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目的构建NL4-3env-EGFP/DsRed2前病毒,观察中国主要流行的HIV-1 CRF01-AE?CRF07/08-BC和B′亚型构造病毒株体外适应性。方法 5份HIV-1B′,CRF07/08-BC,CRF01-AE亚型感染的干血斑样本和1份Zambia C亚型PBMC DNA样本,经单基因组扩增技术获得env全长基因,克隆、亚克隆、转染等步骤构建14个NL4-3env-EGFP/DsRed2前病毒,按顺序进行配对竞争实验,流式细胞仪定量检测被感染细胞的数量。结果 CRF01-AE亚型前病毒适应性最强,适应性值W>1,其次为B′,Zambia C,CRF07-BC/CRF08-BC,P<0.05。结论可利用NL4-3env-EGFP/DsRed2构造前病毒,通过竞争实验比较流行病毒株的体外适应性,推测env基因对适应性的影响。
Objective To construct the NL4-3env-EGFP / DsRed2 provirus and to observe the in vitro adaptability of the major HIV-1 CRF01-AE? CRF07 / 08-BC and B? Isoform strains in China. Methods Five samples of PBMC infected with HIV-1B ’, CRF07 / 08-BC and CRF01-AE subtypes and 1 Zambia C subtype PBMC DNA were obtained. The full-length env gene was cloned, 14 NL4-3env-EGFP / DsRed2 provirus were constructed by subcloning, transfection and other steps, and the competition experiments were carried out in sequence. The number of infected cells was quantitatively determined by flow cytometry. Results The provirus of CRF01-AE subtypes had the strongest adaptability, W> 1, followed by B ’, Zambia C, CRF07-BC / CRF08-BC, P <0.05. Conclusion The prokaryotic expression vector NL4-3env-EGFP / DsRed2 can be used to compare the adaptability of env genes with the adaptability of the virulent strains in vitro.