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应用PCR技术从含有HCV(HepatitisCvirus)全长开放阅读框的质粒pBRTM/HCV1-3011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westernblot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。
The NS5A full-length gene fragment was obtained from the plasmid pBRTM / HCV1-3011 containing the full-length open reading frame of HCV (HepatitisCvirus) by PCR and cloned into the eukaryotic expression vector pcDNA3.1 (-) using gene recombination technology. The NS5A gene was inserted into pcDNA3.1 (-) correctly by restriction enzyme digestion, PCR and DNA sequencing. Hela cells were transfected by lipofectamine. After 48h, the NS5A gene was passaged and neoantibody on pcDNA3.1 (-) Sex genes were added to G-418 for screening. Approximately two weeks later, a stably expressed cell line was obtained. The results of RT-PCR and western blot showed that HCV NS5A gene was expressed in Hela cells. Under exactly the same culture conditions, the Hela cells expressing NS5A gene showed a significantly slower growth rate compared with the pcDNA3.1 (-) transfected cells, with a doubling time of about 35-36 h, which was higher than that of the control cells About 50%, while the pcDNA3.1 (-) transfected cells doubling time and normal Hela cells was no significant difference, both for 23-24h. Which proves that HCV NS5A protein has the effect of inhibiting Hela cell growth.