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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 the structural gene gag of representative strains of subtype E and B of Chinese type Ⅰ human immunodeficiency virus (HIV). Methods Based on the molecular epidemiological survey, we selected 1 representative sample which was judged by partial gp12 0 gene sequencing as E subtype and 1 by partial gp12 0 gene sequencing as B subtype. By using nested polymerase chain (PCR) to amplify the pro virus in peripheral blood. The full-length gag gene of structural gene was obtained and cloned into plin8Pr5 5 and pFastBacl vector. For the first time, we cloned the full-length Chinese subtype gag gene. The entire DNA sequence was determined by dideoxy chain termination. The baculovirus expression plasmid of HIV 1 gag gene in China was constructed by using insect cell baculovirus expression system and a recombinant baculovirus expressing HIV 1E and B subtype gag was obtained. Results The cloned gag genes of E and B subtypes had a complete reading frame without major deletions and insertions. The virus-like particles formed by gag were expressed in insect cells infected with gag recombinant baculovirus and secreted into the supernatant. Westernblot Infected insect cells were shown to express the corresponding gag gene. Ultrathin electron microscopy showed that infection of insect cells with the recombinant baculovirus of the gag gene resulted in the formation of virus-like particles that were hollow in size and located about 100 nm in size, distinctly different from the particles formed by the insect baculovirus itself. Conclusion The cloned gag gene of representative strains of subtype E and subtype B have complete structure and function, and we have obtained the virus-like particle vaccine candidate vaccine against HIV-1 epidemic in our country.