CN1057302C - Transdeoxyoilnucleotide for inhibiting replication of hepatitis B virus - Google Patents
Transdeoxyoilnucleotide for inhibiting replication of hepatitis B virus Download PDFInfo
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- CN1057302C CN1057302C CN98111018A CN98111018A CN1057302C CN 1057302 C CN1057302 C CN 1057302C CN 98111018 A CN98111018 A CN 98111018A CN 98111018 A CN98111018 A CN 98111018A CN 1057302 C CN1057302 C CN 1057302C
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Abstract
The present invention relates to an antisense oligodeoxynucleotide inhibiting the DNA replication of hepatitis B viruses and the gene expression of hepatitis B viruses, which comprises four antisense oligodeoxynucleotides which can be respectively combined with the 1798 position to the 1821 position and the 1807 position to the 1827 position of the hepatitis B viruses, the 1875 position to 1895 position of an epsilon area and an mRNA start codon area of a P gene. Mono phosphorylation can be used to modify the 3' end of the oligodeoxynucleotide in order to improve the stability to enable the oligodeoxynucleotide to be more effective in inhibiting the hepatitis B viruses. A cell experiment proves that the antisense oligodeoxynucleotide can resist the hepatitis B viruses and can be used to prepare medicaments for inhibiting the hepatitis B viruses and treating hepatitis B.
Description
The present invention relates to suppress hepatitis B virus (Hepatitis B Virus, HBV) antisense oligodeoxynucleotideresulted of the expression of dna replication dna and hepatitis B virogene, it also can be by 3 ' single phosphorylation modification improving stability, thereby improve the inhibition effect to HBV.
The hepatitis B that hepatitis B virus (HBV) causes is a kind of serious transmissible disease.Estimate according to the World Health Organization (1988), about 2-3 hundred million people of HBV carrier in the whole world, mainly be distributed in South East Asia and Africa, global all hepatocellular carcinomas (Hepatocellular Carcmoma, HCC) among the patient, have 80% to be HBV the infected, the relative risk that is subjected to the chronically infected crowd of HBV to suffer from HCC increases by 100 times at least.China is the high popular district of hepatitis B, and the crowd of 8-10% (about 100,000,000 populations) is hepatitis B (virus) surface antigen (Hepatitis B (virus) Surface Antigen, positive person HBsAg).According to whole nation investigation in 1980, it is about 2,000 ten thousand that the patient of hepatitis now suffers from China, is chronic hepatitis patient 60% or more wherein, this shows that the relative HCC of hepatitis B that is caused by the HBV infection is one of global main health problem.But, up to the present, still lack the methods of treatment that effectively directly suppresses viral proliferation clinically.Therefore, the new methods of treatment of research is a current urgent task.
HBV is a kind of liver property dna virus of having a liking for, and has very strong host specificity, and its infection can cause acute or chronic hepatitis.The infection of HBV, breeding rely on duplicating and genomic transcript and expression of its DNA.The HBV genome is a partially double stranded ring-shaped DNA molecule, about 3.2kb, it contains 4 main single open reading frames, they encode respectively cAg (preC, C), polysaccharase (P), surface antigen (preS1, preS2 and S) and X protein (X).The cis-regulating element of controlling these genetic expressions has promotor Cp, Sp1, Sp2 and Xp, enhanser ENI and ENII, static son (silencer) and glucocorticoid response element GRE etc.Under their regulation and control, transcribe and produce corresponding mRNA:3.5kb, 2.4kb, 2.1kb and 0.8kb mRNA (as shown in Figure 1).The different 3.5kb mRNA of initiation site has different functions.CAg before long preceding core mRNA translation produces is processed into e antigen (HBeAg) then; And short pregenomic mRNA translation produces C albumen and P albumen, also be the template of HBV dna replication dna simultaneously, produce daughter DNA (Li Zaiping by reverse transcription, Wang Yuan: HBV molecular biology: advancing biological chemistry collection of thesis (two), Shanghai science tech publishing house, 1992,247-256).
The reproduction process of HBV virus clear after deliberation (referring to Robinson WS, Klote L, and AokiN., J.Med.Virol., 1990,31:18-32), comprising:
1, the preceding gene RNA of 3.5kb is packaged to be advanced in the new synthetic capsid, becomes the template of reverse transcription.The recognition site of capsid packing is ε.
2, reverse transcription produces minus-strand dna under the effect of polysaccharase.Primer is the N end of polysaccharase, is combined in the DRI place of 3 ' end.Form DNA:RNA heterozygosis two strands, by the RNA in the RNase H hydrolysis heterozygosis chain in the polysaccharase, stay the dna single chain then.
3, be template with the minus-strand dna, synthesize section of DNA at the DRII place of 3 ' end to DRI, again from the synthetic positive chain DNA of the DRI of 5 ' end.The packaged cell that transports simultaneously in the building-up process, go out cell after positive the synthetic of chain DNA stop the dna molecular of consequently partially double stranded part strand.
As seen, important element comprises direct repeat sequence DRI in the hbv replication process, the recognition site ε of DRII and capsid packing.DRI, DRII are positive and negative DNA chain synthetic starting points.The ε sequence can form higher structure in the preceding gene RNA, is the recognition signal that forms capsid protein.Polysaccharase during the HBV dna replication dna is a P albumen.P albumen is formed from different translation starting point translations by 3.5kb mRNA, is the basic protein that is rich in Histidine of a 95KD, can be formed to the less product of various molecular weights.Now having illustrated proteic 4 functional domains of P is followed successively by: 1. the albumen primer [is also referred to as primase (primase) or terminal protein (terminal protein, TP)] (can be used as viral DNA synthetic primer, and with covalently bound [the Wang G.H. of nascent DNA, Seeger C.Cell 1992,71 (4): 663-670]); 2. transcribed spacer (not influencing the activity of reversed transcriptive enzyme after deletion is most of); 3. reversed transcriptive enzyme; 4. RNA enzyme H (RNaseH).
