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[目的]构建丙型肝炎病毒(HCV)非结构蛋白NS3解旋酶基因原核表达载体,为进一步研究和解析NS3的解旋酶基因对病毒复制的机制准备条件。[方法]将含有NS3基因的pMD-24/HCVNS3质粒转化感受态菌DH-5α并扩增;提取pMD-24/HCVNS3质粒;从pMD-24/HCVNS3质粒中扩增出NS3解旋酶基因;并将其插入到克隆载体pMD-18T中,再与表达载体pGEX-4T-1重组,以得到重组的原核表达载体pGEX-4T-1/NS3解旋酶。[结果]从pMD-24/HCVNS3质粒中扩增出的NS3解旋酶基因片断大小正确,经测序证明其碱基序列为编码目的基因的正确序列;电泳结果证明已将此片段克隆到pGEX-4T-1内。[结论]成功地构建了HCVNS3解旋酶基因的原核表达载体pGEX-4T-1/NS3解旋酶。
[Objective] To construct the prokaryotic expression vector of NS3 helicase gene of non-structural protein of hepatitis C virus (HCV) to prepare and study the mechanism of virus replication by NS3 helicase gene. [Method] The pMD-24 / HCVNS3 plasmid containing NS3 gene was transformed into competent E. coli DH5a and amplified. Plasmid pMD-24 / HCVNS3 was extracted. The NS3 helicase gene was amplified from pMD-24 / HCVNS3 plasmid. Inserted into the cloning vector pMD-18T and recombined with the expression vector pGEX-4T-1 to obtain the recombinant prokaryotic expression vector pGEX-4T-1 / NS3 helicase. [Result] The fragment of NS3 helicase amplified from pMD-24 / HCVNS3 plasmid was correct in size and proved to be the correct sequence encoding the target gene by sequencing. The result of electrophoresis confirmed that the fragment was cloned into pGEX- 4T-1 inside. [Conclusion] The prokaryotic expression vector pGEX-4T-1 / NS3 helicase of HCV NS3 helicase gene was successfully constructed.