Background technology
(1) the treatment present situation of hepatitis B virus (HBV)
Chronic hepatitis B is one of disease occurred frequently of China, is the major cause that causes hepatic fibrosis and liver cancer.If at present the drug main of treatment hepatitis B is based on the immunomodulator of interferon-alpha with based on the nucleoside analog of lamivudine, Adefovir, but result of treatment still is unsatisfied with.HBV DNA to continue to duplicate be the major cause that causes the hepatitis B chronicity.Therefore effectively suppressing the HBV dna replication dna, is the key of treatment chronic hepatitis B and control HBV persistent infection.
(2) RNA interferential present situation
Recently, (RNA interference, the RNAi) appearance of technology have injected new vitality for the treatment of resistant to viral disease in the RNA interference.In decades, RNA is regarded as merely from DNA and obtains genetic information, and information is passed to protein.But discover that recently small fragment RNA (small RNA) is being brought into play the effect of gene regulating, it can close expression of gene, or changes the level [1] of genetic expression.It is verified in mammalian cell that reality is tested research, the double-stranded RNA of 21-23 length of nucleotides (double-stranded RNA, dsRNA), specific RNA can effectively degrade, thereby the expression of blocking protein [2,3], the dsRNA of these length-specifics is called as siRNA (small interfering RNAs).Further mechanism is discovered, siRNA not only can be in the function of post-transcriptional level silence (transcriptionalgene silencing PTGS) specific gene, can also be in DNA and transcriptional level regulatory gene (transcriptional gene silencing, expression TGS) [4].Research now mainly concentrates on post-transcriptional level, after forming, siRNAs at first enters RISC complex body (RNA induced silencingcomplex) in cytoplasm, under the effect of ATP and helicase etc., untwist into strand, antisense strand is attached on the RNA of particular sequence by the base complementrity mode, the specific RNA sequence of cutting under the assistance of RISC complex body, thus degradation of rna makes it to translate into protein, cause this specific gene silence (gene silencing), lose function [4].Simultaneously, RNAi has high degree of specificity, the almost expression of suppressor gene fully of the double-stranded RNA of 19 pairs of Nucleotide (its 3 ' end plug-in 2 uracil sequences [5]), and after one of them coding mutation fallen, it has just disappeared to the restraining effect of gene, this application to siRNA is very important, can avoid siRNA degraded and said target mrna other the mRNA[5 with family].In addition, existing discovering, RNAi is except expressing in the post-transcriptional level regulatory gene, and can make the homologous DNA sequence structure that methylates, thereby has important regulation at transcriptional level, the dsRNA of confirmations such as Mette and NOSPro gene promoter area homologous 23nt can directly cause the methylating of dna sequence dna of goal gene, causes transcribing inactivation [6].
RNAi is as cell " immunity " system on a kind of RNA basis, be applied to anti-virus infection recently and the research of duplicating in, and in the experiment in vitro of HIV, obtained comparatively ideal result, action intensity is considerably beyond antisense nucleic acid and ribozyme technology [7].Rossi etc. with HIV-1 encode rev gene and with its homologous double-stranded RNA cotransfection in 293 cells, the rev expression of gene is significantly suppressed; With retarding effect and the sense-rna of siRNA, ribozyme etc. compare simultaneously, find to have only the siRNA group to suppress the expression of HIVrev-EGFP, its inhibiting rate reaches 90%, and sense-rna, ribozyme group and control group do not have significant difference [7], and this result is confirmed [8] by scholars such as Sharp equally.In RNAi research, there are some researches show: can effectively suppress the expression [10] of duplicating of HCV RNA and viral protein at the siRNA of HCV gene target sequence at virogene of hepatitis.Because what RNA disturb to adopt is double-stranded RNA, it is more stable, be difficult for being degraded, so RNA disturbs not only external, and also has effect preferably in animal body.Therefore, the discovery of RNAi phenomenon is chosen as one of 2002 ten big science achievements by " SCIENCE " magazine and AAAS, has broad application prospects.Consistently now think that the siRNA at virogene is best therapeutic strategy [9].