Suppress duplicating of HBV viral DNA, reduce the quantity of virus, can reach and suppress HBV genetic expression, the final purpose of removing virus.
Antisense OligodeoxynucleotideTechnique Technique (antisense oligonucleotide technology) for suppress the HBV expression of gene provide a kind of new method (Keoagh T, Shaffer JD, Anal.Chem., 1996,68:3405-12).Antisense oligonucleotide is attached on the complementary mRNA by the mode of base pairing, can suppress montage and the translation of RNA.From statistics, a certain 17 specific aggressiveness base sequences only occur once in people's gene, if adopt 20 or 20 antisense oligonucleotides to be used for treatment of diseases with last based composition, hardly repeating sequences can be arranged in genome, therefore, the effect of antisense oligonucleotide is very special, can reduce toxic side effect significantly.Therefore, the characteristics of antisense oligonucleotide are to combine with said target mrna specifically by the mode of base pairing, stop the mRNA translation, thereby suppress the expression of specific gene single-mindedly.Through the abundant and development over more than 10 years, antisense nucleic acid has developed into a proven technique, and it can provide strong instrument (Holt, Mol.Med.Yoday for expression and the viral breeding of inhibition of the function of research specific gene, special inhibition oncogene, 1996,2:184-5; Plenat, Mol.Med.Today, 1996,2:250-7).It also can become expression and the breeding of hepatitis B virus and the medicine of treatment hepatitis B that suppresses hepatitis B virogene.
1978, Zamecnik and stephenson (Zamecnik PC, Stephenson M, Proc.Natl.Acad.Sci.USA, 1978,75:280-284; Stephenson M, Zamecnik PC, Proc.Natl.Acad.Sci.USA, 1978,75:285-289) at first oligonucleotide is used for specificity and suppresses viral gene expression, they prove that first one section oligonucleotide with Rous sarcoma virus RNA complementary 13 Nucleotide length can suppress the breeding of Rous sarcoma virus.1984, the notion of sense-rna is proposed first by Simons, Lzant and Weintraub, from that time, having delivered a large amount of achievement in research proof antisense oligonucleotides can be in the breeding of vitro inhibition virus, for example to vesicular stomatitis virus, herpes simplex virus, influenza virus, human immune deficiency venereal disease poison (HIV), hepatitis C virus (HCV), human papillomavirus (HPV) (Leonetti, Gene, 1988,72:323-327; Smith, Proc.Natl.Acad.Sci.USA, 1986,83:2728-32; Zenal, Nucleic Acids Res., 1987,15:9909-13) etc.Antisense oligonucleotide has also been done more work to the inhibition of HBV genetic expression.Abroad, several families laboratory and company have applied for several patents (WO 95/19433, and WO 96/03152, and WO 96/39502, and WO 97/03211).They are at S, preceding S, and C, preceding C, many antisense oligonucleotides have been designed in gene such as P and ε site.These all adopt thiophosphoric acid to modify all at the ayw hypotype.What their antisense oligonucleotide had has stronger restraining effect to an expression of gene, but to the restraining effect of other genes a little less than.Wherein also there are some antisense oligonucleotides to be used for suppressing duplicating of HBV DNA, for example at the ε site.Domestic, Zhao Xiaoxia etc. (viral journal, 1992,8:125-130), respectively the S of HBV and C gene reverse cloning to the prokaryotic cell prokaryocyte transcription vector, in prokaryotic cell prokaryocyte, studied HBV S and C gene pairs forward clone's the S gene and the restraining effect of C genetic expression of reverse cloning.The strong grade of Yao Zhi studied restraining effect (Acta.Virol.1996, the 40:35-9 of antisense oligonucleotide to HBV genetic expression in external and cell; 1995,39:227-30).But the hepatitis B virus hypotype that they select for use also is the ayw hypotype, and this kind of is less in China, and the adr hypotype is more in China.Therefore, many research still is among the breadboard research so far owing to suppress the not high enough or oligonucleotide poor stability of efficient or be not suitable for Chinese hypotype, does not enter the clinical study stage.
For this reason, the purpose of this invention is to provide and a kind ofly can effectively suppress duplicating and the antisense oligodeoxynucleotideresulted of the expression of hepatitis B virogene of hepatitis B virus (comprising the adr hypotype) DNA, it comprise four difference can with the HBV gene in 1798 to 1821,1807 to 1827, the mRNA initiation codon subregion bonded antisense oligodeoxynucleotideresulted of the P gene of 1875 to 1895 of the ε district and HBV.They can be by 3 ' single phosphorylation modification improving its stability, thereby improve this oligonucleotide restraining effect to hepatitis B virus in cell.This antisense oligodeoxynucleotideresulted can be applicable to the medicine as the treatment hepatitis B.
