以假病毒为基础的HIV-1中和抗体检测体系的建立以及初步应用

Establishment and primary application of pseudovirus-based neutralization assay for HIV-1

目的建立以假病毒为基础的HIV-1中和抗体检测方法，并对其进行初步应用。方法从重组质粒中扩增出gp160基因片段，并克隆到pcDNA3．1质粒上，酶切鉴定得到阳性克隆。将阳性克隆分别和pSG3／Xenv质粒共转染获得假病毒。用假病毒分别检测单克隆抗体和HIV-1抗体阳性血清的中和活性。结果成功地获得了4株假病毒CHB01、CHB02、CHBC03和CHAE04。单克隆抗体4E10可以中和4株假病毒；单克隆抗体2F5不能中和CHBC03假病毒株，但可以中和CHB01、CHB02和CHAE04假病毒株；单克隆抗体IgG1b12可以中和CHBC03、CHB01和CHB02假病毒株，则不能中和CHAE04假病毒株。43份HIV-1抗体阳性血清中针对不同假病毒的中和抗体明显不同。结论所获得的假病毒可以用于中和抗体的检测，但不同假病毒株的中和特性不同。

Objective To establish a pseudovirus-based neutralization assay. Methods The functional gp160 genes were amplified from plasmids containing HIV-1 gene. The products were ligased into pcDNA3.1 plasmid and positive clones were screened by digestion with restriction enzymes. The pseudoviruses were harvested by co-transfection of the positive clone and pSG3Δenv plasmid. The neutralizing activities of monoclonal antibodies and HIV-1 antibody positive plasma were measured by these pseudoviruses. Results The four strains of psedoviruses （ CHB01, CHB02, CHBC03 and CHAE04） had been successfully obtained. Monoclonal antibody 4E10 could neutralize all of 4 pseudoviruses while 2G12 could not neutralize any pesudoviruses. Monoclonal antibody 2F5 could neutralize pseudovirus CHB01, CHB02 and CHAE04 but not CHBC03, while IgGlbl2 could neutralize pseudovirus CHB01, CHB02 and CHBC03 but not CHAE04. The neutralizing activities of 43 of HIV-1 antibody positive plasma against different subtypes of pseudovirus were significant differences and the cross-neutralization effects for some samples exist. Conclusion The harvested pseudoviruses could be used in the neutralization assay. However, the neutralizing characteristics of different pseudoviruses may be different.