Construction of eukaryotic expression vector of hepatitis C virus NS5A gene / 吉林大学学报(医学版)

Objective To construct an eukaryotic 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 pcDNA3.1(+)/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 NS5A gene has been successfully constructed.

Cloning and expression of ns5a gene of hepatitis C virus 1b strain DY in Escherichia coli / 西安交通大学学报(医学版)

Objective To clone and express the ns5a gene of hepatitis C virus(HCV) 1b strain DY.Methods By using the prokaryotic cell gene engineering,HCV ns5a gene was amplified with nested PCR from the plasmid HCV17 of HCV 1b strain DY full-length gene and inserted into the cloning pMD18-T vector.The cloned HCV ns5a gene was separated and subcloned into expression vector pET-28a and induced by IPTG in E.coli.BL21.The expressed product was identified by SDS-PAGE and Western-blot methods.Results Recombinant expression plasmid pet-28a-ns5a was constructed and expressed successfully.Conclusion HCV ns5a gene was cloned and expressed.This might be helpful for further studies on the nature and biological properties of the ns5a gene.