HIV-1型中国株E、B亚型gag基因的克隆与表达

Cloning and expressing of full-length Subtype E and Subtype B gag gene from uncultured PBMCs of HIV-1 infected individuals in China

目的 克隆和表达人免疫缺陷病毒 (HIV)Ⅰ型中国株E、B亚型代表株的结构基因gag。方法 在分子流行病学调查的基础上 ,选择 1份经部分 gp12 0基因测序判定为E亚型和 1份经部分gp12 0基因测序判定为B亚型的代表性样品 ,利用套式聚合酶链反应 (PCR) ,扩增外周血中的前病毒 ,获得了结构基因 gag全长片断 ,并先后克隆到plin8Pr5 5和 pFastBacl载体中 ,我们首次克隆到全长的中国株E亚型 gag基因。采用双脱氧链末端终止法测定全部DNA序列。采用昆虫细胞 杆状病毒表达系统 ,构建了中国HIV 1gag基因的杆状病毒表达质粒 ,得到了表达HIV 1E和B亚型 gag的重组杆状病毒。结果 克隆到的E、B亚型 gag基因具有完整的阅读框架 ,无大的缺失和插入 ,gag重组杆状病毒感染昆虫细胞表达了由gag形成的病毒样颗粒 ,并分泌到上清中 ,Westernblot显示感染的昆虫细胞表达相应的 gag基因。超薄电镜显示用 gag基因的重组昆虫杆状病毒感染昆虫细胞可形成病毒样颗粒 ,这些颗粒为空心的大约 10 0nm颗粒样物定位在胞内 ,明显不同于昆虫杆状病毒本身形成的颗粒。结论 克隆到的E、B亚型代表株的 gag基因具有完整的结构和功能 ,我们得到了针对我国HIV 1流行株的病毒样颗粒候选疫苗抗原。

Objective To clone and express subtype E and subtype B gag gene of the prevalent HIV 1 strain in China.Methods One HIV 1 positive sample, whose subtype had been determined as subtype E and one HIV 1 positive sample, whose subtype had been determined as subtype B by sequence analysis of partial env gene were chosen to be amplified by nested PCR. We got two full length fragments of gag gene and incorporated them first into plin8Pr55 and then into pFastBacl.Results Sequence analysis showed the cloned gag genes have the complete open reading frames and have no major deletion and insertion. Using the Bac to Bac system, we obtained the recombinant baculoviruses containing gag genes. Western blot analysis showed that insect cells infected with gag recombinant baculoviruses expressed HIV 1 gag antigen. Thin section electron microscopy showed that virus like particles(VLPs) formed in insect cells was infected with gag.Conclusion The cloning and expression of gag genes of the prevalent HIV 1 stains in China revealed that the cloned genes have intact structure and function and this paves the way for development of HIV 1 vaccines targeted at the epidemic of HIV 1 in China, also for study of biological functions of gag gene and development of diagnostic kits of HIV 1.