The invention provides a kind of antisense oligodeoxynucleotideresulted that hepatitis B virus DNA duplicates and hepatitis B virogene is expressed that suppresses, this antisense oligodeoxynucleotideresulted and HBV 1798 to 1821,1807 to 1827,1875 to 1895 and the P gene mRNA initiation codon subregion combination of HBV in ε district.This antisense oligodeoxynucleotideresulted comprises 4 antisense oligodeoxynucleotideresulted, i.e. AsC, AsD, AsE and AsP, they respectively with 1798 to 1821 of HBV, 1807 to 1827,1875 to 1895 and the P gene mRNA initiation codon subregion combination of HBV in ε district, their sequence is as follows:
AsC 5’TGC?ATG?GTG?CTG?GTG?AAC?AGA?CCA?3’
AsD 5’AAA?AGT?TGC?ATG?GTG?CTG?GTG?3’
AsE 5’GCC?ACC?CAA?GGC?ACA?GCT?TGG?3’
AsP 5’GGG?CAT?TTG?GTG?GTC?TGT?AAG?CAG?3’。
This antisense oligodeoxynucleotideresulted can be passed through 3 ' single phosphorylation modification, and to improve stability, it is more effective to make it suppress hepatitis B virus, and their structure is as follows:
AsC(3’P) 5’TGC?ATG?GTG?CTG?GTG?AAC?AGA?CCAp?3’
AsD(3’P) 5’AAA?AGT?TGC?ATG?GTG?CTG?GTGp?3’
AsE(3’P) 5’GCC?ACC?CAA?GGC?ACA?GCT?TGGp?3’
AsP(3’P) 5’GGG?CAT?TTG?GTG?GTC?TGT?AAG?CAGp?3’。
We adopt following several strategies when considering the dna replication dna of inhibition HBV:
1),, is selected in 1798 to 1821 and 1807 to 1827 zone design AsC and AsD between DRI and the DRII, 3 ' and 5 ' end of gene RNA before they are positioned at according to the reproduction process of HBV DNA.It also is the position in the cap district of the initiator codon of anterior c gene and preceding gene RNA.1807 to 1827 districts of 3 ' end of gene RNA before 3 ' of gene RNA 1798 to 1821 of holding or AsF were attached to before AsE was attached to, form the DNA:RNA heteroduplex, at first under the effect of RNaseH, RNA in the hydrolysis heterozygosis chain, gene RNA 3 ' end is not with the DRI site before making, and it can not be inverted records into DNA.And AsC and AsD 3 ' end itself can not be as DNA synthetic primer for phosphate group, thereby has suppressed the (see figure 2) of duplicating of viral DNA.In addition, 4 of HBV kinds of mRNA stop transcribing and adding the polyA tail in same site.Promptly 3 ' of 4 kinds of mRNA end all has DRII, DRI, ε element and TATAAA.Between DRII and DRI, AsC is arranged, the binding site of AsD.AsC, AsD combines at the target sequence of 3 ' end with 4 kinds of mRNA, can mRNA be cut off by RNase H.After mRNA was cut the polyA tail, the life-span shortened greatly, caused the minimizing of the expression of all proteins.The AsC site is also at the proteic initial code of preceding C place, and therefore AsD also can suppress preceding C albumen and C albumen, the proteic expression of P also in the cap district of preceding gene RNA.
2), design AsE acts on the ε site of HBV.The ε site of HBV is at 1849 to 1910 of gene, and this section sequence can form higher structure in preceding gene RNA, is the recognition signal that forms capsid protein.1875 to 1895 effects in the ε site of the preceding gene RNA of AsE and HBV, wherein 1880 to 1885 is the ring district.This zone of gene RNA can not form higher structure before the combination of AsE made, and promptly can not form the recognition signal of capsid protein, thereby can suppress the initial of reverse transcription.It also can act on 4 kinds of mRNA.
3), design AsP acts on the initial code zone of the P gene of HBV.The P gene of HBV is since 2310,2292 to 2315 Nucleotide complementations of AsP and P gene, and promptly it can combine with the initiation codon subregion (18～+ 6) of P gene.It is proteic synthetic to suppress P.Suppress HBV P expression of gene with AsP and will suppress duplicating of HBV DNA.
According to above theory and HBV gene order (comprising the adr hypotype), at 1798 to 1821 of HBV, 1807 to 1827, ε site and P gene, the present invention designs corresponding antisense oligodeoxynucleotideresulted: AsC 5 ' TGC ATG GTG CTG GTG AAC AGA CCA 3 ' and HBV1798 to 1821 Nucleotide complementation, AsD 5 ' AAA AGT TGC ATG GTG CTG GTG 3 ' and HBV1807 to 1827 Nucleotide complementation.Be that they can combine with 3 ' the end target of 4 kinds of mRNA.1875 to 1895 Nucleotide complementations of AsE 5 ' GCC ACC CAAGGC ACA GCT TGG 3 ' and HBV.2292 to 2315 Nucleotide complementations of AsP 5 ' GGGCAT TTG GTG GTC TGT AAG CAG 3 ' and HBV.Wherein AsC and AsP be specially at the adr hypotype, AsD, and AsE, then general to each hypotype, compare with existing report, some differences are arranged on the sequence, bigger difference is the bibliographical information chemically modified, the present invention adopts 3 ' single phosphorylation modification.
The mechanism of action of 4 kinds of oligonucleotide that the present invention proposes mainly is conceived to suppress duplicating of viral DNA, rather than suppress the translation of certain RNA merely, especially activating RNaseH with AsC and AsD cuts off preceding gene RNA and AsC in the middle of DRI and DRII, AsD and AsE combine with 4 kinds of mRNA, make it to have been there is no report by this mechanism of RNase H hydrolysis.By suppressing duplicating of viral DNA, reduce viral DNA quantity, and then to cause the expression of virogene be that virus antigen albumen synthetic reduces.The restraining effect that these oligonucleotide are understood in minimizing that can be by detecting the HBV viral DNA in experiment and the proteic minimizing of virus antigen.
Consider to reach the hydrolase nucleic acid that greater activity is arranged in the cell in the serum that wherein be mainly 3 ' → 5 ' exonuclease, it requires 3 ' end is substrate for the nucleic acid of OH group.According to the achievement in research before the inventor (referring to application for a patent for invention numbers 97106495.4, June 28 1997 applying date), the present invention also carries out 3 ' single phosphorylation modification to oligonucleotide, after the OH of oligonucleotide 3 ' group is by phosphorylation, just can not be as the substrate of 3 ' → 5 ' exonuclease, prolonged it and reach the intracellular residence time in serum, therefore can more effectively combine with the complementary sequence of goal gene, restraining effect is also stronger.In addition, the OH group of oligonucleotide 3 ' can not can suppress duplicating of viral DNA as viral DNA synthetic primer after by phosphorylation.Therefore, on dna synthesizer, synthesized the antisense oligodeoxynucleotideresulted AsC (3 ' P) of above-mentioned 43 ' single phosphorylation modifications of the present invention, AsD (3 ' P), AsE (3 ' P) and AsP (3 ' P) have carried out purifying with the method for polyacrylamide gel electrophoresis.
