
d' d' O U Ii M r-IJ.! U r1r-1J.-!
O O
U ~ o I o . ~ ~ 0 0 0 p ~ o ~ I
v O o 'I o M ~ o O o 0 o I ~
X o x M
m U ovII,r-iM r-IU U o~ M ~ M ~ U
D M O
N M ~O r1 r v U1 Ci --fa J-1 r U1 O l0 1.J r ftS r1 ~D M ri (IS l0 -r-1 -r1 .-a ~ r ~o ~r ~
~ D
E H N t11r1 W ~ tf1 .~ .~
-r1 O W ri O Cn ~ r1 V1 v -r1 Ol O O O O
U N ~ U ~C
~ p N (IS
b 0 O o o O C7.n ~ 0 0 0 o O fia -ri 111d' r ri '~ l0 ~-1 -.~i O
O
1J 1~
f~ 01 M r1 H O (fS M M M M M
r1 d~ ~D 00 O O ri OD OD0000 CO
p O1 ~p l0 10 ~ O O O O O
M ~O r N N ~ d' d'd'd' l0 N OD OD COO ~ OD 00N 00 4DO
U 00 00 r r r u1 U T 0101O\ O\tI1 i . . . . U 1 . . . . . U
U U
-I O r1 N M d'H ri O H N M V'H
L~ ~
('-., .('-, .,1 -ri IJ 01 CAD 1~
01 r oo ~ r~ r 01 ~ In m M d~ M O
r O d' t11 ~ U1 r1 00 d~ O ~ H
U N o 0 o U O
U U
(,'' O O O O O M F.~ O O O O O M
~
O
ri ~D N C,' ' r f.,' u1 01 r m O W ~ O
-~
(J) 01 O N O -r1 t31-rl O7 -fi -ri v N
~o ~, .a ~
1-I M M M O ~ ~-1 H
-ri -'1 (C IlS
v r~ O O O '~ ~-1 ~
~.i h .i ,5 -'1 ., -!-'a 0 0 0 0 o Ca ~-~ 0 0 0 0 0 ~1 ~
U
Q 01 ~O O
M N N
~ O O N ~
r1 H
-ri N r1 O
r-i N ,i.,'' N
O
H o o 0 0 0 ~ ~ E-~0 0 0 0 o O
3 M a1 01 3 u1 ~
-I ~ ~ ~ ~ 0 p .-i O P ~
G y~U ~ 0 U
r-I ~-i -~v x .,~N
v v H W o 0 0 0 0 0 ~ cn 0 0 0 0 0 0 g io 3 H 01 d~ d' y~H lfl u1 u1 N Q r M ~, x v ~ r x (A l0 01 r1 C.,''y.~ N V' .f", r1 r-i S1 O O O cd~ ~1 O (a r1 r-i -r-I. . . ,.-~~ -r-~ r-i N v fl-,0 0 0 0 o pp C.,0 0 0 0 o W
O
.d 1J'~ N ODl0O N
S-1 aD N N ~0 O ''1S-1 O 01d'l0 O
r-i r1 ri tn O t11r d' ~ -rl 01 r I!1M 10 r1 ri ri 01 1f1vD r ''1~ rl '-1N N r1 N
H ~ ~ r oo r D ~ H ~ ~ ~ ~ ~ O o H O
~ A
~. ~0 l0O OO N N
O N l0 10 l0 O O O t!101~Dtf1O O A
ri r1 U d' OD N l0 d~d' U OD a0rid' tovO
r-I r-I
v o r r m r r v ~ N M N
W v cn ~ ao 01 m r r ~n t~ N a ~ ao d woo N ~
W O r l0 N O r1 U7 W t11d'a0 O r1p r1 r1 Y-1 d' O ~-IN V~r1 S-1 O d'00l0 ~Or-i r1 r1 -r1 N v-1O ~L1r v -ri v-IM N N r1~ ..
v N
r ~ r r r U C~ ~ ~ ~ ~ ~ U o U
o O 'n H
w -1 ~-f CJ
H X X ~
H
N
- (d U 1-i 1, ' 1~
~ ~
~
~ M ri j ~ M r1 r yJ M rir1~.J-1 M rir1 ,~
M ~ O ~ M .--i0 s ~ ~, O
' ''t~ N M r1?-1',N M r1~-I~
~ J7 al S-a U M r1J-1~~~U M .-I-1.-!FC
~ ~ " FC
-ri C; O O O Fi' Ci O O O .,N
H ~ H ~ cn O O O M r1O I O O O O M riO (Yj a W
H U o1 M ~ M ~ U ~ U 01 M ~ M ~ a x p p x
Claims (16)
1. A recombinant super-compound interferon (rSIFN-co) with changed 3-dimensional structure and improved efficacy which can inhibit the DNA duplication and secretion of HBsAg and HBeAg of HBV.
