乙型肝炎病毒前-S1蛋白反式激活蛋白5基因的克隆化研究

Cloning of human gene 5 trans-activated by pre-S1 protein of hepatitis B virus

目的克隆乙型肝炎病毒(HBV)前-S1蛋白反式激活新基因PS1TP5的cDNA,并应用生物信息学技术初步探讨其结构及功能。方法应用聚合酶链反应(PCR)技术以HepG2细胞的cDNA为模板扩增PS1TP5,以pGEM-T载体进行TA克隆,通过PCR、限制性酶切分析及测序进行鉴定,再将其亚克隆到真核表达载体pcDNATM3.1/myc-HisA,通过PCR、限制性酶切分析进行鉴定,并应用生物信息学技术初步分析其物理化学性质、蛋白质结构和功能。结果PCR成功扩增出PS1TP5基因,并将其分别克隆进pGEM-T和pcDNATM3.1/myc-HisA载体,经PCR、限制性酶切鉴定后测序证实。因其可以被前-S1蛋白反式激活,故命名为前-S1反式激活蛋白5(PS1TP5),已在GenBank中注册,注册号AY427953。生物信息学分析确定其ORF为438个核苷酸(nt),编码产物为145个氨基酸残基(aa)。结论发现了HBV前-S1蛋白反式激活新基因PS1TP5,构建了pcDNATM3.1/myc-HisA真核表达载体,为进一步研究其生物学功能及慢性乙型肝炎发病机制创造了条件。

Objective To clone a new human gene 5 trans-activated by pre-S1 protein of hepatitis B virus （HBV）, PS1TP5, and explore its structure and function by bioinformatics analysis. Methods PS1TP5 was amplified by reverse transcription-polymerase chain reaction （RT-PCR） technique by using HepG2 cDNA as template and inserted into pGEM-T vector by TA cloning. Recombinant eukaryotic expression vector pcDNATM3. 1/myc-His A-PS1TP5 had been constructed by subeloning, followed by restriction enzyme digestion analysis and sequencing. Bioinformatic methods were used to analyze its possible physical and chemical characters, structure, and function. Results PS1TP5 was successfully amplified and cloned into pGEM-T and pcDNATM 3. 1/myc-His A vector by RT-PCR from HepG2 cDNA. The new gene had been confirmed by sequencing after PCR identification and restriction enzyme digestion and named as PS1TP5 because of its trans-active function. The sequence for the PS1TP5 gene had been deposited into GenBank, the accession number was AY427953. Bioinformatics analysis showed that its ORF was 438bp and translated a protein of 145 aa. Conclusion A new gene-PS1TP5 has been recognized, and its recombinant eukaryotic expression vector （pcDNA^TM3. 1/myc-His A-PS1TP5） has been constructed. These results will certainly bring some new clues for the study of the biological function of new gene and pathogenesis of chronic hepatitis B.