CN100441687C - Antisense oligonucleotide structure and use for inhibiting epidermal regulator EREG gene expression - Google Patents
Antisense oligonucleotide structure and use for inhibiting epidermal regulator EREG gene expression Download PDFInfo
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Abstract
The present invention relates to a hepatitis B virus resisting target and corresponding antisense oligonucleotide medicine, particularly to the epidermis regulator of a hepatitis B virus infection resisting medicine acting target, the structure of the antisense oligonucleotide of hepatitis B virus resistance for targeting the epidermis regulator, and the purpose of the antisense oligonucleotide during the preparation of medicine for treating hepatitis B and related diseases.
Description
Technical field:
The present invention relates to the biotechnology pharmaceutical field, sequence, structure and the medicine thereof of the antisense oligonucleotide (ASODN, antisense oligodexynucleotide) that specifically a kind of targeting epidermal regulin (epiregulin) treatment hepatitis B virus (HBV) infects.
Background technology:
Viral hepatitis is the Pandemic infection disease of serious threat human health.Wherein, it is that a kind of meeting causes chronically infected serious viral hepatitis type that HBV infects the hepatitis B that causes, and the patient who transfers chronic viral hepatitis B to exists the high suffering from liver cirrhosis and the danger of liver cancer.At present, the mankind still do not find effective medicine can thoroughly remove HBV and final healing hepatitis B in the body, so novel anti HBV medicine has important social and economic benefit.
Epiregulin is a factor of latest find in Urogastron (EGF) family, has brought into play certain effect in the healthy tissues in various epithelial cells source and tumor tissues.This laboratory finds that by screening and checking but epiregulin has the excellent specificity and the property of medicine in anti HBV infecting, can develop into the latent effect target spot of anti-HBV treatment.
ASODN is the oligonucleotide fragment of a class through synthetic, and length mostly is 15～30 Nucleotide.By the principle of base complementrity, disturb transcribing and translating of genes involved, perhaps duplicating of whole genome, its advantage is its theoretic height target-specific, is that a kind of ideal has accurately optionally gene target medicine.Owing to the high degree of specificity of ASODN effect, therefore be considered to have the antitumor and new antiviral drug of potentiality.One of external more existing famous pharmacy corporations have been developed antisense drug as its new drug research emphasis direction.
The objective of the invention is, according to disclosed epiregulin gene mRNA sequence, design is at the ASODN of epiregulin, by suppressing the expression of epiregulin, stop HBV to infect, suppress hbv replication and expression, for the treatment chronic HBV infection provides new specific medicine.
Summary of the invention:
To the effect that of the present invention: by the nucleic acid sequence data storehouse among the retrieval GeneBank, the epiregulin mRNA reference sequences NM_001432 that selects NCBI to announce, adopt this laboratory patent, based on the neural network prediction algorithm of target gene multiple level predict and the online design software AODesigner of antisense of this lab design (software copyright number: 2005SR12155) designed the ASODN sequence of 5 target epiregulin.By with the online blast sequence alignment of GeneBank, selected target sequence all has excellent specificity, can not disturb the expression of human other normal gene.Synthetic sulfo-Antisensedigonucleotsequence sequence (S-ASODN) on automatic dna synthesizer.Adopt transfection that HBV DNA is arranged, but stably express HBV albumen and complete DaneShi particulate HepG2.2.15 cell model carry out screening active ingredients and evaluation to above-mentioned ASODNs.The result shows among 5 ASODNs, E3 when 0.8 μ mol/L to HBV DNA, HBsAg, HBeAg, epiregulin albumen has the obvious suppression effect, and to HBV DNA, HBsAg, the inhibition activity of HBeAg is greater than the inhibition activity of lamivudine.In 0.2-1.6 μ mol/L concentration range, E3 is to HBV DNA, HBsAg, and HBeAg and epiregulin albumen have specific dose-dependent inhibition activity.
Sulfo-Antisensedigonucleotsequence sequence and character
A: antisense; S: justice
According to the present invention, but the expression specificity of inhibition epiregulin suppresses duplicating and expressing of hepatitis B virus, and epiregulin might become the newtype drug action target spot of treatment and prevention HBV relative disease.