(3) 5 kinds of methods of RNA interferential
At present, be used for RNA interferential method and mainly contain 5 kinds: (1) chemical synthesis: directly by the synthetic dsRNA of chemical process, its advantage be fast, resultant quantity is bigger, but cost an arm and a leg; (2) in-vitro transcription method: with Oligo DNA is template, and by the synthetic siRNAs of in-vitro transcription, the relative chemosynthesis of cost is lower, and can obtain siRNAs sooner than chemical synthesis.What deserves to be mentioned is that the siRNAs toxicity that in-vitro transcription obtains is little, good stability, the efficient height, 1/10 of the general siRNA amount that only needs chemosynthesis just can reach equal effect.But technical difficulty is bigger; (3) RNaseIII degrading dsRNA method: after the method preparation of said target mrna with 200～1000 bases with in-vitro transcription, use RNaseIII in external digestion then, obtain the mixture of multiple siRNA, its advantage is to study the phenotype of certain gene function disappearance fast and economically, but be not suitable for long research project, or need a specific siRNA to study particularly gene therapy; (4) siRNA expression vector method: the carrier of construction expression specific siRNA, import and express siRNA in the eukaryotic cell, have the advantage that can increase in a large number, but have the potentially dangerous of gene integration; (5) siRNA expresses framework (siRNA expressioncassettes) method: the dna profiling that directly obtains expressing siRNA by PCR method, be transcribed into siRNA in vivo after importing eukaryotic cell, its advantage is that method is simple, speed is fast, can be used for screening effective siRNA sequence, but also have the potentially dangerous of gene integration.
The relative merits and the RNA that make a general survey of above-mentioned 5 kinds of methods disturb the experimental study of chronic HBV infection and the prospect in the clinical application of being applied in, and the present invention selects the dsRNA of the synthetic particular target sequence of in-vitro transcription method.
Summary of the invention
The invention provides small interference ribonucleic acid (siRNA) sequence and the preparation method of hepatitis B virus (HBV) gene, SEQ ID NO.1-4 is a dna profiling sequence of transcribing 2 couple of siRNA at HBV (ayw hypotype) S gene provided by the invention, with the synthetic special siRNA of in-vitro transcription method at HBV (ayw hypotype) gene, behind transfectional cell, reach the purpose that suppresses HBV genetic expression.This method can be fast, economic, relatively large the siRNA that obtain anti-HBV, for experimental study and the clinical application of anti-HBV provides technology and a large amount of siRNA.
The invention provides 2 couple of transcribing siRNA dna profiling sequence at HBV (ayw hypotype) S gene:
SEQ?ID?NO.1(Antisense?anti-s?siRNA1?Oligonucleotide?template)
5’-AAACCTTCGGACGGAAATTGCCCTGTCTC-3’
SEQ?ID?NO.2(Sense?anti-s?siRNA1?Oligonucleotide?template)
5’-AAGCAATTTCCGTCCGAAGGTCCTGTCTC-3’
SEQ?ID?NO.3(Antisense?anti-s?siRNA2?Oligonucleotide?template)
5’-AATACCGCAGAGTCTAGACTCCCTGTCTC-3’
SEQ?ID?NO.4(Sense?anti-s?siRNA2?Oligonucleotide?template)
5’-AAGAGTCTAGACTCTGCGGTACCTGTCTC-3’
2 pairs of dna profiling sequences at HBV (ayw hypotype) S gene of the present invention realize by following steps:
(1) design of siRNA
(2) design of transcribing template of specific siRNA and synthetic
(3) annealing
(4) benefit is flat
(5) transcribe
(6) template degraded
(7) cross column purification
Embodiment
The present invention is described further in conjunction with the embodiments.
