CN101063129B - Structure and usage of antisense oligonucleotide inhibiting ABHD2 gene expression - Google Patents
Structure and usage of antisense oligonucleotide inhibiting ABHD2 gene expression Download PDFInfo
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Abstract
The invention relates to a serum hepatitis virus target and relative antisense oligonucleotide medicine, which incorporates the usage of these antisense oligonucleotide to prepare medicine to cure serum hepatitis.
Description
Technical field:
The present invention relates to the biotechnology pharmaceutical field, antisense oligonucleotide (ASODN, sequence antisenseoligodexynucleotide), structure and medicine thereof that specifically a kind of target ABHD2 (abhydrolase domaincontaining2) 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.
This laboratory finds that by screening and checking but ABHD2 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 ABHD2 gene mRNA sequence, design by suppressing the expression of ABHD2, stops HBV to infect at the ASODN of ABHD2, suppresses 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 ABHD2mRNA reference sequences NM_007011 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 ABHD2.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, AB3 when 0.8 μ mol/L to HBV DNA, HBsAg, HBeAg, ABHD2 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 0.2-0.8 μ mol/L concentration range, AB3 is to HBV DNA, HBsAg, and HBeAg and ABHD2 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 ABHD2 suppresses duplicating and expressing of hepatitis B virus, and ABHD2 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 ABHD2mRNA, 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 AB3 determines that according to experiment the present invention has comprised any length oligonucleotide that has identical sequence with AB3.
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 AB3.
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:
Figure 1A BHD2mRNA is in HepG2 cell, HepG2.2.15 cell and the HepG2.2.15 cell expression changing conditions before and after drug treating
Fig. 2 ABHD2 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-ABHD2 Antisensedigonucleotsequence sequence is to the restraining effect of HBV DNA in the HepG2.2.15 cell
Fig. 4 sulfo-ABHD2 Antisensedigonucleotsequence sequence is to the restraining effect of HBsAg in the HepG2.2.15 cell
Fig. 5 sulfo-ABHD2 Antisensedigonucleotsequence sequence is to the restraining effect of HBeAg in the HepG2.2.15 cell
Fig. 6 sulfo-ABHD2 Antisensedigonucleotsequence sequence is to the proteic restraining effect of ABHD2 in the HepG2.2.15 cell
Fig. 7 sulfo-ABHD2 Antisensedigonucleotsequence sequence AB3 is to the influence of HepG2.2.15 cell proliferation
Fig. 8 sulfo-ABHD2 Antisensedigonucleotsequence sequence AB3 is dose-dependently and just sequence A B3s thereof to the proteic restraining effect of ABHD2 in the HepG2.2.15 cell to the proteic restraining effect of ABHD2 in the HepG2.2.15 cell
Fig. 9 sulfo-ABHD2 Antisensedigonucleotsequence sequence AB3 and just sequence thereof are to the restraining effect of HepG2.2.15 emiocytosis HBsAg, HBeAg, HBV DNA
Figure 10 sulfo-ABHD2 Antisensedigonucleotsequence sequence AB3 is dose-dependently to the restraining effect of HepG2.2.15 emiocytosis HBsAg, HBeAg, HBV DNA
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 back 1:3 and goes down to posterity, 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 ABHD2mRNA 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 RNA1 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, DTT2.5 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 ABHD2 upstream primer is 5 '-GCCCCACCTGACCTCTACT-3 ', downstream primer is 5 '-AACGAAAGTGCGGATGTATT-3 ', the amplification fragment length is 324bp.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, Taq1U.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 ABHD2 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 proteins, behind 12% polyacrylamide gel electrophoresis, adopt half-dried transfer printing with albumen go to nitrocellulose filter (
BA-S83Reinforced 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 the anti-people ABHD2 of rabbit antibody (professor Xue Yanning provides) or the anti-people β of rabbit-actin antibody (Sigma) then, TBST washes film 3 times, each 10min, combine 1h with the anti-rabbit two anti-(middle mountain) of horseradish peroxidase mark 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.ABHD2mRNA 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 detect the expression less than ABHD2mRNA in the HepG2 cell, and its expression level is higher in the HepG2.2.15 cell as shown in fig. 1.After 25 μ mol/L lamivudines were handled, ABHD2mRNA expressed obviously downward modulation in the HepG2.2.15 cell.Control represents the relative expression situation of ABHD2mRNA in cellular control unit among Fig. 1; Lamivudine represents the relative expression situation of ABHD2mRNA in lamivudine treatment group cell; HepG2, HepG2.2.15 represent the relative expression situation of ABHD2mRNA in HepG2, HepG2.2.15 cell.