According to the present invention, can specificity suppress duplicating and expressing of hepatitis B virus at the ASODNs of epiregulin mRNA, might become the novel biological engineering medicine of treatment and prevention HBV relative disease.
According to the present invention, the length of antisense oligonucleotide and its cell permeability, relevant with factors such as target sequence binding affinity and effect specificitys, the length of E3 determines that according to experiment the present invention has comprised any length oligonucleotide that has identical sequence with E3.
According to the present invention, for strengthening nuclease resistance, bioavailability and the tissue target tropism etc. of antisense oligonucleotide, the present invention has comprised the thio-modification of E3.
According to the present invention, oligonucleotide of the present invention and modifier thereof can be made into the preparation of parenterai administration by means known in the art.
According to the present invention, the treatment of oligonucleotide of the present invention and modifier thereof is formed can use independent effective constituent or composition forms comprises other antisense oligonucleotides of associating and derivative form thereof.
According to the present invention, treatment of the present invention is formed, comprise pharmacokinetics, pharmacokinetics, mode of administration, route of administration, the receptor's of certain drug age, body weight, hepatic and renal function state, character, degree and the treatment time limit etc. of disease according to different situations, with the appropriate dosage administration.
Enforcement of the present invention has important social benefit and economic benefit to the hepatitis B of serious harm human health and the treatment of relative disease thereof.
Description of drawings:
Fig. 1 epiregulin mRNA is in HepG2 cell, HepG2.2.15 cell and the HepG2.2.15 cell expression changing conditions before and after drug treating
Fig. 2 epiregulin albumen is in HepG2 cell, HepG2.2.15 cell and the HepG2.2.15 cell expression changing conditions before and after drug treating
Fig. 3 sulfo-epiregulin Antisensedigonucleotsequence sequence is to the restraining effect of HBV DNA in the HepG2.2.15 cell
Fig. 4 sulfo-epiregulin Antisensedigonucleotsequence sequence is to the restraining effect of HBsAg in the HepG2.2.15 cell
Fig. 5 sulfo-epiregulin Antisensedigonucleotsequence sequence is to the restraining effect of HBeAg in the HepG2.2.15 cell
Fig. 6 sulfo-epiregulin Antisensedigonucleotsequence sequence is to the proteic restraining effect of epiregulin in the HepG2.2.15 cell
Fig. 7 sulfo-epiregulin Antisensedigonucleotsequence sequence E3 is to the influence of HepG2.2.15 cell proliferation
Fig. 8 sulfo-epiregulin Antisensedigonucleotsequence sequence E3 is dose-dependently and just sequence E3s thereof to the proteic restraining effect of epiregulin in the HepG2.2.15 cell to the proteic restraining effect of epiregulin in the HepG2.2.15 cell
Fig. 9 sulfo-epiregulin Antisensedigonucleotsequence sequence E3 and just sequence thereof are to the restraining effect of HepG2.2.15 emiocytosis HBsAg, HBeAg, HBV DNA
Figure 10 sulfo-epiregulin Antisensedigonucleotsequence sequence E3 is dose-dependently to the restraining effect of HepG2.2.15 emiocytosis HBsAg, HBeAg, HBVDNA
Embodiment:
Embodiment one
Materials and methods
1. medicine preparation
It is 10mmol/L that lamivudine is dissolved to final concentration with PBS.
2. cell cultures
Used cell is the HepG2.2.15 cell strain that hepatoma cell line HepG2 cell strain and transfection have HBV DNA.The HepG2.2.15 cell derives from the HepG2 cell, contains the HBV DNA of integration, sustainable stably secretion DaneShi particle and HBsAg in nutrient solution in cell cultivation process, HBV DNA etc.(the HepG2.2.15 cell is with containing 10% foetal calf serum for FBS, DMEM cell culture fluid cultivation Gibco), and the MEM cell culture fluid of 380 μ g/mlG418 (Promega) is cultivated with containing 10% foetal calf serum for the HepG2 cell.
3. cell dosing
Treat that the HepG2.2.15 cell covers with the back and goes down to posterity at 1: 3, use the MEM nutrient solution that contains 2%FBS after 48 hours instead, add lamivudine.The lamivudine final concentration is 25 μ mol/L, sets up corresponding cellular control unit simultaneously.Changed the cell culture fluid that contains the isoconcentration medicine, the 8th day collecting cell after the dosing in 4 days.