Embodiment one external expression of transcribing synthetic RNA interfering vitro inhibition HBV surface antigen blending gene
The preparation of siRNA
(1) experimental result of siRNA is directly synthesized in the principle of design of the design consideration siRNA of siRNA and utilization, chooses the target cDNA target sequence at 2 siRNA of HBV (ayw hypotype) S gene.Sequence is as follows:
(anti-s?siRNA1)5’-AAACCTTCGGACGGAAATTGC-3’
(anti-s?siRNA2)5’-AATACCGCAGAGTCTAGACTC-3’
(2) design of transcribing template of specific siRNA and synthetic according to above-mentioned cDNA target sequence, the DNA that designs and synthesizes two sections 29-mer is as template, comprise that 8 start in the base (being called homing sequence, leader sequence) of primer 3 ' end coupling and the siRNA sequence of 21 designs with T7.
1. the antisense strand of specificity anti-s siRNA1 and positive-sense strand are as follows:
Antisense?anti-s?siRNA1?Oligonucleotide?template
5’-AAACCTTCGGACGGAAATTGCCCTGTCTC-3’
Sense?anti-s?siRNA1?Oligonucleotide?template
5’-AAGCAATTTCCGTCCGAAGGTCCTGTCTC-3’
2. the antisense strand of specificity anti-s siRNA2 and positive-sense strand are as follows:
Antisense?anti-s?siRNA2?Oligonucleotide?template
5’-AATACCGCAGAGTCTAGACTCCCTGTCTC-3’
Sense?anti-s?siRNA2?Oligonucleotide?template
5’-AAGAGTCTAGACTCTGCGGTACCTGTCTC-3’
(3) annealing mixes these two templates and corresponding T7 promoter primer respectively, the terminal and dna profiling annealed combination of primer.
2μl?T7?promoter?primer
6μl?DNA?Hyb?buffer
The template of 2 μ l justice or antisense
70℃，5min→room?temp，5min
(4) mend flat putting down and become the double-stranded DNA template with the big segment benefit of archaeal dna polymerase Klenow.
In above-mentioned reaction tubes, add:
2μl?10×Klenow?Reaction?buffer
2μl?10×dNTP?Mix
4μl?Nucl?ease-free?water
2μl?Exo-klenow
Soft mixing, 37 ℃, 30min;
(5) transcribe respectively and carry out in-vitro transcription with t7 rna polymerase, product is mixed forming dsRNA, new double-stranded RNA comprises homing sequence and 19 base sequences of middle complementary and 3 ' end two multiple U (UU) of 5 ' end strand.
Add in each responsive transcription pipe:
2 μ l justice or antisense siRNA template
4μl?Nuclease-free?water
10μl?2×NTP?Mix
2μl?10×T7?Reaction?buffer
2μl?T7?Enzyme?Mix
Soft mixing, 37 ℃, 2hr;
Justice and antisense transcription product mix, and place 37 ℃ to spend the night
(6) the template degraded is by DNA enzyme liberating template, use simultaneously the single-minded nuclease digestion of strand 5 ' homing sequence, because RNase can not cut the U base and can not cut double-stranded RNA, double-stranded siRNAs of the 21-mer that so the product that obtains is exactly for we to be needed---19 base complementrities are arranged, and 3 ' end respectively has 2 U outstanding.
Add in the above-mentioned reactant:
6μl?Digestion?buffer
48.5μl?Nuclease-free?water
3μl?RNase
2.5μl?DNase
Behind the mixing, 37 ℃, 2hr;
(7) cross the purifying pillar that column purification provides by test kit, remove impurity such as primer, base salt and protein, the siRNAs that can transform usefulness exactly immediately that obtains, by specification carries out.
Embodiment 2 anti-s siRNA suppress the green fluorescent protein expression of reporter plasmid pGFP-S and siRNA cotransfection HepG2 cell and the experiment of HBV S genetic expression
(1) reporter plasmid pGFP-S and siRNA cotransfection HepG2 cell reporter plasmid pGFP-S are the plasmids of expressing reporter gene green fluorescent protein and HBsAg fusion rotein.The HepG2 cell is according to 1 * 10524 orifice plates are inoculated in/hole, cultivate to reach the 80%-90% fusion rate after 24 hours, and cotransfection pGFP-S and siRNA, concrete steps are with reference to the Lipofectamine2000 of Invitrogen company specification sheets.Change 10%FCS DMEM after 6 hours, the feminine gender group of a transfection pGFP-S carrier is set simultaneously, every group is repeated 3 holes.
