SHIV-XJ02170在中国恒河猴体内传代过程中env基因变异研究

Sequence variation in the env region of SHIV-XJ02170 during in vivo passaging

目的 研究SHIV-XJ02170在中国恒河猴体内传代过程中env基因序列变异的特点.方法 应用聚合酶链反应（PCR）和逆转录聚合酶链反应（RT-PCR）分别对各代动物SHIV-XJ02170病毒载量峰值时间点全血前病毒DNA gp160基因和血浆病毒RNA gp120基因进行扩增.GP160基因PCR产物直接进行测序分析,gp120 RNA扩增产物连接T载体后每份样品挑选18个克隆测序,分析传代过程中病毒的基因距离（divergence,diversity）的变化规律及病毒基因进化的特点.结果 SHIV-XJ02170在传代过程中基因连续进化并且进化的方向和病毒传代的顺序完全一致,基因距离总体上表现为逐步扩大的趋势,病毒传代早期存在明显的＂瓶颈效应＂.氨基酸序列分析发现V3环顶端四肽和辅助受体在传代过程中未发生改变.结论 SHIV-XJ02170经过中国恒河猴体内传代后基因距离出现明显扩大过程,且按照传代的顺序发生连续的基因进化.这从分子水平上部分解释了 SHIV-XJ02170经猴体传代出现毒力增强的原因.

Objective To identify the variation in the Env region of SHIV-XJ02170 during passaging in Chinese origin Rhesus Macaques.Methods Fragments of the SHIV-XJ02170 gp160 and gp120 gene were amplified by PCR and RT-PCR separately from the blood samples of SHIV-XJ02170 infected animals at the peak viral load time point.Purified RT-PCR product was ligated into T easy vector and transformed into JM109 competent cells,18 clones were selected by PCR method and sequenced by ABI 3730DNA sequencers.The gene distances（divergence,diversity）were calculated using DISTANCE.Results In all,the SHTV-XJ02170 gp120 gene evolved forward along the virus passaging.It could be found that viral divergence from the founder strain serially enhanced during in vivo passaging,but in the early phase of each passage,SHIV-XJ02170 gp120 gene evolved toward ancestral state upon transmission to a new host.All of the SHIV-XJ02170 strains had V3 loop central motif（GPGQ）and were predicted to be using CCR5 on the basis of the critical amino acids within V3 loop.Conclusion There was significant increase in the genetic distance during serial passaging,and SHIV-XJ02170 gp120 gene evolved forward along passaging.This could partly explain why the virus infectivity was enhanced during in vivo passaging.