HIV-1包膜蛋白2G12和2F5中和表位的改造对假病毒形成及中和活性的影响

The impact of the modification of 2G12 and 2F5 neutralizing epitopes of the HIV-1 envelope （Env） on its assembly of functional pseudovirus and neutralizing activity

目的研究HIV-1膜蛋白（Env）特定中和表位的改造对功能性假病毒形成及中和活性的影响。方法采用环形诱变及DpnⅠ筛选的方法对Env进行定点突变，将2G12和2F5两个中和表位整合人不含该表位的BC亚型的Env上，比较改造对假病毒的形成情况及对2G12和2F5单抗的中和活性的影响。结果对5株假病毒（BC02、BC03、BC04、BC05和BC12）的Env特定中和表位进行改造，其中BC04和BC12的2G12表位改造后，不能形成假病毒，BC02、BC03和BC05增加2G12和2F5两个表位后，仍能够形成假病毒，且假病毒滴度较改造前尤明显变化，改造后的BC03假病毒较改造前对单抗2G12和2F5的中和活性均有所提高，而改造后的BC02和BC05假病毒较改造前对单抗2F5的中和活性增强，而对单抗2G12的中和活性尤变化。结论2G12中和表位部分位点的改造影响假病毒的形成，中和表位的增加能够提高单抗2G12的中和活性，为免疫原的优化提供了新思路。

Objective To study the influence of the modification of the special neutralizing epitopes of the HIV-1 envelope （ Env ） on its assembly of functional pseudovirus and neutralizing activity. Methods Site-directed mutations were performed using cycling mutagenesis and selection of mutants with Dpn Ⅰ . With this method, the 2G12 and 2F5 neutralizing epitopes were integrated into Env of subtype BC which was without the two epitopes, then the capability of forming pseudovirus and the neutralizing activity against 2G12 and 2F5 were compared with pre-modified Env. Results The special Env neutralizing epitopes of five HIV pseudovirus （BC02, BC03, BC04, BC05 and BC12） were modified. Among the five pseudovirus, BC04 and BC12 pseudovirus can＇t be formed after the 2G12 epitope was modified, whilst the BC02, BC03 and BC05 pseudovirus can be formed after the 2G12 and 2F5 epitopes were added, and there was no variation of the pseudovirus titer; On the aspect of neutralizing activity, BC03 pseudovirus against 2G12 and 2F5 was enhanced, BC02 and BC05 pseudovirus against 2F5 was enhanced while which against 2G12 was not changed. Conclusion The modification of 2G12 epitope influences the forming of pseudovirus and the addition of neutralizing epitopes can enhance the neutralizing activity of pseudovirus, which offers new approach for the optimization of HIV immunogen.