HIV-1包膜基因变异的体外研究
HIV-1包膜基因变异的体外研究
中国病毒学 2000年第2期第15卷 研究报告
作者：樊卫平 李敬云 鲍作义 吕富双 王宏霞
单位：樊卫平(山西医科大学微生物免疫教研室，太原 300050)；李敬云 鲍作义 吕富双 王宏霞(军事医学科学院微生物流行病研究所，北京 100071)
关键词：HIV-1；传代；包膜基因；变异
摘要：采用亚型测定、核苷酸和氨基酸序列测定和同源性分析等方法，观察了HIV-1 ⅢB毒株在实验室长期传代过程中包膜基因变异的情况。研究的毒株包括：经过实验室8年多连续使用而获得的毒株、在MT4细胞长期连续传代而获得的每间隔10代的毒株样品及多次更换宿主细胞传代而获得的毒株。主要结果有：(1)各种毒株包膜基因变异均不显著，核苷酸序列同源性均大于92%，变异距离均小于7.5%，且随着传代数增加核苷酸趋于稳定，代间同源性由92%上升至99%，而变异距离由7.5%下降为0.6%。(2)在传代过程中HIV-1亚型保持稳定，各种毒株均为HIV-1 B3亚型。结果显示HIV在体外长期传代培养的过程中变异不大，遗传性状稳定，可能是体外生长的环境十分稳定，缺乏机体免疫学压力。结果也提示体外长期传代HIV毒株仍然适用于各种HIV的应用研究，用长期传代的方法发展HIV-减毒活疫苗的可能性不大。
中图分类号：R512.91 文献标识码：A
文章编号：1003-5125(2000)02-0127-09
Study on the Variation of the env Gene of HIV-1 in vitroFANG Wei-ping
(Institute of Microbiology and Epidemiology,Academy of Military Medical Science,Beijing 100071,China)
LI Jing-yun,BAO Zuo-yi,LU Fu-shuang,WANG Hong-xia
(Department of Microbiology and Immunology,Shanxi Medical University,Taiyuan 030001,China)
Abstract:In order to understand the characteristics and rules of HIV diversity when passaged in vitro,the systematic study on the variation of env gene of HIV strain introduced from American in 1988 was carried out.Taking all the following strains as the objects:the 96-strain derived from the primary strain by continuous freeze-thaw-usage in laboratory for more than eight years; the 42h strain derived from primary strain by serial passaging with continuous changing of host cell in MT4 and HelaCD4+ and the strains sampled from serial passaged primary strain in MT4 cell every ten generations,The pre-DNA fragments of these HIV env gene were amplified by nested PCR.The fragment of 1200 bp originated from the V1-V5 region was analyzed.The subtypes of these HIV strains were identified by Heteroduplexes Mobility Assay (HMA).The main results were:(1) the env gene diversity of all the strains were indistinctive,all the nucleotide homogeneous were greater than 92% and all the gene diversity distance were less than 7.5%,moreover,the gene was seem to becoming fixed as the passage going on,the homogeneous between generation risen from 92% to 99%,where gene distance decreased from 7.5% to 0.59%.(2) All strains's subtypes were HIV-1 B3,the genetic type maintained stable during the passage.It meant that the variation of env gene of HIV in vitro was similar to that of infecting terminal period in vivo.The reasons may lie in that the more adaptive and higher virulence HIV strain,with the simple culturing circumstances and without the selective pressure of anti-HIV humoral and cellular immune existing in body.It is concluded that the characteristics of heredity maintained stable when HIVs were serial passaged in vitro,so passaged HIVs in vitro fit for use of HIV applied studies.But it is futile to want to get attenuated HIV vaccine by long term passages.
Key words:HIV; Serial passage; env gene; Diversity
人免疫缺陷病毒(HIV)的显著特征之一是其高度的变异性，这是HIV逃脱机体免疫监视的主要原因，也是HIV疫苗和治疗研究的主要障碍。观察HIV在体外培养条件下的变异情况对于评估机体内环境在HIV变异中的作用以及评价体外长期传代HIV毒种的实用性具有一定的意义。为此，我们对体外长期传代HIV毒株env基因的变异情况进行了系统的观察，对本室保存的长期传代HIV毒株以及为本实验目的而连续传代的毒株样品进行了HIV亚型测定，同时进行核苷酸和氨基酸序列测定和同源性分析。