2. The interferon of claim 1, wherein the 3-dimensional change was the result of changes of its production techniques, and efficacy gains not seen in interferon described in U.S. Patent Nos. 4,695,623 and 4,897,471.
3. A super-compound interferon of claim 1 or claim 2, wherein it has its unique secondary and tertiary structure which elicit its special efficacies.
4. A super-compound interferon of claim 1 or claim 2, produced by a highly efficient express system which is constructed with a special promoter.
5. The super-compound interferon of claim 4, wherein the promoter is P BAD.
6. The super-compound interferon of claim 4, wherein its gene is artificially synthesized cDNA, adjusted according to codon preference of E. Coli.
7. A process for production of recombinant super-compound interferon recited in claim 1 or 2.
8. The process for production of claim 7, comprising extraction of super-compound interferon from fermentation broth, collection of inclusion body, denaturation and renaturation of the harvested protein.
9. The process of claim 7, wherein the process maintains the high efficacy even when the super-compound interferon is used with an agent and in a particular concentration.
10. The process of claim 7, comprising separation and purification of the super-compound interferon.
11. The process of claim 7, comprising lyophilization of purified super-compound interferon.
12. The process of claim 7, comprising production of liquid injection of super-compound interferon.
13. Uses of super-compound interferon in preparing medicines for inhibition of HBV-DNA, HBsAg and HBeAg, wherein the virus diseases comprising hepatitis A, hepatitis B, hepatitis C, other types of hepatitis, infections of viruses such as: Epstein-Barr virus, HIV, herpes viruses (Epstein-Barr virus, Cytomegalovirus, herpes simplex viruses), papovaviruses, poxviruses, picornaviruses, adenoviruses, rihnoviruses, human T cell leukaemia viruses I, or human T cell leukaemia viruses II.
14. Uses of claim 1 and 2, wherein the super-compound interferon selected for interferon is .alpha., .beta., .gamma. such as, IFN-1a, IFN-2b or other mutants.
15. Uses of claim 13, wherein super-compound interferon was administered via oral, vein injection, muscle injection, subcutaneous injection, nasal, or mucosal administration.
16. Uses of claim 13, wherein super-compound interferon was administered following the protocol as follows:
injection 9 µg or 15 µg per day, 3 times a week, total 24 weeks.
injection 9 µg or 15 µg per day, 3 times a week, total 24 weeks.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN01104367.9 | 2001-02-28 | ||
CNB011043679A CN1245215C (en) | 2001-02-28 | 2001-02-28 | Recombination high efficiency composite interferon used as hepatitis B surface antigen and e antigen inhibitor |
PCT/CN2002/000128 WO2002080958A1 (en) | 2001-02-28 | 2002-02-28 | Recombination super compound interferon used as hepatitis b surface antigen and e antigen inhibitor |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2439503A1 true CA2439503A1 (en) | 2002-10-17 |
Family
ID=4653854
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002439503A Abandoned CA2439503A1 (en) | 2001-02-28 | 2002-02-28 | Recombination super compound interferon used as hepatitis b surface antigen and e antigen inhibitor |
Country Status (10)
Country | Link |
---|---|
US (3) | US7364724B2 (en) |
EP (1) | EP1371373B1 (en) |
JP (1) | JP4617058B2 (en) |
CN (1) | CN1245215C (en) |
AT (1) | ATE446104T1 (en) |
AU (1) | AU2003248419B2 (en) |
CA (1) | CA2439503A1 (en) |
DE (1) | DE60234085D1 (en) |
HK (1) | HK1061201A1 (en) |
WO (1) | WO2002080958A1 (en) |
Families Citing this family (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060035327A1 (en) * | 2001-02-28 | 2006-02-16 | Guangwen Wei | Recombinant super-compound interferon and uses thereof |
US20050079579A1 (en) * | 2001-02-28 | 2005-04-14 | Guangwen Wei | Uses of spatial configuration to modulate protein function |
