Transactivating effect of complete S protein of hepatitis B virus and cloning of genes transactivated by complete S protein using suppression subtractive hybridization technique

AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection. METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BamH I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). Suppression subtractive hybridization and bioinformatics techniques were used. The mRNA of HepG2 cells transfected with pcDNA3.Incomplete S and pcDNA3.1(-) empty vector was isolated, and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR) method, and cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification. RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of β-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete s was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86 positive clones. Colony PCR showed that 86 clones contained DNA inserts of 200-1 000 bp, respectively. Sequence analysis was performed in 35 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 33 coding sequences were obtained. These cDNA sequences might be target genes transactivated by complete S protein of HBV. Moreover, two unknown genes were discovered, full length coding sequences were obtained by bioinformatics techniques, one of them was named complete S transactivated protein 1 (CSTP1) and registered in GenBank (AY553877). CONCLUSION: The complete S gene of HBV has a transactivating effect on SV40 early promoter. A subtractive cDNA library of genes transactivated by HBV complete S protein using SSH technique has been constructed successfully. The obtained sequences may be target genes transactivated by HBV complete S protein among which some genes coding proteins are involved in cell cycle regulation, metabolism, immunity, signal transduction, cell apoptosis and formation mechanism of hepatic carcinoma.

Upreguiation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

AIM： To study the changes of human telomerase reverse transcriptase （hTERT） mRNA expression in human hepatocarcinoma cell lines （HepG2） and cholangiocarcinoma cell lines （QBC939） after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis. METHODS： HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein （EGFP） coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting. RESULTS： Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene. CONCLUSION： HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection. 2005 The WJG Press and Elsevier Inc. All rights reserved

Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy

AIM To detect hyper-conserved regions in the hepatitis B virus(HBV) X gene(HBX) 5' region that could be candidates for gene therapy.METHODS The study included 27 chronic hepatitis B treatmentnaive patients in various clinical stages(from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeA g-negative and HBeA g-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/m L, the HBX 5' end region [nucleotide(nt) 1255-1611] was PCRamplified and submitted to next-generation sequencing(NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences(haplotypes) obtained by NGS. Conservation at the HBx protein amino acid(aa) level was also analyzed.RESULTS NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample(2487-9279, IQR 2817). In 14/27 patients(51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region(nt 1255-1286) and the other in the 5' end coding region(nt 1519-1603). This last region coded for a conserved amino acid region(aa 63-76) that partially overlaps a Kunitz-like domain.CONCLUSION Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.

Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy.

HIGN LEVEL EXPRESSION OF HEPATITIS C VIRUS CORE GENE IN E.COLI

Objective To express the hepatitis C virus (HCV) core gene in E . coli on a high level. Methods The cDNA coding for HCV core protein was amplified by polymerase chain reaction (PCR). The PCR product was purified and digested with restriction enzymes and inserted into the downstream of P RP L promoter of a high level expression vector pBV220 . HCV core gene was expressed in E . coli in a non fused form. The expression protein was analysed by SDS PAGE , and its immunoactivity was tested by ELISA . Results Sequence analysis of the amplified PCR products confirmed that we have successfully cloned and expresssed the intact core protein of HCV. SDS PAGE showed that a specific protein with a molecular weight of 21kDa at a level of 14.0% of the total bacterial proteins appeared in bacteria harboring pBV/HCVCore, while this protein was absent in the control bacteria harboring pBV220. The results of enzyme immunoassay analysis showed that this protein could be specifically recognized by the HCV positive sera from patients with hepatitis C .Conclusion The intact HCV core protein was successfully expressed in  E . coli  in a non fused form on a high level, and its immunoactivity was high.