For four antisense oligodeoxynucleotideresulted studying the present invention design restraining effect for HBV dna replication dna and expression of gene, the HepG2 cell that the present invention adopts HBV plasmid DNA (p1.2II) transfection to originate to liver is studied the effect of antisense oligodeoxynucleotideresulted as model.Plasmid (p1.2II) contains HBV (adr hypotype) gene clone of 1.2 double-length degree, comprise the HBV genome of total length and 1403～1983 overlap, it can express all HBV mRNA, transfection can HBsAg expression and HBeAg and produce the complete virion that infective activity is arranged after the HepG2 cell in liver source.Because it is the adr hypotype, thereby can be used as the inhibiting model of four antisense oligodeoxynucleotideresulted of research the present invention design to hepatitis B.Measure examining the shellfish number and can observing the restraining effect of antisense oligodeoxynucleotideresulted of HBV DNA in the cell to the HBV dna replication dna.Can measure the expression amount of HBsAg and HBeAg with existing mensuration test kit, can observe after antisense oligodeoxynucleotideresulted suppresses the HBV dna replication dna, viral DNA is reduced, and then the restraining effect to HBV genetic expression that produces.Result of study proof synthetic antisense oligodeoxynucleotideresulted AsC of the present invention (3 ' P), AsD (3 ' P), AsE (3 ' P) and AsP (3 ' P) can suppress the expression with anti-source of the S of HBV and the anti-source of E of duplicating of HBV viral DNA.To express antisense oligodeoxynucleotideresulted of the present invention in the following embodiments all has the obvious suppression effect to hepatitis B virus dna replication dna and genetic expression in the HepG2 cell, proves that antisense oligodeoxynucleotideresulted of the present invention can be used for preparing the medicine that suppresses hepatitis B virus and treatment hepatitis B.
The invention has the advantages that:
1, a kind of antisense oligodeoxynucleotideresulted is provided, it can cut off before the template of viral dna replication gene RNA or suppress the initial of viral dna replication or suppress the synthetic of varial polymerases by RNaseH, reach and suppress duplicating of hepatitis B virus DNA, reduce viral DNA quantity, and then suppress the breeding of virus and the expression of hepatitis B virogene.This is that a kind of direct inhibition hepatitis B virus DNA duplicates and the material of the expression of virogene, for the treatment of hepatitis B provides new method.
2, have the high characteristics of specificity with antisense oligodeoxynucleotideresulted inhibition HBV viral dna replication and expression of gene, and can not exert an influence human body cell itself.
3, antisense oligodeoxynucleotideresulted provided by the invention can also add that a phosphoric acid improves its stability in vivo by 3 ' end in antisense oligodeoxynucleotideresulted.Make them more obvious to the restraining effect of hepatitis B virus.Because the present invention designs the synthetic antisense oligodeoxynucleotideresulted and do not contain non-natural ornamental equivalent, its degraded product can not produce toxic side effect to human body.
The present invention is further elaborated by the following drawings and embodiment, but does not place restrictions on scope of the present invention.
The genomic structure of Fig. 1 HBV, transcription product, effect of antisense site.
Fig. 2 HBV virus replication and AsC or the diagram of AsD restraining effect.
Fig. 3 dot blot detects HBV DNA
Among the figure 1, the contrast deoxy-oligonucleotide; 2, do not add deoxy-oligonucleotide; The antisense oligodeoxynucleotideresulted AsD of 3,3 ' single phosphorylation modification (3 ' P); The antisense oligodeoxynucleotideresulted AsC of 4,3 ' single phosphorylation modification (3 ' P); The antisense oligodeoxynucleotideresulted AsE of 5,3 ' single phosphorylation modification (3 ' P); The antisense oligodeoxynucleotideresulted AsP of 6,3 ' single phosphorylation modification (3 ' P).
The restraining effect that the antisense oligodeoxynucleotideresulted of Fig. 4 different concns is expressed HBeAg
■ among the figure, the antisense oligodeoxynucleotideresulted AsC of 3 ' single phosphorylation modification (3 ' P); ◆, the antisense oligodeoxynucleotideresulted AsD of 3 ' single phosphorylation modification (3 ' P); ●, the antisense oligodeoxynucleotideresulted AsE of 3 ' single phosphorylation modification (3 ' P); ▲, the antisense oligodeoxynucleotideresulted AsP of 3 ' single phosphorylation modification (3 ' P); *, contrast deoxy-oligonucleotide (NC).
The restraining effect that the antisense oligodeoxynucleotideresulted of Fig. 5 different concns is expressed HBsAg
■ among the figure, the antisense oligodeoxynucleotideresulted AsC of 3 ' single phosphorylation modification (3 ' P); ◆, the antisense oligodeoxynucleotideresulted AsD of 3 ' single phosphorylation modification (3 ' P); ●, the antisense oligodeoxynucleotideresulted AsE of 3 ' single phosphorylation modification (3 ' P); ▲, the antisense oligodeoxynucleotideresulted AsP of 3 ' single phosphorylation modification (3 ' P); *, contrast deoxy-oligonucleotide (NC).
Fig. 6 antisense oligodeoxynucleotideresulted is to the time curve of HBeAg expression inhibiting
A492 represents 492 millimicrons of absorbance values among the figure.■, the antisense oligodeoxynucleotideresulted AsC of 3 ' single phosphorylation modification (3 ' P); ◆, the antisense oligodeoxynucleotideresulted AsD of 3 ' single phosphorylation modification (3 ' P); ●, the antisense oligodeoxynucleotideresulted AsE of 3 ' single phosphorylation modification (3 ' P); ▲, the antisense oligodeoxynucleotideresulted AsP of 3 ' single phosphorylation modification (3 ' P); *, contrast deoxy-oligonucleotide (NC); *, do not add deoxy-oligonucleotide.
Fig. 7 antisense oligodeoxynucleotideresulted is to the time curve of HBsAg expression inhibiting
Antisense oligodeoxynucleotideresulted or contrast deoxy-oligonucleotide concentration are 10umol/L.