2.ABHD2 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 ABHD2 albumen relative expression quantity in the HepG2.2.15 cell is higher, and almost detects the proteic expression less than ABHD2 in the HepG2 cell.ABHD2 albumen was obviously reduced after 25 μ mol/L lamivudines were handled the HepG2.2.15 cell.
Conclusion
ABHD2 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 ABHD2 mRNA reference sequences NM 007011 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) the ASODN sequence of 5 target ABHD2 of design.By with the online blast sequence alignment of GeneBank, selected target sequence all has excellent specificity, can not disturb the expression (seeing sequence table 1-5) 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 PureII (Oligo Prep OP120, 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 hours, after waiting to grow to 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, 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 ABHD2 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 AB1-AB5 of target ABHD2mRNA, 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
The 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.AB1-AB5 represents five antisense sequences restraining effect to HBV DNA when 0.8 μ mol/L, can show from figure, and AB3 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 AB1-AB5 of target ABHD2mRNA, 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%.AB1-AB5 represents the restraining effect of five antisense sequences pair cells secretion HBsAg, and wherein the average inhibiting rate of AB3 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 AB1-AB5 of target ABHD2mRNA, 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%.AB1-AB5 represents the restraining effect of five antisense sequences pair cells secretion HBeAg, and wherein the average inhibiting rate of AB3 is greater than 50%, 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 ABHD2
Five antisense sequences AB1-AB5 of target ABHD2mRNA, 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 ABHD2 expression amount by Western Blot method.As seen from Figure 6, AB3 can significantly suppress the proteic expression of ABHD2, AB5 to ABHD2 albumen restraining effect a little less than, AB1, AB2, AB4 does not have obvious restraining effect to ABHD2 albumen.
Conclusion
1. the expression that suppresses ABHD2 can suppress duplicating and expressing of HBV in the HepG2.2.15 cell
2. in five sulfo-ABHD2 Antisensedigonucleotsequence sequences, AB3 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 AB3 preferably, synthetic its positive MODN (seeing sequence table 6).Synthesizing of all G 3139s with embodiment two.
2. the cytotoxicity of sulfo-Antisensedigonucleotsequence sequence AB3 detects
The Hep2.2.15 cell is cultivated in 37 ℃, 5%CO2 incubator in the MEM substratum that contains 10% foetal calf serum (Gibco) and 380 μ g/ml.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 hours, after waiting to grow to 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 AB3 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 AB3 is investigated
With 0.8 μ mol/L transfection HepG2.2.15 cell, transfection method is with embodiment two respectively for sulfo-Antisensedigonucleotsequence sequence AB3 and just sequence A B3s 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 AB3 and the just sequence pair cell nutrient solution thereof, AB3 and just sequence thereof are to the proteic restraining effect of ABHD2.
4. the dose-dependently of sulfo-Antisensedigonucleotsequence sequence AB3 detects
Sulfo-Antisensedigonucleotsequence sequence AB3 is with 0.2 μ M, 0.4 μ M, 0.8 μ M transfection HepG2.2.15 cell, and transfection method is with embodiment one.Collect nutrient solution, extract total protein,, detect HBsAg in the AB3 different concns pair cell nutrient solution, HBeAg, the restraining effect of HBV DNA and the proteic restraining effect of ABHD2 with reference to the method for embodiment one, example two.
The result
1. sulfo-antisense oligonucleotide AB3 is to the influence of HepG2.2.15 cell proliferation
Sulfo-antisense oligonucleotide AB3 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 AB3 and normal cell contrast are basic identical.
