摘要
目的：构建丙型肝炎病毒单链丝氨酸蛋白酶（scNS4A／NS3）的真核表达载体；建立重组质粒稳定转染的HepG2细胞克隆．方法：根据文献报道设计扩增scNS4A／NS3编码基因的引物，从HCV阳性患者血清中提取病毒RNA，RT-PCR方法扩增出scNS4A／NS3基因片段，BamH Ⅰ／HindⅢ双酶切后连接到经同样酶切的真核表达载体pcDNA3，1（-），转化菌株JM109感受态细胞，获得重组质粒pcDNA3．1（-）～scNS4A／NS3，经酶切鉴定及序列测定．将阳性重组质粒用脂质体法转染HepG2细胞，经持续G418压力选择和克隆化获得稳定转染的细胞系，用RT-PCR，IFA，Western-blot证实该稳定细胞系可以表达单链丝氨酸蛋白．结果：成功构建了真核表达载体pcDNA3．1（-）-scNS4A／NS3；建立了稳定转染的HepG2细胞克隆，命名为scpHepG2．结论：获得稳定的scpHepG2细胞克隆可表达单链丝氨酸蛋白，为下一步建立以细胞为基础的评价抗HCV丝氨酸蛋白酶药物系统奠定基础．
AIM： To construct a eukaryotic expression vector of HCV single chain serine protease （scNS4A/NS3） and to obtain its stably transfected HepG2 cell line. METHODS： According to the sequence of HCV scNS4A/NS3 gene from the literature, the primers amplifying the gene coding seNS4A/NS3 protease were designed. HCV RNA was extracted from the HCV positive serum and the gene coding scNS4A/NS3 protease was amplified via RTPCR. The PCR product was digested by BamH Ⅰ/Hind Ⅲ and purified by gel extraction. This fragment was inserted into pcDNA3.1 （ - ） with T4 ligase and transformed into E. coli JM109. The positive recombinant plasmid was selected and identified via sequence assay and restrictive enzyme digestion. The recombinant plasmid was then transfeeted into HepG2 cell by LipofectAMINE2000. The cells containing stable transformants were selected by the ability of resistance to G418. The stably transfected cell line was identified by RT-PCR and IFA and Western-blot. RESULTS： The eukaryotic expression vector named pcDNA3. 1 （ -）-scNS4A/NS3 was successfully constructed and the stably transfected HepG2 cell line which expressed seNS4A/NS3 protease was obtained. CONCLUSION： The stably transfected HepG2 cell line expressing single chain serine protease facilitates the establishment of cell-based system in evaluating anti-HCV serine protease drug.
出处
《第四军医大学学报》
北大核心
2006年第1期42-45,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金(30371280)
关键词
丙型肝炎病毒
单链丝氨酸蛋白酶
转染
脂质体法
细胞克隆
hepatitis C virus
single chain serine protease
lipofeetamine, transfection
colone cells