A492 represents 492 millimicrons of absorbance values among the figure.■, the antisense oligodeoxynucleotideresulted AsC of 3 ' single phosphorylation modification (3 ' P); ◆, the antisense oligodeoxynucleotideresulted AsD of 3 ' single phosphorylation modification (3 ' P); ●, the antisense oligodeoxynucleotideresulted AsE of 3 ' single phosphorylation modification (3 ' P); ▲, the antisense oligodeoxynucleotideresulted AsP of 3 ' single phosphorylation modification (3 ' P); *, contrast deoxy-oligonucleotide (NC); *, do not add deoxy-oligonucleotide.
Synthesizing of embodiment 1 oligonucleotide
4 antisense oligodeoxynucleotideresulted AsC have been synthesized, AsD, AsE and AsP; Their structure is:
AsC 5’TGC?ATG?GTG?CTG?GTG?AAC?AGA?CCA?3’
AsD 5’AAA?AGT?TGC?ATG?GTG?CTG?GTG?3’
AsE 5’GCC?ACC?CAA?GGC?ACA?GCT?TGG?3’
AsP 5’GGG?CAT?TTG?GTG?GTC?TGT?AAG?CAG?3’。
And the antisense oligodeoxynucleotideresulted AsC (3 ' P) of 43 ' single phosphorylation modifications, AsD (3 ' P), AsE (3 ' P) and AsP (3 ' P); Their structure is:
AsC(3’P) 5’TGC?ATG?GTG?CTG?GTG?AAC?AGA?CCAp?3’
AsD(3’P) 5’AAA?AGT?TGC?ATG?GTG?CTG?GTGp?3’
AsE(3’P) 5’GCC?ACC?CAA?GGC?ACA?GCT?TGGp?3’
AsP(3’P) 5’GGG?CAT?TTG?GTG?GTC?TGT?AAG?CAGp?3’。
Article 1, non-complementary contrast deoxy-oligonucleotide:
NC： 5’ GAT?CAT?GGC?GCG?CTT?TGA?GGA?3’
3 '-synthetic use 0.2 μ mole 3 ' phosphoric acid solid phase column of single phosphorylation oligonucleotide (3 '-phosphateCPG Glen Research company product, full name 2-[2-(4,4 '-dimethyl, three benzyloxies) second sulphonyl] ethyl-(2-[2-(4 for succinyl chain alkyl amine-micropore glass pearl, 4 '-Dimethoxytrityloxy) ethylsulfomyl] ethyl-succinoyl long chain alkylamino-CPG), synthetic with the synthetic order of 0.2 μ mole on ABI 391EP dna synthesizer.Non-modified antisense deoxy-oligonucleotide and contrast deoxy-oligonucleotide are used 0.2 μ mole dG solid phase column and dA solid phase column (Glen Research product) respectively, and be synthetic with identical synthetic order.Product polyacrylamide gel electrophoresis purifying.
One, experimental procedure
(1) transfection of plasmid DNA and antisense oligodeoxynucleotideresulted
HepG
2Cell cultures in 1 * DMEM (containing 10% foetal calf serum) nutrient solution, 37 ℃, 5%CO
2Cultivate, transfection the day before yesterday, inoculating cell is in 12 orifice plates, overnight incubation, and transfection renewed bright nutrient solution in preceding 5 hours.Prep solution A: plasmid p1.2II (professor Wang Yuan of Shanghai Inst. of Biochemistry, Chinese Academy of Sciences provides) 10 μ g, antisense oligodeoxynucleotideresulted (the antisense oligodeoxynucleotideresulted AsC (3 of 3 ' single phosphorylation modification, P), 3, the antisense oligodeoxynucleotideresulted AsD (3 ' P) of single phosphorylation modification, the antisense oligodeoxynucleotideresulted AsE of 3 ' single phosphorylation modification (3 ' P), the antisense oligodeoxynucleotideresulted AsP of 3 ' single phosphorylation modification (3 ' P)) or each 10 μ mol/L of contrast deoxy-oligonucleotide, add DMEM to the 100 μ l that does not contain serum, mixing; Solution B: 6 μ l lipofectin (Gibco BRL product), 94 μ l do not contain the DMEM of serum, mixing, room temperature was placed 30 minutes.Solution A and solution B mixing, room temperature was placed 30 minutes.Wash cell twice with the DMEM that does not contain serum, add above-mentioned transfection mixed solution (each sample repeats 3 times), 37 ℃, 5%CO
2Cultivated 5 hours, and added the DMEM substratum that 2ml contains 10% foetal calf serum, continue at 37 ℃ 5%CO
2Cultivated 120 hours.
(2) extracting of cell DNA
10 μ mol/L oligonucleotide effects are after 5 days, and cell merges the cell of triplicate after trysinization, 4 ℃ with 3000g centrifugal 2 minutes, collecting cell adds 1ml TRIzol reagent in the 1.5ml centrifuge tube, abundant mixing, room temperature left standstill 5 minutes, added the 0.2ml chloroform, vibrated 15 seconds, room temperature was placed 2～3 minutes, centrifugal 15 minutes of 4 ℃ of 1200g are transferred to water in another 1.5ml centrifuge tube, are used for the cell total rna extracting.In the middle level and the lower floor organic phase add the 0.3ml dehydrated alcohol, mixing, room temperature was placed 5 minutes, then 10, centrifugal 5 minutes of 000g abandons supernatant, add 1ml 0.1mol/L Trisodium Citrate, 10% ethanolic soln, room temperature was placed 30 minutes, per 5 minutes mixings once, then in 4 ℃, 10, centrifugal 5 minutes of 000g, repeat Trisodium Citrate with 1ml 0.1mol/L, 10% ethanolic soln is washed once, is suspended in 75% ethanol, and room temperature was placed 20 minutes, shook once in per 5 minutes, in 4 ℃, 10, centrifugal 5 minutes of 000g, after placing air drying, add 100 μ l 8mmol/L NaOH dissolving.
(3) preparation of HBV gene probe
Plasmid p1.2II with the BamHI enzyme cut, electrophoresis, be purified into the complete linear HBVDNA that contains 3.2kb length, be equipped with the HBV dna probe with Hexanucleotide primer legal system at random, (referring to molecular cloning experiment guide, second edition, J.Sambrook; , E.F.Fritsch and T.Maniatis work, Jin Dongyan etc. translate, Science Press, 1986,502-504).