4.RT-PCR detect epiregulin mRNA at HepG2, HepG2.2.15 cell and the expression in the HepG2.2.15 of drug treating cell
Treat that HepG2 cell, HepG2.2.15 cell cover with or the dosing of HepG2.2.15 cell after the 8th day, nutrient solution is removed in suction, after twice of PBS cleaning, extract cell total rna according to Trizol test kit (Invitrogen) specification sheets, ultraviolet spectrophotometer is quantitative, OD260/280 is between 1.8-2.0, and RNA denaturing formaldehyde electrophoresis showed does not have degraded.Carry out reverse transcription then, concrete grammar is as follows: respectively get total RNA 1 μ g, OligodT (15) 0.5 μ g, RNA enzyme inhibitors (40U) 0.1 μ l, totally 10.3 μ l, mixing, 70 ℃ of incubation 10min, cooled on ice immediately.Add the first chain reaction damping fluid, 5 μ l, DTT 2.5 μ l, RNA enzyme inhibitors 0.7 μ l, dNTP (A, G, C, T10mM) 1.0 μ l, mixing, 42 ℃ of reaction 2min, add reversed transcriptive enzyme superscriptII0.5 μ l, 42 ℃ of incubation 1h, last 70 ℃ of sex change 15min.Reverse transcription product is got the template of 0.5 μ l as pcr amplification, carries out double PCR with house-keeping gene GAPDH as interior mark.Target gene epiregulin upstream primer is 5 '-TCCATCTTCTACAGGCAGTCC-3 ', downstream primer is 5 '-TCACGGTCAAAGCCACATA-3 ', the amplification fragment length is 300bp.The upstream primer of GAPDH is 5 '-ACCACAGTCCATGCCATCAC-3 ', and downstream primer is 5 '-TCCACCACCCTGTTGCTGTA-3 ', amplification segment 452bp.PCR reaction system cumulative volume 20 μ l, upstream and downstream primer final concentration are 1 μ M, Mg
2+Concentration 1.5mM adds reverse transcription product 0.5 μ l, Taq 1U.The PCR loop parameter is 94 ℃ of pre-sex change 2min, 94 ℃ of 30s, and 55 ℃ of 30s, 72 ℃ of 30s circulate 30 times, and last 72 ℃ are extended 2min.2% sepharose (Sigma) electrophoresis detection.
5.Western the trace method detects epiregulin at HepG2 cell, HepG2.2.15 cell and the expression in the HepG2.2.15 of drug treating cell
Treat that HepG2 cell, HepG2.2.15 cell cover with or the dosing of HepG2.2.15 cell after the 8th day, inhale and remove nutrient solution, clean twice with PBS after, RIPA-PICT (Pharmacia) method is extracted total protein of cell, behind the protein quantification each extract is transferred to same concentrations.Every hole 30-50 μ g total protein, behind 12% polyacrylamide gel electrophoresis, adopt half-dried transfer printing with albumen go to nitrocellulose filter (
BA-S 83 Reinforced NC, Schleicher﹠amp; Schuell) on.4 ℃ of sealings are spent the night, and confining liquid is formed: 5% skim-milk, 1 * TBST.Combine 1h with goat-anti people epiregulin antibody (Santa cruz) or the anti-people β of rabbit-actin antibody (Sigma) then, TBST washes film 3 times, each 10min, combine 1h with the anti-sheep two anti-(middle mountain) of horseradish peroxidase mark, anti-rabbit two anti-(middle mountain) again, TBST washes film 3 times, each 10min, the colour developing of ECL (Pharmacia) Color Appearance System, the exposure of X-ray sheet.
The result
1.Epiregulin mRNA is at HepG2 cell, HepG2.2.15 cell and the expression in the HepG2.2.15 of drug treating cell
Behind the HepG2 cell of equivalent, the HepG2.2.15 cell total rna reverse transcription PCR, 2% agarose gel electrophoresis detects expression, and house-keeping gene GAPDH is as interior mark.The result almost detects the expression less than epiregulin mRNA as shown in fig. 1 in the HepG2 cell, and its expression level and GAPDH expression level are approaching in the HepG2.2.15 cell.After 25 μ mol/L lamivudines were handled, epiregulin mRNA expressed obviously downward modulation in the HepG2.2.15 cell.Control represents the relative expression situation of epiregulin mRNA in cellular control unit among Fig. 1; Lamivudine represents the relative expression situation of epiregulin mRNA in lamivudine treatment group cell; HepG2, HepG2.2.15 represent the relative expression situation of epiregulin mRNA in HepG2, HepG2.2.15 cell.