(2) fluorescence microscope cell green fluorescent protein was expressed transfection after 48 hours, and (DMIRB Leica) observes the expression of green fluorescent protein in the HepG2 cell down at inverted fluorescence microscope.The result shows that compare with the feminine gender group of pGFP-S carrier, the fluorescence intensity of the HepG2 cell of transfection pGFP-S and anti-S siRNA weakens, and the fluorocyte number reduces.
(3) flow cytometer observation of cell green fluorescent protein was expressed transfection after 48 hours, conventional digestion HepG2 cell, and centrifugal suspension is resuspended in PBS, and flow cytometer detects the positive cell number ratio and the average fluorescent strength of express fluorescent protein.The result shows, compares with the feminine gender group of pGFP-S carrier, and the positive cell number ratio of the HepG2 of transfection pGFP-S and siRNA reduces, and average fluorescent strength reduces.
(4) reverse transcription-real-time fluorescence quantitative PCR method detects the expressed rna preparation of HBV S gene RNA: transfection was gathered in the crops the HepG2 cell after 72 hours, Trizol method extracting RNA, and method is with reference to Invitrogen company specification sheets.Reverse transcription, process is with reference to the MBI process specifications.Fluorescence dye standard measure PCR, upstream primer: 5 '-CTCACAATACCGCAGAGTC-3 '; Downstream primer: 5 '-TAAACTGAGCCAGGAGAAA-3 '; Internal reference GAPDH, upstream primer: 5 '-ACAGTCAGCCGCATCTTCTTT-3 ', downstream primer: 5 '-GCAACAATATCCACTTTACCAGAG-3 ' .PCR condition is: 95 ℃ of 3min, 94 ℃ of 30s again, 56 ℃ of 30s, 72 ℃ of 45s, 25 circulations, 72 ℃ of fluoroscopic examination point selection.Do positive control (being template group directly) and negative control (not adding template group) simultaneously with the pGFP-S plasmid DNA.Real-time quantitative PCR detects in 25 circulation fixed points and shows that compare with negative control, anti-S siRNA1 and anti-SsiRNA2 all reach more than 50% the inhibiting rate of S gene.
Embodiment 3anti-s siRNA suppresses the experiment to the HBsAg of siRNA transfection HepG2.2.15 cell and HbeAg expression
(1) siRNA transfection HepG2.2.15 cell HepG2.2.15 cell is according to 1 * 104Inoculate 24 orifice plates, cultivate and reach the 30%-40% fusion rate after 24 hours, each 0.84 μ g of transfection siRNA, concrete steps are referring to the OLIGOFECTAMINE of Invitrogen company reagent specification sheets.Irrelevant contrast siGFP group (FEBS Letters 543 (2003) 51-54) is set: sense RNA 5-GGCUACGUCCAGGAGCGCACC-3, anti-sense RNA 5-UGCGCUCCUGGACGUAGCCTT-3.Change 10%FCS DMEM after 6 hours.Establish 3 multiple holes for every group.
(2) 48h behind radioimmunology detection HBsAg and the HBeAg change in concentration transfection HepG2.2.15 cell, difference collecting cell nutrient solution, centrifugal removal cell debris, supernatant carry out radioimmunology and detect, and detection method illustrates referring to test kit.Behind transfection anti-S siRNA1 and the anti-S siRNA2, HBsAg in the HepG2.2.15 cell conditioned medium and the concentration of HBeAg are low than the HepG2.2.15 cell, and anti-SsiRNA1 and anti-S siRNA2 all reach more than 60% the inhibiting rate of HBsAg and HBeAg.