CN1245215C (en) | 2001-02-28 | 2006-03-15 | 四川省生物工程研究中心 | Recombination high efficiency composite interferon used as hepatitis B surface antigen and e antigen inhibitor |
US8551469B2 (en) | 2001-02-28 | 2013-10-08 | Superlab Far East Limited | Treatment of tumors and viral diseases with recombinant interferon alpha |
US7335496B2 (en) * | 2003-06-05 | 2008-02-26 | Ajinomoto Co., Inc. | Method for producing target substance |
US7585647B2 (en) | 2003-08-28 | 2009-09-08 | Guangwen Wei | Nucleic acid encoding recombinant interferon |
KR20150103335A (en) * | 2003-08-28 | 2015-09-09 | 수퍼랩 파 이스트 리미티드 | Uses of Interferons With Altered Spatial Structure |
AU2011202683B2 (en) * | 2003-08-28 | 2012-08-09 | Superlab Far East Limited | Uses of interferons with altered spatial structure |
EP2325202B1 (en) * | 2003-08-28 | 2014-10-22 | Superlab Far East Limited | Uses of interferons with altered spatial structure |
CN1740197B (en) * | 2004-08-26 | 2010-05-12 | 辉阳科技美国公司 | Recombination interferon with new space conformation and enhanced effect, its preparing method and application |
CN101137391B (en) * | 2005-03-09 | 2012-07-18 | 魏光文 | Uses of recombinant super-compound interferon |
CN101525381B (en) * | 2008-03-04 | 2012-04-18 | 北京百川飞虹生物科技有限公司 | Novel recombinant consensus interferon and construction of a high-efficiency expression vector thereof |
CN102101886A (en) | 2009-12-18 | 2011-06-22 | 四川辉阳生命工程股份有限公司 | Variable-conformation recombinant interferon crystal, and three-dimensional structure and use thereof |
WO2013055811A1 (en) * | 2011-10-12 | 2013-04-18 | Hitachi Chemical Co., Ltd. | Ex vivo methods of predicting responsiveness of a subject to interferon therapy |
US8492386B2 (en) | 2011-10-21 | 2013-07-23 | Abbvie Inc. | Methods for treating HCV |
CA2811250C (en) | 2011-10-21 | 2015-08-11 | Abbvie Inc. | Methods for treating hcv |
US8466159B2 (en) | 2011-10-21 | 2013-06-18 | Abbvie Inc. | Methods for treating HCV |
DK2583677T1 (en) | 2011-10-21 | 2015-01-19 | Abbvie Inc | Methods for treatment of HCV comprising at least two direct-acting antiviral agents ribavirin, interferon but not |
TWI726291B (en) | 2013-01-07 | 2021-05-01 | 英屬維爾京群島商遠東超級實驗室有限公司 | Methods and compositions for treatment of bone, skin, subcutaneous, mucosal and/or submucosal cancer by percutaneous and/or transmucosal administration of interferon |
WO2015070751A1 (en) | 2013-11-13 | 2015-05-21 | Superlab Far East Limited | Methods of determining interferon having direct inhibitory effects on tumors and uses thereof |
CA2985524C (en) | 2015-05-12 | 2023-10-17 | Superlab Far East Limited | Methods of determining interferon having direct inhibitory effects on tumors and uses thereof |
EA201892448A1 (en) | 2016-04-28 | 2019-06-28 | Эмори Юниверсити | ALKYN-CONTAINING NUCLEOTIDE AND NUCLEOSIDE THERAPEUTIC COMPOSITIONS AND RELATED APPLICATION METHODS |
Family Cites Families (36)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4179337A (en) | 1973-07-20 | 1979-12-18 | Davis Frank F | Non-immunogenic polypeptides |
US4672108A (en) | 1981-12-07 | 1987-06-09 | Hoffmann-La Roche Inc. | Crystalline human leukocyte interferon |
DE3269727D1 (en) | 1981-12-07 | 1986-04-10 | Hoffmann La Roche | Crystalline human leukocyte interferon |
US6936694B1 (en) * | 1982-05-06 | 2005-08-30 | Intermune, Inc. | Manufacture and expression of large structural genes |
US4462940A (en) | 1982-09-23 | 1984-07-31 | Cetus Corporation | Process for the recovery of human β-interferon-like polypeptides |
US4681930A (en) | 1983-09-20 | 1987-07-21 | Hoffmann-La Roche Inc. | Immune interferon and a method for its extraction and purification |
US5372808A (en) | 1990-10-17 | 1994-12-13 | Amgen Inc. | Methods and compositions for the treatment of diseases with consensus interferon while reducing side effect |
US5441734A (en) | 1993-02-25 | 1995-08-15 | Schering Corporation | Metal-interferon-alpha crystals |
EP0626448A3 (en) | 1993-05-26 | 1998-01-14 | BOEHRINGER INGELHEIM INTERNATIONAL GmbH | Process for preparing and purifying alpha-interferon |