(4) detect HBV DNA with dot blot (Dot Blot)
With the suction filtration sample injector the DNA sample spot of sex change to nylon membrane, with UV-crosslinked on film, then with
32The HBV dna probe hybridization of P mark.Concrete grammar is referring to the modern molecular biology experimental technique, Lu Shengdong chief editor, Higher Education Publishing House,, 209-210 in 1993; The molecular cloning experiment guide, second edition, J.Sambrook ﹠amp; , E.F.Fritsch and T.Maniatis work, Jin Dongyan etc. translate, Science Press,, 483-490 in 1986.
Two, experimental result and analysis
As can be seen from Figure 3, antisense oligodeoxynucleotideresulted AsC (3 ' P) with 3 ' single phosphorylation modification, the antisense oligodeoxynucleotideresulted AsD of 3 ' single phosphorylation modification (3 ' P), the HepG of antisense oligodeoxynucleotideresulted AsP (3 ' P) effect of the antisense oligodeoxynucleotideresulted AsE of 3 ' single phosphorylation modification (3 ' P), 3 ' single phosphorylation modification
2In the cell, its HBV DNA examines the shellfish number and will be less than and do not add oligonucleotide and add not HepG with HBV RNA paired contrast deoxy-oligonucleotide
2, in the cell HBV DNA examine the shellfish number, it is less to show as the hybridization spot diameter, the hybridization point is more shallow.Illustrate that antisense oligodeoxynucleotideresulted can suppress duplicating of HBV DNA.
The inhibition test that embodiment 3 different concns antisense oligodeoxynucleotideresulted are expressed hepatitis B virogene
One, experimental procedure
(1) plasmid DNA (p1.2II) and antisense oligodeoxynucleotideresulted transfection HepG
2Cell
HepG
2Cell cultures in 1 * DMEM (containing 10% foetal calf serum) nutrient solution, 37 ℃, 5%CO
2Cultivate, transfection the day before yesterday, inoculating cell is in 96 orifice plates, overnight incubation, and transfection renewed bright nutrient solution in preceding 5 hours.Obtain solution A:0.8 μ g plasmid p1.2II, and the deoxy-oligonucleotide of the antisense oligodeoxynucleotideresulted of different concns and contrast usefulness, they are 0,40,80,120,160 respectively, 200pmol.When the transfection volume was 20 μ l, the final concentration of deoxy-oligonucleotide was respectively 0,2,4,6,8,10 μ mol/L), add DMEM to 10 μ l, mixing; Solution B: 0.3 μ l lipofectin, 9.7 μ lDMEM, mixing, room temperature was placed 30 minutes.Solution A and solution B mixing, room temperature was placed 30 minutes.Wash cell twice with DMEM, add above-mentioned transfection mixed solution (do 6 times in each sample hole, the supernatant of 3 samples replication HBsAg wherein, the supernatant of three samples is used for replication HBeAg in addition), 37 ℃, 5%CO
2Cultivated 5 hours, and added the DMEM substratum that 100 μ l contain 10% foetal calf serum, continue to cultivate 120 hours, get the mensuration that 100 μ l cells and supernatant are used for HBsAg and HBeAg.
(2) mensuration of HBsAg and HBeAg in the cells and supernatant
Added 100 μ l sample to be measured at bag by anti-HBs or HBe Sptting plate, and establish HBsAg or HBeAg positive control 2 holes, negative control 2 holes, blank 1 hole, every then hole adds enzyme conjugates 100 μ 1 (blank does not add), abundant mixing, shrouding.Hatched 1 hour for 37 ℃.Discard liquid in the reaction plate hole, fill with every hole, left standstill for 20 seconds, dry 5 times repeatedly with washings.Every then hole adds substrate solution 100 μ 1, shrouding.Hatched 15 minutes for 37 ℃, add stop buffer 50 μ l.At last, on microplate reader,, read 492 millimicrons of photoabsorption A of each hole reaction solution with blank well school zero
492Value.
(3) antisense oligodeoxynucleotideresulted is as follows to the method for calculation of HBsAg and HBeAg inhibiting rate:
Two, experimental result and analysis
(1) the antisense oligodeoxynucleotideresulted AsC of anti-HBV (3 ' P), AsD (3 ' P), AsE (3 ' P), AsP (3 ' P) is to the restraining effect of HBV antigen presentation
As Fig. 4, shown in Figure 5, the antisense oligodeoxynucleotideresulted AsC of anti-HBV (3 ' P), AsD (3 ' P), AsE (3 ' P), AsP (3 ' P) had both suppressed the inhibition that also shows HBV genetic expression of duplicating of HBV DNA.The antisense oligodeoxynucleotideresulted AsC of 3 ' single phosphorylation modification (3 ' P) can obviously suppress the expression of HBeAg and HBsAg since 2 μ mol/L, raising along with antisense oligodeoxynucleotideresulted AsC (3 ' P) concentration of 3 ' single phosphorylation modification, restraining effect to HBeAg and HBsAg expression also strengthens gradually, during to 10 μ mol/L, be respectively 78.3% and 84.0% to the inhibiting rate of HBeAg and HBsAg; AsD (3 ' P) can obviously suppress the expression of HBeAg and HBsAg since 2 μ mol/L, raising along with antisense oligodeoxynucleotideresulted AsD (3 ' P) concentration of 3 ' single phosphorylation modification, restraining effect to HBeAg and HbsAg expression also strengthens gradually, during to 10 μ mol/L, be respectively 82.2% and 88.0% to the inhibiting rate of HBeAg and HBsAg; When AsE (3 ' P) concentration is increased to 10 μ mol/L, be respectively 80.1% and 87.0% to the inhibiting rate of HBeAg and HBsAg; The antisense oligodeoxynucleotideresulted AsP of 3 ' terminal single phosphorylation modification (3 ' P) also can obviously suppress HBeAg and HBsAg since 2 μ mol/L to express, raising along with antisense oligodeoxynucleotideresulted AsP (3 ' P) concentration of 3 ' terminal single phosphorylation modification, restraining effect to HBeAg and HBsAg expression also strengthens gradually, during to 10 μ mol/L, be respectively 47.6% and 33.8% to the inhibiting rate of HBeAg and HBsAg.