Hepatitis E virus chimeric DNA vaccine elicits immunologic response in mice

AIM： To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and ORF3 chimeric gene and to assess its ability to elicit specific immunologic response in mice. METHODS： The gene encoding the structural protein of HEV ORF2 fragment and full-length ORF3 was amplified by PCR. The PCR products were cloned into an eucaryotic expression plasmid pcDNA3. The resulting plasmid pcHEV23 was used as a DNA vaccine to inoculate BALB/c mice intramuscularly thrice at a dose of 100 or 200 ug. Mice injected with empty pcDNA3 DNA or saline served as control and then specific immune responses in the mice were detected. RESULTS： After 2-3 times of inoculation, all mice injected with pcHEV23 had anti-HEV IgG seroconversion and specific T lymphocyte proliferation. The lymphocyte stimulation index in the group immunized with pcHEV23 （3.1＋0.49） was higher than that in the control group （0.787±0.12, P〈0.01）. None in the control group had a detectable level of anti-HEV IgG. CONCLUSION： DNA vaccine containing HEV ORF2 and ORF3 chimeric gene can successfully induce specific humoral and cellular immune response in mice.

Immunogenicity and protective efficacy of DHBV DNA vaccines expressing envelope and capsid fusion proteins in ducks delivered by attenuated Salmonella typhimurium

Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

Cloning and Expression of HBV Infection Related Novel Gene C12orf49

To clone,express and purify C12orf49 recombinant protein.To prepare rabbit anti-C12orf49 protein polyclonal antibody and further elucidate its biological function.Methods PCR was applied to amplify the gene C12orf49 in vitro.pET-32a(+)-C12orf49,the recombinant protein prokaryotic expression vector,was transformed into E.coli.IPTG was used as an inductive agent to obtain C12orf49 recombinant protein,then the recombinant protein was analyzed with sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE)and Western blot.Specific polyclonal antibody was derived from rabbits that was immunized by recombinant protein.ELISA and Western blot were used to detect its titer and specificity,respectively.MTT cell proliferation experiment was carried out to observe the effect of protein on proliferation of HepG2 cells.Results The C12orf49 recombinant protein was expressed in a large quantity.Data of ELISA indicated that the titer of polyclonal antibody was higher than 1:1 280 000.And a good specificity of the antibody was confirmed by Western blot.C12orf49 recombinant protein might have an advanced effect on the proliferation of HepG2 cells.Conclusions Using C12orf49 recombinant protein,we can obtain the polyclonal antibody with great titer and good specificity.Human novel gene C12orf49 encoded protein could promote the proliferation of HepG2 cells.

A Novel Fusion Protein Possessing Both Antiviral and pHSA-binding Activities Composed of Hepatitis B Virus Pre-S2 Peptide and Human α2a-interferon

The human α-interferon(IFNα) is a group of proteins possessing activity to render cells resistant to viral infection. It is one of the most promising and well documented recombinant proteins used in biotherapy. A product of recombinant human α2a-interferon(IFN-α2a) first developed in our laboratory has been put into clinical trials upon the approval of the China National Committee for Drug Administration. Data show that

HBV包膜-核心蛋白融合基因DNA疫苗诱导小鼠持久的细胞免疫应答

构建编码HBV包膜 核心蛋白融合基因的DNA疫苗pSC、pSS1S2C和编码HBV包膜蛋白或核心蛋白基因的DNA疫苗 pHBs、pHBc ,分别肌肉注射免疫BALB/c小鼠 ,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应 ,比较融合基因DNA疫苗与单基因DNA疫苗诱生免疫应答的强度 ,发现融合基因DNA疫苗诱生抗体的效率明显不及单基因DNA疫苗 ,但其能诱导更强、更持久的细胞免疫应答 ,表明HBV包膜 核心蛋白融合基因DNA疫苗对于治疗慢性乙型肝炎可能比单基因DNA疫苗更为有效 .

