目的为分析中国SHIV／猕猴AIDS模型的病毒载量变化趋势，建立一种实时、灵敏、特异的针对人／猴免疫缺陷病毒的定量检测方法。方法体外转录制备RNA标准品，利用TaqMan EZ RT-PCR试剂盒的反应体系和针对SHIV gag保守区91个碱基的TaqMan探针和引物，建立一步法实时荧光定量RT-PCR。提取126份来自SHIV-CN97001感染恒河猴血浆病毒RNA并定量检测。结果利．用梯度稀释的RNA标准品对反应体系进行优化，标准曲线下限达到2×10^2拷贝／ml，相关性（r〉0.99）及重复性（CV=4．14％）均能达到测定要求。病毒载量的检测结果表明SHIV—CN97001在猴体内传代过程中病毒载量有先升后降的趋势，病毒载量通常在接种病毒或感染猴的全血后第14天达到高峰。血浆载量可达到10^5—10^6拷贝／ml。结论成功地建立了一步法定量SHIVRNA的实时荧光定量RT-PCR，为SHIV／恒河猴AIDS模型的建立与应用提供了灵敏的病毒载量检测方法。SHIV—CN97001的体内繁殖能力在猴体内传代过程中有所增强。
Clone N3 and C from Human immunodeficiency virus(HIV) gp41 gene were expressed using the pET expression system. When induced by IPTG at 37℃, both two clones did not express in E.coli BL21(DE)3. Howerver, when induced at 16℃, the two clones were both overexpressed, and the amount of the product was about 20% of the total bacteria protein. In Western blotting test, the protein product could react with HIV-positive serum. After IPTG induction, E. coli cells had much higher death rate at 37℃ than at 16℃; [3H]uridine release assay also showed that after IPTG induction, E. coli had a higher release at 37℃. The results suggested that overexpression of the two proteins was due to their decreased toxicity at lower temperature.
