河南省分离的HIV-1 B-Thai亚型流行株全基因组克隆及序列分析

Cloning and characterization of a full-length HIV-1 genome of a prevalent subtype B-Thai strain in Henan Province

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, identify and phylogenetically characterize a clade B-Thai HIV isolate representing the most prevalent virus in Henan province. Methods Peripheral blood mononuclear cells (PBMCs) from an HIV-1 infected patient in Henan Province were separated, and co-cultivated with phytohemagglutinin-stimulated healthy donor PBMCs. Proviral DNA was extracted from productively infected PBMCs. The full-length HIV-1 genome was amplified by using the LA Tag long template PCR system . Primers were positioned in conserved regions within the HIV-1 long terminal repeats. Purified PCR products were T-A ligated into a pWSK29-T vector(CNHN 24 clone). Three recombinant clones containing virtually full-length HIV-1 genome were identified by PCR. The full-length genome was sequenced by using the primer-walking approach. Nucleotide sequence similarities were calculated by the local-homology algorithm. Phylogenetic trees of gag, pol and env reading frames were constructed using the Phylip software. Results HIV-1 C3V4 sequences indicate that the epidemic in this area was B-Thai subtype. V3 loop multiple amino acid sequence alignments showed amino acid alterations at nine positions. The 9 010 bp genomic sequence derived from isolate CNHN 24 contained all known structural and regulatory genes of an HIV-1 genome. No major deletions, insertions, or rearrangements were found. The highest homologies of the gag, pol, vpr, and vif reading frames to the corresponding clade B-Thai RL 42 sequences were 95.42%-97.08%. Phylogenetic trees showed the closest relationship of CNHN 24 and RL 42. Conclusion The cloning and characterization of a virtually full-length HIV-1 B-Thai subtype in central China was completed in our laboratory. The data should be helpful to future studies on the genetic diversity of HIV-1.