HCV5A基因在Hela细胞中的稳定表达及对细胞生长的抑制现象  

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

作　　者：杨小骏[1] 叶林柏[1] 郜金荣[1] 刘静[1] 阳帆[1] 叶力[1] 佘应龙[1] 廖庆娇[1] 吴正辉[1] 郑义[1] 

机构地区：[1]武汉大学生命科学学院病毒研究所,湖北武汉430072

出　　处：《中国病毒学》2004年第4期340-344,共5页Virologica Sinica

基　　金：国家博士点基金项目(20010486015)

摘　　要：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。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.

关 键 词：丙型肝炎病毒 HCV PCR技术 5A基因 HELA细胞 表达 细胞生长 

分 类 号：R346[医药卫生—基础医学]