2.Epiregulin albumen is at HepG2 cell, HepG2.2.15 cell and the expression in the HepG2.2.15 of drug treating cell
Show among Fig. 2 that epiregulin albumen relative expression quantity in the HepG2.2.15 cell is higher, and almost detects the proteic expression less than epiregulin in the HepG2 cell.Epiregulin albumen was obviously reduced after 25 μ mol/L lamivudines were handled the HepG2.2.15 cell.
Conclusion
Epiregulin infects the back up-regulated at HBV, and expresses obviously downward modulation after drug intervention, therefore may become the newtype drug action target spot of treatment and prevention HBV relative disease.
Embodiment two
Materials and methods
1.S-ASODN design and synthetic
Nucleic acid sequence data storehouse among the retrieval GeneBank, the epiregulin mRNA reference sequences NM_001432 that selects NCBI to announce, adopt this laboratory patent, based on the neural network prediction algorithm of target gene multiple level predict and the online design software AODesigner of antisense of this lab design (software copyright number: 2005SR12155) designed the ASODN sequence of 5 target epiregulin.By with the online blast sequence alignment of GeneBank, selected target sequence all has excellent specificity, can not disturb the expression (seeing 1-5 in the sequence table) of human other normal gene.All oligonucleotide all adopt ABI8909 type automatic dna synthesizer to synthesize and carry out thio-modification when synthetic, process is as follows: with sulfo-reagent (Beaucage regent, Transgenomic) be dissolved in that to make its final concentration in the anhydrous acetonitrile be 1g/100ml, place the AUX position of dna synthesizer, adopt the DNA sulfuration program that provides on the synthesizer to carry out synthetic automatically G 3139.Synthetic 55 ℃ of cuttings of strong aqua and the deprotection of finishing be after 15 hours, through the anti-phase purification column of Micro Pure II (Oligo PrepOP120, SAVANT) purifying, the quantitative final vacuum drying of ultraviolet ,-20 ℃ of preservations are standby.
2.ASODN transfection
The Hep2.2.15 cell is in the MEM nutrient solution that contains 10% foetal calf serum (Gibco) and 380 μ g/ml, in 37 ℃, 5%CO
2Cultivate in the incubator.The observation of cell growth conditions is good, is cultured to the logarithmic growth after date, with Hep2.2.15 cell inoculation 6 orifice plates, 1.5 * 10
5Cells/well, 37 ℃, 5%CO2 was hatched 48-72 hour, after waiting to grow to the 40-60% cell and converging, under the serum-free state, adopted liposome Lipofectin (Invitrogen, 1mg/ml) reagent and with reference to specification sheets operation carrying out transfection.The concentration of antisense oligonucleotide is respectively 0.2 μ M, 0.4 μ M, 0.8 μ M and establishes cell contrast, liposome contrast.After the transfection 22 hours, change normal cell nutrient solution (the MEM cell culture fluid that contains 10%FBS), 37 ℃, 5%CO2 was hatched 72 hours.The collecting cell nutrient solution ,-20 ℃ of preservations are standby.(Trizol RNA extracts test kit, and Invitrogen) and the Hep2.2.15 total protein of cell ,-20 ℃ of preservations are standby to extract the Hep2.2.15 cell total rna.
