Ⅰ型人类免疫缺陷病毒的核酸异质性及准种特点的研究

目的研究Ⅰ型人类免疫缺陷病毒(HIV-1)膜蛋白基因(env)的准种与变异特点.方法采用巢式PCR方法对5例艾滋病患者血浆HIV-1env基因C2～V3区进行PCR扩增,产物经纯化后克隆至pMD-18T载体中,每例患者分别挑取5～8个克隆,共34株,完成阳性克隆的鉴定、抽提纯化和测序,将获得的DNA序列及氨基酸序列进行分析,并计算其氨基酸同源替换率(ds)与非同源替换率(dn)的比值.结果同一患者体内HIV准种株平均为83.5%,各克隆株之间的核苷酸同源性在88%～99%之间;不同患者之间的核苷酸同源性在75%～91%;34株序列的V3区平均变异频率明显高于C2～C3区(P＜O.05);而V3区和C2～C3区的ds/dn比值均＞1.结论艾滋病患者体内有大量HIV准种存在,基因的变异在V3区发生较频繁,免疫选择压力在该区变异中不起主要作用.

Observation of gene heterogeneity and quasispecies of HIV-1

Objective] To investigate the HIV quasispecies groups in the patients with AIDS. Methods] Blood samples were obtained from 5 patients with AIDS, the C2~C3 region of HIV-1 envelope gene(env) was amplified by nested polymerse chain reaction(PCR) from plasma of each patient, and then subject to sequencing, clonal-quasispecies analysis, and the accumulation of synonymous(ds) and nonsynonymous(dn) substitutions as well as ds/dn ratios were calculated. Results] The average quasispecies groups were 83.5%in each patient, the homology of the nucleic acid sequence from same patient and other patients were 88%~99% and 75%~91%, respectively, further sequence analysis on env clones for each patient showed significantly greater HIV mutation frequencies in V3 than C2~C3 region(P <0.05); the ds/dn ratio in the V3 and C2~C3 region are higher than 1.0. Conclusions] There are HIV quasispecies groups in AIDS patients. The mutation always occurred in V3 region, the variability of the V3 region may be not related to immune selective pressure.