(2) anti-HBV antisense oligodeoxynucleotideresulted AsC (3 ' P), AsD (3 ' P), AsE (3 ' P), AsP (3 ' P) is to the comparison of HBV genetic expression influence
The antisense oligodeoxynucleotideresulted AsC of 3 ' single phosphorylation modification (3 ' P), the antisense oligodeoxynucleotideresulted AsD of 3 ' single phosphorylation modification (3 ' P), the antisense oligodeoxynucleotideresulted AsE of 3 ' single phosphorylation modification (3 ' P), and the antisense oligodeoxynucleotideresulted AsP (3 ' P) of 3 ' single phosphorylation modification all can suppress the HBV expression of gene.But the antisense oligodeoxynucleotideresulted AsD of 3 ' single phosphorylation modification (3 ' P) is respectively 0,31.4%, 51.8%, 67.1%, 72.9% and 82.2% to the inhibiting rate that HBeAg expresses when 0,2,4,6,8,10 μ mol/L; The inhibiting rate that HBsAg is expressed is respectively 0,31.4%, 51.9%, 76.3%, 81.7% and 88.0%.The antisense oligodeoxynucleotideresulted AsC of 3 ' single phosphorylation modification (3 ' P) is respectively 0,28.3%, 47.8%, 62.7%, 76.0% and 78.3% to the inhibiting rate that HBeAg expresses when 0,2,4,6,8,10 μ mol/L; The inhibiting rate that HBsAg is expressed is respectively 0,28.2%, 46.8%, 73.8%, 78.0% and 84.0%.The antisense oligodeoxynucleotideresulted AsE of 3 ' single phosphorylation modification (3 ' P) is respectively 0,29.7%, 49.3%, 63.8%, 73.7% and 80.1% to the inhibiting rate that HBeAg expresses when 0,2,4,6,8,10 μ mol/L; The inhibiting rate that HBsAg is expressed is respectively 0,30.1%, 49.8%, 76.3%, 80.2% and 87.0%.And the antisense oligodeoxynucleotideresulted AsP of 3 ' single phosphorylation modification (3 ' P) is respectively 0,17.9%, 26.6%, 32.9%, 45.2% and 47.6% to the inhibiting rate that HBeAg expresses when 0,2,4,6,8,10 μ mol/L; The inhibiting rate that HBsAg is expressed is respectively 0,10.0%, 23.8%, 29.4% and 33.8%.The antisense oligodeoxynucleotideresulted AsD of 3 ' single phosphorylation modification (3 ' P), AsE (3 ' P) and AsC (3 ' P)
To exceed about one times the restraining effect of HBV gene than the antisense oligodeoxynucleotideresulted AsP (3 ' P) of 3 ' single phosphorylation modification the restraining effect of HBV genetic expression.
Embodiment 4 antisense oligodeoxynucleotideresulted suppress the time effect that hepatitis B virogene is expressed
One, experimental procedure
HepG
2Cell cultures in 1 * DMEM (containing 10% foetal calf serum) nutrient solution, 37 ℃, 5%CO
2Cultivate, transfection the day before yesterday, inoculating cell is in 12 orifice plates, overnight incubation, and transfection renewed bright nutrient solution in preceding 5 hours.Prep solution A: plasmid p1.2II 10 μ g, antisense oligodeoxynucleotideresulted (the antisense oligodeoxynucleotideresulted AsD (3 ' P) of the antisense oligodeoxynucleotideresulted AsC of 3 ' single phosphorylation modification (3 ' P), 3 ' single phosphorylation modification, the antisense oligodeoxynucleotideresulted AsE of 3 ' single phosphorylation modification (3 ' P), the antisense oligodeoxynucleotideresulted AsP of 3 ' single phosphorylation modification (3 ' P)) or each 10 μ mol/L of contrast deoxy-oligonucleotide, add DMEM to the 100 μ l that does not contain serum, mixing; Solution B: 6 μ l lipofectin, 94 μ l do not contain the DMEM of serum, mixing, room temperature was placed 30 minutes.Solution A and solution B mixing, room temperature was placed 30 minutes, washed cell twice with the DMEM that does not contain serum, added above-mentioned transfection mixed solution (each sample repeats 3 times), and 37 ℃, 5%CO
2Cultivated 5 hours, and added the DMEM nutrient solution that 2ml contains 10% foetal calf serum, continue at 37 ℃ 5%CO
2Cultivate, and got the mensuration that 200 μ, 1 cell culture fluid is used for HBsAg and HBeAg every 12 hours, concrete steps are with embodiment 2.
Two, experimental result and analysis
Can see that from Fig. 6 and Fig. 7 behind the HBV gene transfection HepG2 cell, when not adding oligonucleotide, HBeAg and HBsAg began at second day to express, along with the increase of time, expression amount progressively raises.As the antisense oligodeoxynucleotideresulted AsC (3 ' P) that adds 10 μ mol/L, 3 ' single phosphorylation modification or the antisense oligodeoxynucleotideresulted AsD (3 ' P) of 3 ' single phosphorylation modification, the antisense oligodeoxynucleotideresulted AsE of 3 ' single phosphorylation modification (3 ' P), or during the antisense oligodeoxynucleotideresulted AsP of 3 ' single phosphorylation modification (3 ' P), when not adding antisense oligodeoxynucleotideresulted, to obviously not reduce the expression ratio of HBeAg and HBsAg, especially the antisense oligodeoxynucleotideresulted AsD (3 ' P) that adds 3 ' single phosphorylation modification, when AsE (3 ' P) and AsC (3 ' P), restraining effect to HBeAg and HBsAg expression is especially remarkable, and this restraining effect continues to preceding 4 days of experiment end.The expression amount of HBeAg and HBsAg rises very slow, and after the 4th day, the expression amount of HBeAg and HBsAg just has slightly significantly rising.Illustrate that antisense oligonucleotide may persist to after the DNA transfection the 5th day to the restraining effect of HBV genetic expression.Simultaneously, when adding can not contrast deoxy-oligonucleotide with the HBVmRNA complementary, also some reduced the expression amount of HBeAg and HBsAg, and this may be because the deoxy-oligonucleotide in the DNA transfection system has influenced the transfection efficiency of HBV DNA, therefore, the expression amount of HBeAg and HBsAg descends to some extent.But HBeAg and HBsAg expression amount decline scope will be significantly less than the restraining effect of antisense oligodeoxynucleotideresulted to HBeAg and HBsAg expression.