3.ASODN the restraining effect to Hep2.2.15 emiocytosis HBV DNA detects
Get cell culture fluid, 100 ℃ are boiled 15min, the centrifugal 10min of 12000r/min, get the template of supernatant as quantitative fluorescent PCR, experimentation is operated (He Yunyan, Wang Shengqi etc., Chinese hepatopathy magazine by the method for the combined probe PCR detection by quantitative HBV that this laboratory is set up, 2001, V9N6:376-377).The primer sequence of detection by quantitative HBV DNA is: P1:5 '-GGAGTA TGG ATT CGC ACT CCT C-3 '; P2:5 '-TTG TTG TTG TAG GGG ACC TGC CT-3 '; Fluorescent probe sequence F:5 '-ACT TCC GGA AAC TAC TGT TAG ACG A-3 '; Cancellation probe sequence Q:5 '-GTA GTTTCC GGA AGT-3 '.20 μ l reaction systems contain the 200nmol/L primer, 670nmol/L fluorescent probe F, 180nmol/L cancellation probe, 200 μ mol/LdNTP, the Mg of 4.0mmol/L
2+, 2 μ l templates are put into the automatic PCR instrument of iCycle with each reaction tubes with the typical curve reaction tubes behind the mixing and are increased, and amplification condition is: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, totally 40 circulations, reaction finish the back and calculate quantitative result automatically by computer.
4.ASODN to Hep2.2.15 emiocytosis HBsAg, the restraining effect of HBeAg detects
Get the good cell culture fluid of collection, according to HBsAg, HBeAg ELISA detection kit (magnificent company) specification sheets operation steps detects.Get in the 96 antigen coated orifice plates of 50 μ l cell culture fluids adding hepatitis B surface antigen or e, every hole adds the enzyme mark binding substances of 50 μ l surface antigens or e antigen correspondence, 37 ℃ hatch 1h after, wash plate 5 times with washing lotion, add colour developing liquid A50 μ l, add colour developing liquid B50 μ l again, hatch 15min for 37 ℃, add stop buffer 50 μ l, at multiple labeling inspection enzyme-linked immunosorbent assay instrument (VICTOR
TMWallac 1420 Multilabel Counter Wallac) go up the mensuration 450nm 1s of place light absorption value A.According to IR=(A450
Contrast-A450
Administration)/A450
ContrastCalculate inhibiting rate.
5.ASODN the proteic restraining effect of epiregulin in the Hep2.2.15 cell is detected
RIPA-PICT (Pharmacia) extracts total protein of cell, behind the protein quantification each extract is transferred to same concentrations.Carry out the western Blot experiment then.Experimental technique reference example one.
The result
1.ASODN restraining effect to Hep2.2.15 emiocytosis HBV DNA
Five antisense sequences E1-E5 of target epiregulin mRNA, handle the Hep2.2.15 cell with 0.8 μ mol/L administration respectively, set up the cell contrast simultaneously, liposome contrast and positive drug lamivudine control group, collecting cell nutrient solution after 72 hours, fluorescence quantitative PCR detection is respectively organized excretory HBV DNA copy number in the cell, according to formula IR=(C
The dosing group-C
The cell control group)/C
Cell Control groupCalculate inhibiting rate, IR represents inhibiting rate in the formula, and C represents HBV DNA copy number in the detected cell culture fluid.The triplicate experiment, the mean value of calculating inhibiting rate.C represents the cell control group among Fig. 3, and LIP represents the liposome control group, and it near 0, does not have the obvious suppression effect to the inhibiting rate of HBV DNA.LAM25 represents the restraining effect of 25 μ mol/L lamivudines to HepG2.2.15 emiocytosis HBV DNA respectively.E1-E5 represents five antisense sequences restraining effect to HBV DNA when 0.8 μ mol/L, can show from figure, and E3 is close to the restraining effect of HBV DNA in the restraining effect of HBV DNA and the 25 μ mol/L lamivudine pair cell nutrient solutions.
2.ASODN restraining effect to Hep2.2.15 emiocytosis HBsAg
Five antisense sequences E1-E5 of target epiregulin mRNA, handle the HepG2.2.15 cell with 0.8 μ mol/L dosing respectively, set up cell contrast, liposome contrast and positive drug lamivudine control group simultaneously, collecting cell nutrient solution after 72 hours, HBsAg ELISA detection kit detects the expression of HBsAg in the Hep2.2.15 cell culture fluid, according to formula IR=(A
The dosing group-A
The cell control group)/A
The cell control groupCalculate inhibiting rate, IR represents inhibiting rate in the formula, and the A representative respectively detects the absorbancy of hole at 450nm.The triplicate experiment, the mean value of calculating inhibiting rate is seen Fig. 4.LIP represents the restraining effect of liposome control group pair cell secretion HBsAg among the figure, and showing does not have obvious restraining effect.LAM25 represents the restraining effect of 25 μ mol/L lamivudine pair cells secretion HBsAg, and inhibiting rate is less than 25%.E1-E5 represents the restraining effect of five antisense sequences pair cells secretion HBsAg, and wherein the average inhibiting rate of E3 is greater than 50%, and greater than the restraining effect of 25 μ mol/L lamivudine pair cells secretion HBsAg.