DE4329756A1 (en) | 1993-09-03 | 1995-03-09 | Boehringer Ingelheim Int | Process for the preparation and purification of alpha-interferon |
US5766582A (en) | 1994-10-11 | 1998-06-16 | Schering Corporation | Stable, aqueous alfa interferon solution formulations |
JP2758154B2 (en) | 1995-04-06 | 1998-05-28 | エフ・ホフマン−ラ ロシユ アーゲー | Liquid preparations containing interferon |
EP0861321B1 (en) | 1995-10-13 | 2006-05-31 | President And Fellows Of Harvard College | Phosphopantetheinyl transferases and uses thereof |
US5972331A (en) | 1995-12-22 | 1999-10-26 | Schering Corporation | Crystalline interferon alpha for pulmonary delivery and method for producing the same |
US5874304A (en) | 1996-01-18 | 1999-02-23 | University Of Florida Research Foundation, Inc. | Humanized green fluorescent protein genes and methods |
US5980884A (en) | 1996-02-05 | 1999-11-09 | Amgen, Inc. | Methods for retreatment of patients afflicted with Hepatitis C using consensus interferon |
US6532437B1 (en) | 1996-10-23 | 2003-03-11 | Cornell Research Foundation, Inc. | Crystalline frap complex |
CN1186120A (en) | 1996-12-24 | 1998-07-01 | 深圳科兴生物制品有限公司 | Recombinative human interferon protein as secretory gene in colibacillus |
US6114145A (en) * | 1997-12-05 | 2000-09-05 | Human Genome Sciences, Inc. | Synferon, a synthetic interferon |
US6087478A (en) | 1998-01-23 | 2000-07-11 | The Rockefeller University | Crystal of the N-terminal domain of a STAT protein and methods of use thereof |
BR9907964A (en) | 1998-02-06 | 2000-10-17 | Ilexus Pty Limited | Three-dimensional structures and models of fc receivers and their uses |
CN1062565C (en) | 1998-06-29 | 2001-02-28 | 深圳九先生物工程有限公司 | Recombination human alpha type composite interferon, prepn. method and use therefor |
KR100399156B1 (en) | 1999-11-19 | 2003-09-26 | 주식회사 엘지생명과학 | Liquid Formulation of α-Interferon |
CN1175901C (en) | 1999-12-06 | 2004-11-17 | 天津华立达生物工程有限公司 | Stable water solution of interferon |
US20020043262A1 (en) | 2000-08-22 | 2002-04-18 | Alan Langford | Spray device |
US7544354B2 (en) | 2000-10-27 | 2009-06-09 | Novartis Vaccines And Diagnostics | Methods of protein purification and recovery |
EP1355939A2 (en) | 2000-11-03 | 2003-10-29 | Pestka Biomedical Laboratories, Inc. | Interferons, uses and compositions related thereto |
CN1245215C (en) | 2001-02-28 | 2006-03-15 | 四川省生物工程研究中心 | Recombination high efficiency composite interferon used as hepatitis B surface antigen and e antigen inhibitor |
US6546074B1 (en) | 2001-03-27 | 2003-04-08 | Astex Technology Limited | Protein crystal structure and method for identifying protein modulators |
CN1375502A (en) | 2001-10-25 | 2002-10-23 | 南京药科大学 | Polyglycol modified recombinant human interferon |
US6807478B2 (en) * | 2001-12-27 | 2004-10-19 | Koninklijke Philips Electronics N.V. | In-building navigation system |
CN1176946C (en) | 2002-05-16 | 2004-11-24 | 中国人民解放军第二军医大学 | New-type alpha-interferon |
CN1202861C (en) | 2003-07-18 | 2005-05-25 | 中国科学院微生物研究所 | Use of compound interferon in the treating of SARS disease |
EP2325202B1 (en) | 2003-08-28 | 2014-10-22 | Superlab Far East Limited | Uses of interferons with altered spatial structure |
WO2005067963A1 (en) | 2003-12-23 | 2005-07-28 | Intermune, Inc. | Use of polyethylene glycol-modified interferon-alpha in therapeutic dosing regimens |
CN101137391B (en) | 2005-03-09 | 2012-07-18 | 魏光文 | Uses of recombinant super-compound interferon |
-
2001
- 2001-02-28 CN CNB011043679A patent/CN1245215C/en not_active Expired - Lifetime
-
2002
- 2002-02-28 DE DE60234085T patent/DE60234085D1/en not_active Expired - Lifetime
- 2002-02-28 WO PCT/CN2002/000128 patent/WO2002080958A1/en active Application Filing
- 2002-02-28 EP EP02702211A patent/EP1371373B1/en not_active Expired - Lifetime
- 2002-02-28 AT AT02702211T patent/ATE446104T1/en not_active IP Right Cessation
- 2002-02-28 JP JP2002578997A patent/JP4617058B2/en not_active Expired - Lifetime
