丙型肝炎病毒NS5A基因真核表达载体的构建

Construction of eukaryotic expression vector of hepatitis C virus NS5A gene

目的:构建HCV NS5A/pCI-neo真核表达载体,转染Huh-7细胞系,为体外高效表达NS5A蛋白奠定基础。方法:用PCR方法从带有丙型肝炎病毒NS5A基因的pC DNA 3.1(+)/HCV NS345质粒中扩增出HCV NS5A基因,然后TA克隆于pGEM-T载体中,转化大肠杆菌JM109。阳性克隆经测序鉴定,再经双酶切消化后插入真核表达载体pCI-neo上,经酶切鉴定与测序证实。结果:用PCR方法扩增出HCV NS5A基因全序列,因其cDNA的G+C含量高直接测序未成功,后经TA克隆,筛选得到的阳性克隆经测序鉴定,与设计的HCV NS5A基因序列完全一致。经酶切连接并成功插入pCI-neo上。结论:成功构建了高效表达丙型肝炎病毒NS5A基因的pCI-neo真核表达载体。

Objective To construct an eukaryotie expression vector of the hepatitis C virus （HCV） NS5A gene. and obtain a stable transfected Huh-7 cell line which provides a basis for further investigation of the hepatitis virus C NS5A protein. Methods The HCV NS5A gene from the peDNA3.1 （＋） /HCV NS345 plasmid with HCV NS5A gene was amplified by PCR, and cloned to pGEM-T vector, and transformed into E. coli JM109. The positive colonies were first confirmed by restriction enzyme digestion and sequencing and then were inserted to eukaryotic expression vector pCI-neo, and verified by enzyme digestion and sequencing. Results After TA colon of the HCV NS5A was amplified by PCR, the positive colonies were finally verified by sequencing, which were totally in line with the designed coding sequence of HCV NS5A gene. Conclusion Eukaryotic expression vector of HCV NSSA gene has been successfully constructed.