丙型肝炎病毒非结构蛋白NS5A反式激活基因NS5ATP9的克隆化研究

目的 筛选并克隆丙型肝炎病毒 (HCV)非结构蛋白 5A(NS5A)反式激活新型靶基因 ,研究HCVNS5A反式激活作用的分子生物学机制。方法 应用抑制性消减杂交技术 (SSH)及生物信息学技术 ,以HCVNS5A表达质粒pcDNA3 1(-) -NS5A转染HepG2细胞 ,以空载体pcDNA3 1(-)为平行对照 ,提取mRNA并进行抑制性消减杂交分析。对于所获基因片段序列分析表明 ,其中之一为新型基因片段 ,与GenBank中注册的已知功能基因序列没有同源性 ,利用表达序列标签 (EST)序列数据库的搜索和比对 ,进行电子拼接 ,根据基因起始密码子的Kozak规则和终止密码子下游保守的多聚腺苷酸信号序列 ,确定新型基因序列。从HepG2细胞提取总RNA ,以逆转录多聚酶链反应 (RT -PCR)技术扩增获得该新基因的全长序列 ,并测序证实 ,命名为NS5ATP9,在GenBank中注册 ,注册号为AF5 2 93 70。结果 NS5ATP9基因的编码序列全长为 3 3 6个核苷酸 (nt) ,编码产物由 111个氨基酸残基 (aa)组成 ,并成功的克隆化。结论 HCVNS5A反式激活新型靶基因NS5ATP9的筛选与克隆 。

HBsAg/截短型HCV核心蛋白融合基因对番茄的遗传转化

[目的]本研究旨在探索乙肝病毒表面抗原/截短型HCV核心蛋白融合基因在植物中表达乙肝丙肝双价抗原的可能性,以期为进一步研制植物乙肝丙肝双价口服疫苗打下基础。[方法]应用重组PCR技术将乙肝病毒(HBV)S基因连接在丙肝病毒(HCV)截短型核心蛋白序列的5'端,2者通过柔性肽(Gly4Ser)2序列相连,构建成融合基因BC。将融合基因BC克隆到植物双元表达载体pBin438上,获得pBin438BC,然后用冻融法将其转入根癌农杆菌EHA105中,侵染丽春番茄子叶和下胚轴,经预培养、共培养、脱菌培养、筛选培养、生根培养,提取抗性植株基因组DNA,进行PCR及PCR-Southern检测。[结果]获得了9株抗卡那霉素的番茄植株,5株获得阳性信号。[结论]初步表明目的基因已整合到番茄基因组中。

乙型肝炎病毒(adr亚型)preS2-S基因转基因小鼠的建立

目的 :建立可表达乙型肝炎病毒 (adr亚型 )包膜中蛋白的转基因小鼠品系。 方法 :通过原核显微注射法制备带有乙型肝炎病毒 (adr亚型 ) pre S2 - S基因的转基因小鼠。应用 PCR方法筛选乙型肝炎病毒 pre S2 - S基因转基因首建者 (founder)小鼠 ,再以免疫组织化学法分析乙型肝炎病毒蛋白在这些小鼠中的表达特性 ,PCR和免疫组化均阳性的转基因小鼠与正常同系异性小鼠交配用于传代培育 ,并用 PCR法检测其子代小鼠。结果 :共注射受精卵 6 9枚。选取存活受精卵 5 8枚分别植入 4只假孕小鼠 ,产仔并存活 2 3只。经尾组织 DNA PCR筛选得到 5只整合 pre S2 - S基因的 founder小鼠 ,免疫组织化学法发现其中3只小鼠的肝脏中表达 HBs Ag,进而对其中表达较强的阳性鼠进行保种。结论 :所建立的乙型肝炎病毒 (adr亚型 ) pre S2 - S基因转基因小鼠品系具有表达乙肝病毒中蛋白的生物学特性 。

HCV核心基因cDNA真核表达载体的构建及其表达

丙型肝炎病毒(HCV)感染具有明显的慢性化倾向。与肝细胞癌(HCC)发生关系密切,是肝癌的病因之一.近年来HCV 核心蛋白的作用受到重视,被认为具有基因调节功能.我们采用基因重组技术构建 HCV 核心基因 cDNA真核表达质粒,导入 HepG2细胞株,以期获得永久性表达 HCV核心蛋白的细胞,为进一步探讨 HCV

