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

目的 克隆、鉴定自河南省分离的HIV-1 B-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LA Tag长链扩增系统扩增HIV-1全长基因，纯化的PCR产物与pWSK29-T载体连接，成功克隆CNHN24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-1 Env、Gag和Pol基因的进化树。结果 V3V4环序列分析表明：本次克隆的CNHN24株病毒为HIV-1 B-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9010bp，全长HIV-1基因的CNHN24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RL42株的同源率达到95．42％～97．08％，进化分析显示，CNHN24株与RL42株的遗传距离最近。结论 HIV-1 CNHN24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。

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

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.