3.ASODN restraining effect to Hep2.2.15 emiocytosis HBeAg
Five antisense sequences E1-E5 of target epiregulin mRNA, handle the Hep2.2.15 cell with 0.8 μ mol/L administration respectively, collecting cell nutrient solution after 72 hours, HBeAg ELISA detection kit detects the expression of HBeAg in the Hep2.2.15 cell culture fluid, according to formula IR=(A
The dosing group-A
The cell control group)/A
The cell control groupCalculate inhibiting rate, IR represents inhibiting rate in the formula, and the A representative respectively detects the absorbancy of hole at 450nm.The triplicate experiment, the mean value of calculating inhibiting rate is seen Fig. 5.Among the figure, LIP represents the restraining effect of liposome control group pair cell secretion HBeAg, and showing does not have obvious restraining effect.LAM25 represents the restraining effect of 25 μ mol/L lamivudine pair cells secretion HBeAg, and inhibiting rate is less than 25%.E1-E5 represents the restraining effect of five antisense sequences pair cells secretion HBeAg, and wherein the average inhibiting rate of E3 is greater than 35%, and greater than the restraining effect of 25 μ mol/L lamivudine pair cells secretion HBeAg.
4.ASODN the proteic restraining effect of pair cell epiregulin
Five antisense sequences E1-E5 of target epiregulin mRNA, handle the Hep2.2.15 cell with 0.8 μ mol/L administration respectively, extract total protein behind the 72h, behind 12% polyacrylamide gel electrophoresis, adopt half-dried transfer printing that albumen is gone on the nitrocellulose filter, with β-actin (43kD) is contrast, detects the influence of sulfo-antisense oligonucleotide to target protein epiregulin expression amount by Western Blot method.As seen from Figure 6, E3 can significantly suppress the proteic expression of epiregulin, E1, and E2, E4 does not have obvious restraining effect to epiregulin albumen, and E5 is to epiregulin albumen unrestraint effect.
Conclusion
1. the expression that suppresses epiregulin can suppress duplicating and expressing of HBV in the HepG2.2.15 cell
2. in five sulfo-epiregulin Antisensedigonucleotsequence sequences, E3 has the effect of hbv replication and expression in the obvious suppression HepG2.2.15 cell.
Embodiment three
Materials and methods
1.ASODN design with synthetic
According to embodiment two results, select effect Antisensedigonucleotsequence sequence E3 preferably, synthetic its positive MODN (see in the sequence table 6).Synthesizing of all G 3139s with embodiment two.
2. the cytotoxicity of sulfo-Antisensedigonucleotsequence sequence E3 detects
The Hep2.2.15 cell is in the MEM substratum that contains 10% foetal calf serum (Gibco) and 380 μ g/ml, in 37 ℃, 5%CO
2Cultivate in the incubator.The observation of cell growth conditions is good, is cultured to the logarithmic growth after date, inoculates 96 orifice plates, 0.75 * 10
5Cells/well, 37 ℃, 5%CO2 was hatched 48-72 hour, after waiting to grow to the 40-60% cell and converging, under the serum-free state, adopted liposome Lipofectin (Invitrogen, 1mg/ml) reagent and with reference to specification sheets operation carrying out transfection.The concentration of antisense oligonucleotide E3 is 0.2 μ M, 0.4 μ M, 0.8 μ M, 1.6 μ M, 5 μ M and establishes the cell contrast that every concentration repeats three holes.After the transfection 22 hours, change normal cell nutrient solution (the MEM cell culture fluid that contains 10%FBS), 37 ℃, after 5%CO2 is hatched 72 hours, with reference to MTS (Promega) working instructions, every hole adds MTS20 μ l/100 μ l nutrient solution, and 37 ℃ of lucifuges were hatched 1.5 hours, and 490nm detects absorbancy at multiple labeling inspection enzyme-linked immunosorbent assay instrument.Simultaneously, behind the transfection ASODN every day observation of cell form under inverted microscope.
