HIV-1黑龙江省分离株CHNHLJ03009包膜克隆及分析

Cloning and analysis of HIV-1 envelope isolated from an infected individual in Heilongjiang province

目的分析黑龙江省Ⅰ型人免疫缺陷病毒(human immunodeficiency virus type1,HIV21)原代分离株包膜糖蛋白的变异性及表型特征。方法从一名HIV阳性但未发病的感染者外周血单个核细胞提取DNA,采用保守引物进行HIV包膜直接克隆,进行序列分析及系统树分析。构建了一株带该包膜蛋白的伪病毒并利用表达CXCR4和CCR5的靶细胞进行了感染实验,观察该病毒包膜利用辅助受体的表型特征。结果共获得2个有功能全长env克隆,分别命名为CHNHLJ03009c34(Gen-Bank序列号为AY905493)和CHNHLJ03009c33。用全长env氨基酸序列与国内外分离株进行同源性比较分析发现,与分离株CHNHLJ03009c34的包膜同源性最高的病毒为云南的HIV-1B′亚型分离株RL42,同源性为91.52%。系统发育分析结果表明,该株为泰国B′亚型,与云南分离株RL42的遗传距离最近。对包膜蛋白结构分析结果显示,分离株CHNHLJ03009c34包膜在抗原性和亲水性上与RL42没有明显差别。感染性检测结果显示该病毒只能感染U87.CD4.CCR5细胞,不能感染U87.CD4.CX2CR4细胞。结论本研究从黑龙江省一名HIV21阳性者克隆到HIV-1CHNHLJ03009病毒的包膜基因,该病毒属于B′亚型(泰国亚型),为R5亲嗜性毒株。该包膜基因克隆为首次报道的黑龙江分离株。

Objective To analyze the variability and phenotype of envelope glycoprotein（env） of human immunodeficiency virus prevalent in Heilonjiang province. Methods Fun-length ew gene cloning from PBMC of a HIV-1 positive individual in Heilonjiang province, China was performed with conserved region primers. The amplified PCR products were cloned into a plasmid vector and sequenced. Phylogenetic tree analysis was done upon full length env amino acid sequence. Subsequently, a HIV-1 pseudotyped virus with the envelope protein was constructed and the infectivity was examined with U87 cell Lines expressing CD4 with either CCR5 or CXCR4. Refits Two functional env clones named CHNHLJ03009c34（GenBank Accession No： AY905493） and CHNHLJ03009c33 were obtained. The homology between CHNHLJ03009c34 and a HIV-1 B＇ subtype RL42 strain from Yunnan province is 91.52% through comparing and analyzing full length env amino acid sequence from domestic and abroad isolates. Phylogenetic analysis indicated that CHNHLJ03009c34 had an intimate molecular relation to strain RL42, while it became an independent arm upon the analysis of C2-V3 region. The secondary structure analysis of the envelope protein showed that the antigenicity and hydropathy of the strain did not have definite difference from RL42. Infectivity examination showed that the pseudovirus could only infect U87. CD4. CCR5 cells indicating that it comes from RS-tropic HIV-1. Conclusion HIV-1 primary isolate envelopes CHNHLJ03009 have been cloned from a HIV-1 positive individual in Heilongjiang province. The strain is R5 Thailand B＇ subtype. This is the first analysis of primary isolates in Heilongjiang.