2. the specificity of sulfo-Antisensedigonucleotsequence sequence AB3
Sulfo-Antisensedigonucleotsequence sequence AB3 and just sequence A B3s 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 (AB3s) the treatment group cell culture fluid of AB3.And sulfo-Antisensedigonucleotsequence sequence AB3 has good restraining effect (HBsAg, HBeAg, HBV DNA inhibiting rate 〉=50%).Equally, after sulfo-Antisensedigonucleotsequence sequence AB3 and just sequence A B3s 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 AB3s handles groups of cells ABHD2 protein expression and control group basically identical, and AB3 demonstrates the effect (see figure 8) of good restraining ABHD2 protein expression.
3. sulfo-Antisensedigonucleotsequence sequence AB3 is to the dose-dependently of hbv replication and expression influence.
After sulfo-Antisensedigonucleotsequence sequence AB3 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 AB3 pair cell nutrient solution, HBeAg, the restraining effect of HBV DNA is successively decreased along with the increase of sulfo-Antisensedigonucleotsequence sequence AB3 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 AB3 handle cell 72 hours, extract total protein, the western engram technology detects the proteic expression of ABHD2, the result as shown in Figure 8, along with the increase of AB3 concentration, the ABHD2 expressing quantity reduces gradually, 0.8 during μ mol/L, AB3 handles groups of cells and almost detects the proteic expression less than ABHD2.
Conclusion
1. sulfo-Antisensedigonucleotsequence sequence AB3 is to the not significantly influence of propagation of HepG2.2.15 cell
2. sulfo-Antisensedigonucleotsequence sequence AB3 has the effect of duplicating and expressing of HBV in the sequence-specific inhibition HepG2.2.15 cell
3. in 0.2-0.8 μ mol/L concentration range, AB3 is to HBV DNA, HBsAg, and HBeAg and ABHD2 albumen have specific dose-dependent inhibition activity.
Sequence table
＜110〉INST OF EMISSION ﹠ RADIATION M
＜120〉structure and the purposes of the antisense oligonucleotide of inhibition ABHD2 genetic expression
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<160>6
<170>PatentIn?version3.3
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<211>20
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<213>
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<210>3
<211>20
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<213>
<400>3
<210>4
<211>20
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<213>
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<213>
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Claims (6)
1.ABHD2 drug target target purposes as anti-hepatitis B virus infective.
2. with claim 1 described target ABHD2 non-coding region or coding region complementary antisense oligonucleotide, the sequence of described antisense oligonucleotide be selected from one of following:
1)AB1：5’-CGT?GCA?GCC?ATA?GGT?GAA?CA-3’；
2)AB2：5’-TAG?GTG?AAC?ATG?CGT?GGC?GA-3’；
3)AB3：5’-TAA?AAT?CCC?CAG?GCT?CCT?TC-3’；
4)AB4：5’-TCC?CAC?GTG?CAG?CCA?TAG?GT-3’；
5)AB5：5’-TTT?CAT?GCA?CCA?ACG?GAT?CG-3’。
3. antisense oligonucleotide according to claim 2 is characterized in that described Antisensedigonucleotsequence sequence is as follows:
1)AB3：5’-TAA?AAT?CCC?CAG?GCT?CCT?TC-3’。
4. according to claim 2 or 3 described antisense oligonucleotides, it is characterized in that this antisense oligonucleotide is through chemically modified.
5. antisense oligonucleotide according to claim 4, its chemically modified are thio-modification.
6. the purposes of the arbitrary antisense oligonucleotide described in the claim 2,3,4 or 5 in the medicine of preparation treatment hepatitis B.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006100759775A CN101063129B (en) | 2006-04-26 | 2006-04-26 | Structure and usage of antisense oligonucleotide inhibiting ABHD2 gene expression |
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| WO2017023861A1 (en) * | 2015-08-03 | 2017-02-09 | The Regents Of The University Of California | Compositions and methods for modulating abhd2 activity |
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