HCV 5A基因在Hela细胞中的稳定表达及对细胞生长的抑制现象

应用PCR技术从含有HCV(Hepatitis Cvirus)全长开放阅读框的质粒pBRTM／HCV 1-3011中获得NS5A全长基因片断，利用基因重组技术将其克隆至真核表达载体pcDNA3．1(-)中。通过酶切、PCR及测序鉴定NS5A基因已正确插入到pcDNA3．1(-)中，再利用脂质体介导转染Hela细胞，48h后传代并利用pcDNA3．1(-)质粒上的neo抗性基因加入G-418进行筛选。大约两周后，获得稳定表达的细胞株。经RT-PCR及western blot验证，证实HCV的NS5A基因在Hela细胞中已经获得了表达。在培养条件完全一致的条件下，表达NS5A基因的Hela细胞与pcDNA3．1(-)转染的细胞相比，生长速度明显变慢，其倍增时间约为35-36h，比对照组细胞增加了约50％，而转染pcDNA3．1(-)的细胞的倍增时间与正常Hela细胞则无明显差别，都为23-24h。从而证明HCV的NS5A蛋白具有抑制Hela细胞生长的作用。

Stable Expression of NS5A Gene of HCV in Hela Cells and Inhibition to the Growth of Hela Cells

Full-length NS5A gene of Hepatitis C virus(HCV)was amplified by PCR, using the plasmid pBRTM/HCV 1-3011 containing HCV full-length open reading frame(ORF)as template, and cloned into the eukaryotic expressing plasmid pcDNA3.1(-)by DNA recombination technique. The recombin- ant vector was identified by digestion with restriction enzymes and polymerase chain reaction and by directly sequencing. Then both the recombinant vector pcDNA3.1(-)-NS5A and the control vector pcDNA3.1(-)were transfected Hela cells using LipoVecTM. The cells expressing NS5A stablely were selected by G-418 and further proved by RT-PCR and Western blot analysis. We found the growth of Hela cells expressing NS5A was slower than the cells transfected by pcDNA3.1(-)in the same culture condition, and the population doubling time of Hela cells expressing NS5A gene is increased about 50%(about 35-35 hours). There was no significant difference between the control cells and the cells transfected with pcDNA3.1(-) (about 23-24 hours). The results indicate that NS5A can inhibit the proliferation of Hela cells.