丙型肝炎病毒非结构蛋白NS5A反式激活基因NS5ATP3的克隆化研究

目的 筛选并克隆丙型肝炎病毒 (HCV)非结构蛋白 5A(NS5A)反式激活新型靶基因。方法 以HCVNS5A表达质粒pcD NA3.1( ) NS5A转染HepG2细胞 ,以空载体pcDNA3.1( )为平行对照 ,提取mRNA并进行抑制性消减杂交分析 ,应用生物信息学方法对所获基因片段序列进行分析发现 ,其中有新型基因片段 ,与GenBank中注册的已知功能基因序列没有同源性。通过序列同源性搜索、比对和电子拼接 ,根据基因起始密码子的Kozak规则和终止密码子下游保守的多聚腺苷酸信号序列 ,确定新型基因序列。从转染了pcD NA3.1( ) NS5A的HepG2细胞提取总RNA ,以逆转录聚合酶链反应 (RT PCR)技术扩增 ,获得阳性克隆之后 ,进行鉴定并对克隆的基因及其编码产物的序列进行分析。结果 该新基因的编码序列全长为 15 72nt,编码产物由 5 2 4aa组成 ,并测序证实 ,命名为NS5ATP3,在GenBank中注册 ,注册号为AF5 2 936 4。结论 分子生物学技术与生物信息学技术相结合 ,发现并鉴定、克隆了HCVNS5A反式激活作用的新型靶基因NS5ATP3。

HCV核心蛋白DNA疫苗诱导的小鼠体液免疫应答

目的：观察丙型肝炎病毒（ＨＣＶ）核心蛋白ＤＮＡ疫苗诱导ＢＡＬＢ／ｃ小鼠体液免疫应答的效力，为研制应用于人类的ＨＣＶＤＮＡ疫苗提供实验依据。方法：将全长ＨＣＶ核心蛋白基因插入真核表达质粒载体ｐｃＤＮＡ３．１，构建ＨＣＶ核心蛋白重组表达质粒ｐｃＤＮＡ３．１ＨＣＶｃｏｒｅ，肌注ＢＡＬＢ／ｃ小鼠，以ＥＬＩＳＡ法检测小鼠血清ＨＣＶ抗体。以此重组质粒转染小鼠ＮＩＨ３Ｔ３细胞，以ＨＣＶ抗体阳性小鼠血清为捕获抗体进行Ｗｅｓｔｅｒｎｂｌｏｔ，检测ＤＮＡ疫苗编码的ＨＣＶ核心蛋白的表达。结果：３０％（３／１０）免疫小鼠产生ＨＣＶ核心蛋白抗体，该抗体能检测到重组质粒在ＮＩＨ３Ｔ３细胞表达的ＨＣＶ核心蛋白。

丙型肝炎病毒非结构蛋白HS5A反式激活基因HS5ATP6的克隆化研究

目的 应用抑制性消减杂交 (SSH)技术及生物信息学技术 (bioinformatics)筛选并克隆丙型肝炎病毒 (HCV)非结构蛋白5A(NS5A)反式激活新型靶基因。方法 以HCVNS5A表达质粒pcDNA3 .1(-) NS5A转染HepG2细胞 ,以空载体pcDNA3 .1(-)为平行对照 ,提取mRNA并进行抑制性消减杂交分析。对于所获基因片段序列分析表明 ,其中之一为新型基因片段 ,与GenBank中注册的已知功能基因序列没有同源性 ,利用表达序列标签 (EST)序列的搜索和比对 ,进行电子拼接 ,根据基因起始密码子的KOZ2ak规则和终止密码子下游保守的多聚腺苷酸信号序列 ,确定新型基因序列。结果 从HepG2细胞提取总RNA ,以逆转录多聚酶链反应(RT PCR)技术扩增获得该新基因的全长序列 ,并测序证实 ,命名为NS5ATP6,在GenBank中注册 ,注册号为AF5 2 93 67。NS5ATP6基因的编码序列全长为 15 72个核苷酸 (nt) ,编码产物由 5 2 4个氨基酸残基 (aa)组成。结论 HCVNS5A反式激活新型靶基因NS5ATP6的筛选与克隆 。