3. the specificity of sulfo-Antisensedigonucleotsequence sequence E3 is investigated
With 0.8 μ mol/L transfection HepG2.2.15 cell, transfection method is with embodiment two respectively for sulfo-Antisensedigonucleotsequence sequence E3 and just sequence E3s thereof.Collect nutrient solution, extract total RNA and total protein, with reference to the method for embodiment one, example two, detect the restraining effect of HBsAg, HBeAg, HBV DNA in E3 and the just sequence pair cell nutrient solution thereof, E3 and just sequence thereof are to the proteic restraining effect of epiregulin.
4. the dose-dependently of sulfo-Antisensedigonucleotsequence sequence E3 detects
Sulfo-Antisensedigonucleotsequence sequence E3 is with 0.2 μ M, 0.4 μ M, 0.8 μ M, 1.6 μ M transfection HepG2.2.15 cells, and transfection method is with embodiment one.Collect nutrient solution, extract total protein,, detect HBsAg in the E3 different concns pair cell nutrient solution, HBeAg, the restraining effect of HBV DNA and the proteic restraining effect of epiregulin with reference to the method for embodiment one, example two.
The result
1. sulfo-antisense oligonucleotide E3 is to the influence of HepG2.2.15 cell proliferation
Sulfo-antisense oligonucleotide E3 handles in the cell processes with 0.2 μ M, 0.4 μ M, 0.8 μ M, 1.6 μ M, 5 μ M, and every day is the observation of cell form under inverted microscope, and dosing group cellular form and cellular control unit form do not have noticeable change.The cell proliferation experiment detected result is seen Fig. 7.Show among the figure that in 0.2 μ M-5 μ M scope, each dosage group OD value of E3 and normal cell contrast are basic identical.
2. the specificity of sulfo-Antisensedigonucleotsequence sequence E3
Sulfo-Antisensedigonucleotsequence sequence E3 and just sequence E3s thereof are respectively with 0.8 μ mol/L transfection HepG2.2.15 cell, collect nutrient solution, the ELISA detection kit detects the expression of HBsAg, HBeAg in the nutrient solution, the copy number of HBV DNA in the fluorescence quantitative PCR detection nutrient solution.If the content of HBsAg, HBeAg, HBV DNA is 100% in the control cells nutrient solution, the content of HBsAg, HBeAg then is in the experimental group: A
Experimental group/ A
The cell control group* 100%, wherein A is illustrated in the absorbancy of 450nm.The content of HBV DNA then is in the experimental group: C
Experimental group/ C
The cell control group* 100%, wherein C represents the copy number of HBV DNA in the cell culture fluid, the results are shown in Figure 9.Show among the figure that HBsAg, HBeAg, HBV DNA contrast no significant difference (P 〉=0.05) with normal cell in just G 3139 sequence (E3s) the treatment group cell culture fluid of E3.And sulfo-Antisensedigonucleotsequence sequence E3 has good restraining effect (HBsAg, HBV DNA inhibiting rate 〉=50%, HBeAg inhibiting rate 〉=35%).Equally, after sulfo-Antisensedigonucleotsequence sequence E3 and just sequence E3s thereof are respectively with 0.8 μ mol/L transfection HepG2.2.15 cell 72 hours, extract total protein, the western detected result shows that E3s handles groups of cells epiregulin protein expression and control group basically identical, and E3 demonstrates the effect (see figure 8) of good restraining epiregulin protein expression.
3. sulfo-Antisensedigonucleotsequence sequence E3 is to the dose-dependently of hbv replication and expression influence.
