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目的 克隆、鉴定自河南省分离的HIV 1B Thai亚型流行株并进行系统发育分析。方法收集河南省 1份HIV 1感染者全血后分离的淋巴细胞 ,在植物血凝素存在下与健康献血员淋巴细胞共培养 ,从培养的淋巴细胞中提取前病毒DNA。在HIV 1基因的长末端重复序列的保守区设计引物 ,利用LATag长链扩增系统扩增HIV 1全长基因 ,纯化的PCR产物与pWSK2 9 T载体连接 ,成功克隆CNHN 2 4株。对鉴定的 3个克隆进行全长测序 ,利用局部同源法计算同源率 ,同时采用Phylip软件绘制HIV 1Env、Gag和Pol基因的进化树。结果 V3V4环序列分析表明 :本次克隆的CNHN 2 4株病毒为HIV 1B Thai亚型 ,V3环区氨基酸序列比对发现在 9个位点发生氨基酸替换。在含有 90 10bp ,全长HIV 1基因的CNHN 2 4株克隆中未发现明显的缺失、插入和重排现象 ,Gag、Pol、Vpr和Vif基因与RL 4 2株的同源率达到 95 4 2 %～ 97 0 8% ,进化分析显示 ,CNHN 2 4株与RL 4 2株的遗传距离最近。结论 HIV 1CNHN 2 4株为国内实验室完成的HIV 1全长基因克隆 ,为进一步研究HIV 1流行特征提供了基础。
Objective To clone and identify the epidemic strains of HIV subtype 1BTB from Henan Province and to make phylogenetic analysis. Methods Lymphocytes isolated from one HIV-1 infected whole blood in Henan Province were collected and co-cultured with healthy blood donors in the presence of phytohemagglutinin to obtain proviral DNA from cultured lymphocytes. Primers were designed based on the conserved regions of the long terminal repeats of HIV 1 gene. The full-length HIV 1 gene was amplified by LATag long-chain amplification system. The purified PCR product was ligated into pWSK2 9 T vector, and 4 CNN-2 strains were successfully cloned. The three clones were sequenced, the homology was calculated by homology method, and Phylip software was used to draw the phylogenetic tree of HIV 1 Env, Gag and Pol genes. Results The V3V4 loop sequence analysis showed that the 4 strains of CNHN 2 cloned in this study were HIV 1B Thai subtypes. The amino acid sequence alignment of V3 loop found that amino acid substitution occurred at 9 sites. No significant deletion, insertion and rearrangement were found in CNHN 2 4 clones containing 90 bpp and full-length HIV 1 genes. The homology of Gag, Pol, Vpr and Vif genes to RL 4 2 reached 95 4 2 According to the evolutionary analysis, the genetic distance between CNN 2 4 and RL 4 2 strains was the closest. Conclusion HIV 1CNHN 2 4 clones are full-length HIV 1 gene clones from domestic laboratories, providing the basis for further study of HIV 1 epidemiology.