早期HIV-1感染env基因的动态变异研究

目的 追踪观察HIV-1新发感染者体内CRF07_BC重组毒株膜蛋白基因的变异性.方法 从HIV-1感染者血浆中提取总RNA,通过逆转录多聚酶链反应(RT-PCR)获得HIV-1 gp120全长及C2-C5区段基因.纯化后装入T载体,转化至Top10大肠埃希菌内增殖,通过蓝白斑筛选获得阳性克隆,运用PCR方法进行鉴定,最后对所获得的目的克隆测序.结果从两个感染者血浆样品中获得感染后半年到2年半间多个时间点的gp120基因克隆共135个及gp120基因C2-C5区段克隆15个,分析显示这些克隆均为HIV-1 CRF07_BC亚型.随感染时间的延长,HIV-1 env基因的离散率与多样性均有增加的趋势.env基因同义突变与非同义突变比较结果显示,C1、C3与V4区段非同义突变比率比gp120基因的其他区域都高.基因多态性分析的结果显示,同一时间点内不同毒株基因在不同区段的变异是不同的,基因多样性介于0与0.066±0.028之间.结论 随着患者感染时间的推移,HIV-1膜蛋白基因的变异逐渐增大.C1、C3和V4区的高突变率提示,该基因区可能是HIV-1病毒生存与宿主免疫压力相互作用的主要位点.

Dynamic Variation of HIV-1 CRF07-BC env gene in early HIV-1 infection

Objective To understand the evolution of HIV-1 CRF07_ BC envelope, we performed a longitudinal study on two patients during their early HIV-1 infection. Methods RNA was extracted from the plasma of the individuals and the 42-45 fragments of the gp120 gene of HIV-1 were amplified by RT-PCR. Purified DNA segments were inserted into T easy vector and transformed into E. Coli Top10 competent cells. Positive clones were identified by blue-white screening, confirmed by PCR and sequenced by ABI 3700. Results The samples were collected from the patients every 6 months from seroconversion time. The genetic diversity and divergence in env gene showed consistent increases over time. Our sequence analysis also revealed obvious non-synonymous change in env C1,C3 and V4 regions among these samples. Conclusion The results support the concept that the consistent pattern of viral evolution existed during early phase of HIV-1 infection. 41, 43 and V4 region of env gene may be mainly immunological target during AIDS progression.