在哺乳动物细胞中稳定表达丙型肝炎病毒E2糖蛋白

Stable Expression of E2 Glycoprotein of Hepatitis C Virus in Mammalian Cell

利用ＤＮＡ重组技术，将Ⅲ型中国株ＨＣＶＥ２／ＮＳ１基因片段插入真核表达载体，然后转染哺乳动物细胞ＮＩＨ３Ｔ３以表达Ｅ２糖蛋白．检测显示来自３月以上培养的细胞克隆中表达产物分子量为７０ｋＤ，经Ｗｅｓｔｅｒｎｂｌｏｔ证实该表达产物能与抗ＨＣＶ阳性血清进行特异性反应．以上表明首次在哺乳动物细胞中成功表达Ⅲ型中国株ＨＣＶ的Ｅ２糖蛋白，并建立相应的稳定表达细胞系．

E2/NS1 gene derived from a Chinese genotype Ⅲ isolate of hepatitis C virus was amplified by reverse transcription polymerase chain reaction method and inserted into mammalian expression vector pcDNA3 for E2 glycoprotein expression.The recombinant plasmid p3W14 containing nearly full length E2/NS1 gene downstream of CMV promoter and upstream of BGH polyA was transfected into mammalian cell NIH3T3 by calcium phosphate method.Cells were selected by G418.The G418 resistant clonal cells were cultured more than three months.Cell lysates were analyzed by SDS PAGE and Western Blot.The results showed that the new protein from the mammalian cell was 70 kD,reacting specifically to anti HCV sera,and proved to be E2 glycoprotein of hepatitis C virus.E2/NS1 gene also could be detected in the genomic DNA from neomycin resistance cells by PCR amplification.Additionally,the expression level of E2 glycoprotein had no significant change compared with that of three months before.It was the first time for successfully expression E2 glycoprotein derived from a Chinese HCV isolate lonely and establishment of cell lines for stable expression.