HIV-1感染者外周血单个核细胞缺损病毒基因的研究

目的:研究HIV-1感染者缺损HIV-1 DNA的特性.方法:用长片段PCR法(LD-PCR)研究分析HIV-1感染者外周血单个核细胞(peripheral blood mononuclear, PBMCs)和体外培养感染淋巴细胞中HIV-1基因特征,使用位于HIV-1 DNA链两端的LTR(U5)、LTR(R)为引物,插入有全长HIV-1基因片段的大肠杆菌质粒PNL4-3等为标准对照,并对部分标本克隆测序.结果:经扩增9.1 kb是LD-PCR的主要产物,但在10例HIV-1感染者中有9例PBMCs检测出大小不一、范围较广的缺失HIV-1基因片段,经用基因探针杂交,发现近HIV-1基因中心部位缺失频率增加,缺失结合点常存在3～4个核苷酸短片段直接重复,体外培养中HIV-1基因缺损量减少,完整和缺失基因的存在与培养中病毒分离的时间密切相关.结论:HIV-1感染者PBMCs中存在大量HIV-1基因重组和缺损片段.

Character of viral genomes in peripheral blood mononuclear of HIV-1 infected persons

Objective: To study the character of defective HIV 1 DNA genomes of HIV 1 infected persons. Methods: HIV 1 DNA was amplified by long distance PCR (LD PCR) from lymphocytes infected in vitro and peripheral blood mononuclears (PBMCs) in individuals infected. As primers located between both long terminal repeats LTR(U5) LTR(R), controls were pNL4 3 with full length HIV 1 genomes which was inserted to plasmid prepared in E.coli and several examples were cloned and sequenced etc. Results: 9.1 kb band was the major LD PCR product. However,defected HIV 1 genomes of variable size were extensively detected in PBMCs of 9/10 HIV 1 infected persons. Using oligonucleotide probes the frequency of deletions was found to increase with their proximity to the center of the HIV 1 genome. Short direct repeat of three or four nucleotides was present at the deletion junctions in analyzing further detail; the quantity of defective HIV 1 genomes decreased after in vitro ; persistence of defective genomes and full length in vitro correlated with the time of isolation of infectious virus. Conclusion: All this showed a great number of intragenomic rearrangements in HIV 1 genomes and defective genomes accumulated in PBMCs of persons infected.