从未经培养的HIV-1阳性外周血克隆及系统树分析HIV-1中国株E亚型gp120基因

目的:研究中国HIV-1 E亚型代表株的主要结构基因及其功能。方法:选择3份根据HIV-1 的外膜基因(C2-V3)的序列分析判定为E亚型的阳性血样,采用克隆和系统树分析,通过套式PCR得到全长的gp120 基因片段,并插入到pFastBacl载体中,以双脱氧末端终止法测定全部的DNA 序列。结果:来自广东和广西的样品的gp120 基因归于E亚型,E亚型中国株内的遗传距离为2.56% ～5.87% ,与16 株国际标准株比较,遗传距离为3.60% ～27.98% 。所有克隆到的gp120 基因都具有完整的阅读框架,无大的缺失和插入。结论:HIV-1 E亚型进入中国的时间不长;克隆到的E亚型代表株的gp120 基因具有完整的结构和功能,适合用来构建E亚型的亚单位疫苗表达载体

Cloning and Phylogenetic Analysis of Subtype E gp120 Genes of China HIV 1 Strains from Uncultured HIV 1 positive Peripheral Blood

Objective:To clone and phylogenetically analyze the subtype E gp120 structural genes of China HIV 1 prevalent strains.Methods:Based on the molecular epidemiology survey,three HIV 1 positive blood samples which were determined as subtype E by analysis of partial env genes (C 2 V 3) sequences of HIV 1,were chosen to be amplified by nested PCR,thereby the full length fragments of gp120 genes were obtained and inserted into pFastBac 1.All the DNA sequences were determined by double deoxidation termination method.Results:The phylogenetic analysis showed that the gp120 genes of the samples from Guandong and Guanxi were classified as subtype E.China strains of subtype E have the genetic distance of 2 56%～5 87% between themselves,they have greater genetic distance (3 60%～27 98%) from the 16 international standard strains,and they are close to Thailand strains of subtype E,but far away from African strains of subtype E.All the Cloned gp120 genes have the complete open reading frames,and no major deletion and insertion.Conclusion:The subtype E of HIV 1 was introduced into China recently.The cloned gp120 genes of the prevalent strains of subtype E have the intact structure and function,and can be used to construct the expression vectors of subunit vaccine of subtype E.