丙型肝炎病毒非结构蛋白5A反式激活基因NS5ATP13的克隆化研究

目的 应用微矩阵 (microarray)技术 ,结合生物信息学 (bioinformatics)技术筛选并克隆丙型肝炎病毒 (HCV)非结构蛋白5A(NS5A)的反式激活基因 ,阐明慢性HCV感染、肝纤维化及肝细胞癌 (HCC)的等相关疾病的发病机制。方法 根据HCV H病毒株序列设计、合成序列特异性的引物。以含有全长HCV H株cDNA的pBRTM 3 0 11质粒DNA作为模板 ,进行多聚酶链反应 (PCR)扩增 ,获得的HCVNS5A编码基因片段克隆到TA载体中进行核苷酸序列的测定 ,构建真核表达载体pcDNA3 1(-) NS5A。以pcD NA3 1(-) NS5A转染肝母细胞瘤细胞系HepG2 ,提取总RNA ,逆转录为cDNA后进行表达谱基因芯片分析。应用分子生物学技术 ,结合生物信息学技术 ,克隆HCVNS5A反式激活作用的新的靶基因。结果 构建了真核表达载体pcDNA3 1(-) NS5A ,经过限制性内切酶作图分析和核苷酸序列分析证实正确无误。以pcDNA3 1(-) NS5A转染HepG2后提取总RNA ,逆转录后进行表达谱基因芯片技术分析。应用分子克隆技术结合生物信息学技术克隆NS5A反式激活的新型靶基因 ,命名为NS5ATP13 ,在GenBank中登录 ,登录号为D2 12 62。NS5ATP13基因的编码序列全长为 2 10 3个核苷酸 (nt) ,编码产物由 70 0个氨基酸残基 (aa)组成。结论 丙型肝炎病毒NS5A基因产物具有显著的反?

麻疹病毒融合蛋白(F)和血凝素(HA)在痘苗病毒中的表达

将麻疹病毒F和HA基因插入到痘苗病毒中,分别处于痘苗启动子P7.5与P11控制下,获得重组病毒vLmF和vCmH。用抗F多肽抗体和HA单抗进行ELISA检测,结果表明,两株重组病毒均能表达相应的麻疹蛋白。蛋白印迹显示重组病毒表达产物在分子大小,蛋白切割和糖化方面与麻疹病毒糖蛋白一致。两株重组病毒分别免疫家兔都能产生较高滴度的麻疹抗体,这些抗体具有中和作用和血溶抑制作用。此外,vCmH产生的抗体还具有血凝抑制作用。

丙型肝炎病毒非结构蛋白5A反式激活基因2的克隆化研究

目的:对丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)蛋白反式激活靶基因2(NS5A-TP2)的基因作克隆化研究。方法:依据我室构建的HCVNS5A反式激活基因差异表达的cDNA消减文库筛选结果,利用分子生物学与生物信息学技术相结合的方法获得新基因NS5-ATP2的编码序列,对其氨基酸序列进行分析比较,并对其进行克隆化研究。结果:NS5A-TP2基因编码区为615核苷酸(nt),编码产物为204氨基酸残基(aa)。经核苷酸序列数据库(GenBank)和蛋白质一级结构序列数据库(SwissProt)同源序列的搜寻,与已知基因序列和蛋白序列之间没有显著同源性,说明我们克隆的NS5A-TP2基因属于未知功能新基因。结论:发现了HCV NS5A反式激活作用的新的靶基因,这一发现,为研究新基因的生物学功能及慢性丙型肝炎发病机制提供新的思路。