After sulfo-Antisensedigonucleotsequence sequence E3 handles the HepG2.2.15 cell with 0 μ mol/L, 0.2 μ mol/L, 0.4 μ mol/L, 0.8 μ mol/L administration, collect nutrient solution, the copy number of HBV DNA in the fluorescence quantitative PCR detection nutrient solution, the expression that the ELISA detection kit detects HBsAg, HBeAg in the nutrient solution calculates inhibiting rate respectively according to the formula among the embodiment two.The triplicate experiment, the mean value of calculating inhibiting rate.As Figure 10, HBsAg in the E3 pair cell nutrient solution, HBeAg, the restraining effect of HBV DNA is successively decreased along with the increase of sulfo-Antisensedigonucleotsequence sequence E3 concentration, demonstrates dose-dependence clearly.Equally, after 0 μ mol/L, 0.2 μ mol/L, 0.4 μ mol/L, 0.8 μ mol/L, 1.6 μ mol/LE3 handle cell 72 hours, extract total protein, the western engram technology detects the proteic expression of epiregulin, the result as shown in Figure 8, along with the increase of E3 concentration, the epiregulin expressing quantity reduces gradually, 0.8 when μ mol/L, 1.6 μ mol/L, E3 handles groups of cells and almost detects the proteic expression less than epiregulin.
Conclusion
1. sulfo-Antisensedigonucleotsequence sequence E3 is to the not significantly influence of propagation of HepG2.2.15 cell
2. sulfo-Antisensedigonucleotsequence sequence E3 has the effect of duplicating and expressing of HBV in the sequence-specific inhibition HepG2.2.15 cell
3. in 0.2-1.6 μ mol/L concentration range, E3 is to HBV DNA, HBsAg, and HBeAg and epiregulin albumen have specific dose-dependent inhibition activity.
Sequence table
＜110〉radiation of Chinese People's Liberation Army's car thing Academy of Medical Sciences and radiation medicine institute
＜120〉structure and the purposes of the antisense oligonucleotide of inhibition epidermal regulator EREG gene expression
<130>
<160>6
<170>PatentIn?version?3.3
<210>1
<211>20
<212>DNA
<213>
<400>1
ctaattgcat?tattgtttta 20
<210>2
<211>20
<212>DNA
<213>
<400>2
tttttcaagt?acattaaaat 20
<210>3
<211>20
<212>DNA
<213>
<400>3
actgttatcc?ctgcccataa 20
<210>4
<211>20
<212>DNA
<213>
<400>4
agtttgggag?gctgtggagg 20
<210>5
<211>20
<212>DNA
<213>
<400>5
ttgagaggct?tacaataaat 20
<210>6
<211>20
<212>DNA
<213>
<400>6
ttatgggcag?ggataacagt 20
Claims (6)
1. epidermal regulator EREG is as the application of target in the preparation anti-hepatitis B virus infective medicament.
With the described purposes of claim 1 in target EREG non-coding region or coding region complementary oligonucleotide, the sequence of described oligonucleotide be selected from following it-:
1)E1：5’-CTA?ATT?GCA?TTA?TTG?TTT?TA-3’：
2)E2：5’-TTT?TTC?AAG?TAC?ATT?AAA?AT-3’；
3)E3：5’-ACT?GTT?ATC?CCT?GCC?CAT?AA-3’；
4)E4：5’-AGT?TTG?GGA?GGC?TGT?GGA?GG-3’；
5)E5：5’-TTG?AGA?GGC?TTA?CAA?TAA?AT-3’。
3. oligonucleotide according to claim 2 is characterized in that described oligonucleotide sequence structure is as follows:
1)E3：5’-ACT?GTT?ATC?CCT?GCC?CAT?AA-3’。
4. according to claim 2 or 3 described oligonucleotide, it is characterized in that this oligonucleotide is through the different chemical modification.
5. oligonucleotide according to claim 4, its chemically modified are thio-modification.
6. the purposes of the arbitrary oligonucleotide described in the claim 2,3,4,5 in preparation treatment hepatitis B and diseases related medicine thereof.
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| US5985662A (en) * | 1995-07-13 | 1999-11-16 | Isis Pharmaceuticals Inc. | Antisense inhibition of hepatitis B virus replication |
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| US5985662A (en) * | 1995-07-13 | 1999-11-16 | Isis Pharmaceuticals Inc. | Antisense inhibition of hepatitis B virus replication |
| CN1587408A (en) * | 2004-08-23 | 2005-03-02 | 中国人民解放军军事医学科学院放射医学研究所 | Structure of anti-hypatitis B virus antisense oligonucleotide of target de-sialoglycoprotein receptor |
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| 人epiregulin在大肠杆菌中的表达. 席全胜等.生物化学与生物物理学报，第32卷第3期. 2000 * |
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