- 2002-02-28 CA CA002439503A patent/CA2439503A1/en not_active Abandoned
-
2003
- 2003-08-28 US US10/650,365 patent/US7364724B2/en not_active Expired - Lifetime
- 2003-09-26 AU AU2003248419A patent/AU2003248419B2/en not_active Expired
-
2004
- 2004-06-15 HK HK04104296.9A patent/HK1061201A1/en not_active IP Right Cessation
-
2008
- 2008-04-18 US US12/105,455 patent/US8114395B2/en not_active Expired - Fee Related
-
2011
- 2011-02-01 US US13/019,044 patent/US8425896B2/en not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
CN1311035A (en) | 2001-09-05 |
US20080305080A1 (en) | 2008-12-11 |
JP4617058B2 (en) | 2011-01-19 |
WO2002080958A1 (en) | 2002-10-17 |
HK1061201A1 (en) | 2004-09-10 |
EP1371373A4 (en) | 2005-05-04 |
US8425896B2 (en) | 2013-04-23 |
AU2003248419B2 (en) | 2007-01-04 |
CN1245215C (en) | 2006-03-15 |
JP2005508848A (en) | 2005-04-07 |
AU2003248419A1 (en) | 2003-11-06 |
US20040202641A1 (en) | 2004-10-14 |
EP1371373B1 (en) | 2009-10-21 |
US20110189128A1 (en) | 2011-08-04 |
ATE446104T1 (en) | 2009-11-15 |
US7364724B2 (en) | 2008-04-29 |
DE60234085D1 (en) | 2009-12-03 |
US8114395B2 (en) | 2012-02-14 |
EP1371373A1 (en) | 2003-12-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2439503A1 (en) | Recombination super compound interferon used as hepatitis b surface antigen and e antigen inhibitor | |
CN111500615B (en) | Recombinant expression vector for expressing LL-37 polypeptide, recombinant lactococcus lactis, antiviral drug, construction method and application | |
CN1882682B (en) | Method of preparation of pharmaceutically grade plasmid DNA | |
EP2749290A2 (en) | Uses of recombinant super-compound interferons | |
JP2005508848A6 (en) | Application of consensus interferon as an inhibitor of hepatitis B surface antigen and e antigen | |
CN1683399A (en) | Wasp antibiotic peptide and its preparing method and use | |
CN1786017A (en) | Renaturation of reconstituted human bone protein-1 and making method of its preparation | |
CN108948163B (en) | Macadamia nut plant defensin and application thereof | |
IE45285B1 (en) | Polyribonucleotides capable of promoting the genesis of leucocytes and blood platelets | |
CN101298468B (en) | Method for removing endotoxin from protein medicine in one step | |
CN102212547A (en) | Intestinal trefoil factor recombinant expression vector and preparation method of intestinal trefoil factor | |
CN101875691B (en) | Scorpion arialgesic antitumoral peptide mutant and preparation method thereof | |
CN104262480B (en) | The structure and the preparation method of method of modifying and its freeze dried injection of the long-acting alpha interferon of Recombinant Swine | |
CN113995845B (en) | Ivermectin preparation and preparation method and application thereof | |
DK171419B1 (en) | Microbially Prepared Human Interleukin-1, Methods of Preparation thereof, and Pharmaceutical Preparations Containing It, and Its Use in Treatment and Prophylaxis | |
CN115501185B (en) | Compound for treating osteosarcoma and preparation method thereof | |
WO2022142977A1 (en) | Use of hrpz-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals | |
CN117482252A (en) | Pharmaceutical composition and preparation method and application thereof | |
CN1087748C (en) | Microbial polysaccharide, its preparation method and medicine composite containing it | |
EP0207700B1 (en) | Hypocholesterolemically and/or hypotriclyceridemically active rna fractions | |
CN1273976A (en) | Process for preparing recombined morphogenetic protein (rhOP-1) of human bone | |
CN117122568A (en) | Radix scutellariae powder injection and preparation method thereof | |
Marcia | The multiple dimensions of lncRNAS: how 3D structure determines meg3 function | |
Traore et al. | TcpA from the Clostridium perfringens plasmid pCW3 is more closely related to the DNA translocase FtsK than to coupling proteins | |
CN116200282A (en) | Hepatitis B vaccine and its production method |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Discontinued |