Claims (3)
1. one kind is suppressed the antisense oligodeoxynucleotideresulted that hepatitis B virus DNA duplicates and hepatitis B virogene is expressed, it is characterized in that this antisense oligodeoxynucleotideresulted comprises 4 antisense oligodeoxynucleotideresulted, be AsC, AsD, AsE and AsP, they respectively with 1798 to 1821 of HBV, 1807 to 1827,1875 to 1895 and the P gene mRNA initiation codon subregion combination of HBV in ε district, their sequence is as follows:
AsC 5’TGC?ATG?GTG?CTG?GTG?AAC?AGA?CCA?3’
AsD 5’AAA?AGT?TGC?ATG?GTG?CTG?GTG?3’
AsE 5’GCC?ACC?CAA?GGC?ACA?GCT?TGG?3’
AsP 5’GGG?CAT?TTG?GTG?GTC?TGT?AAG?CAG?3’。
2. antisense oligodeoxynucleotideresulted according to claim 1 is characterized in that this antisense oligodeoxynucleotideresulted is that their structure is as follows by 3 ' single phosphorylation modification:
AsC(3’P) 5’TGC?ATG?GTG?CTG?GTG?AAC?AGA?CCAp?3’
AsD(3’P) 5’AAA?AGT?TGC?ATG?GTG?CTG?GTGp?3’
AsE(3’P) 5’GCC?ACC?CAA?GGC?ACA?GCT?TGGp?3’
AsP(3’P) 5’GGG?CAT?TTG?GTG?GTC?TGT?AAG?CAGp?3’。
3. one kind is suppressed that hepatitis B virus DNA duplicates and the application of the antisense oligodeoxynucleotideresulted that hepatitis B virogene is expressed, and it is characterized in that in this antisense oligodeoxynucleotideresulted any one: AsC, AsD, AsE, AsP, AsC (3 ' P), AsD (3 ' P), AsE (3 ' P), AsP (3 ' P) suppress application in the medicine of hepatitis B virus and treatment hepatitis B in preparation.
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| CN100365009C (en) * | 2003-08-06 | 2008-01-30 | 北京三诺佳邑生物技术有限责任公司 | Anti HBV infection and hepatitis B-preventing nucleotide sequence and its use |
| CN101979554B (en) * | 2010-10-28 | 2015-08-26 | 百奥迈科生物技术有限公司 | A kind of siRNA molecule and application thereof disturbing HBV gene |
| CN102021170B (en) * | 2010-10-28 | 2015-07-01 | 百奥迈科生物技术有限公司 | Small interfering ribonucleic acid (siRNA) molecule of interfering hepatitis B virus gene and application thereof |
| CN101979553B (en) * | 2010-10-28 | 2015-07-01 | 百奥迈科生物技术有限公司 | siRNA molecule for interfering HBV gene and antiviral application thereof |
| RU2667524C2 (en) * | 2011-04-21 | 2018-09-21 | Ионис Фармасьютикалз, Инк. | Modulation of hepatitis b virus (hbv) expression |
| CN113430196A (en) * | 2011-06-30 | 2021-09-24 | 箭头药业股份有限公司 | Compositions and methods for inhibiting gene expression of hepatitis b virus |
| CN109234275A (en) * | 2018-08-30 | 2019-01-18 | 山东省立医院 | A kind of GEM 132 that can inhibit hepatitis B virus DNA and hepatitis B surface antigen simultaneously |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996003152A1 (en) * | 1994-07-28 | 1996-02-08 | Georgetown University | Antisense oligonucleotides against hepatitis b viral replication |
| CN1122138A (en) * | 1993-01-21 | 1996-05-08 | 海布里顿公司 | Foldback triplex-forming oligonucleotides |
| WO1996039502A1 (en) * | 1995-06-06 | 1996-12-12 | Hybridon Inc. | Oligonucleotides specific for hepatitis b virus |
| WO1996040710A1 (en) * | 1995-06-07 | 1996-12-19 | Hybridon, Inc. | Triplex-forming antisense oligonucleotides having abasic linkers targeting nucleic acids comprising mixed sequences of purines and pyrimidines |
| WO2000019433A1 (en) * | 1998-09-29 | 2000-04-06 | Mitsubishi Denki Kabushiki Kaisha | Corner part reinforcing device of disc device chassis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1122138A (en) * | 1993-01-21 | 1996-05-08 | 海布里顿公司 | Foldback triplex-forming oligonucleotides |
| WO1996003152A1 (en) * | 1994-07-28 | 1996-02-08 | Georgetown University | Antisense oligonucleotides against hepatitis b viral replication |
| WO1996039502A1 (en) * | 1995-06-06 | 1996-12-12 | Hybridon Inc. | Oligonucleotides specific for hepatitis b virus |
| WO1996040710A1 (en) * | 1995-06-07 | 1996-12-19 | Hybridon, Inc. | Triplex-forming antisense oligonucleotides having abasic linkers targeting nucleic acids comprising mixed sequences of purines and pyrimidines |
| WO2000019433A1 (en) * | 1998-09-29 | 2000-04-06 | Mitsubishi Denki Kabushiki Kaisha | Corner part reinforcing device of disc device chassis |
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