共表达中国株HIV-1gp120与hIL-6的重组鸡痘病毒研究

Construction and identification of recombinant fowlpox virus of coexpressing HIV-1_(CN) gp120 and hIL-6

目的 构建共表达中国株HIV 1gp12 0与人白细胞介素 6 (IL 6 )的重组鸡痘病毒。方法分别将HIV 1gp12 0基因和hIL 6基因插入到鸡痘病毒表达载体pUTAL复合启动子ATI P7.5和P7.5串联启动子下游 ,构建重组鸡痘病毒表达质粒pUTA GP IL6。利用脂质体法将重组质粒和鸡痘病毒2 82E4株共转染鸡胚成纤维细胞。经BUdR加压筛选 3次后 ,重组病毒分别用PCR、间接免疫荧光试验和Westernblot进行鉴定 ,并进行小鼠免疫研究。结果 重组病毒基因组中可扩增出 1.4kb大小片段 ,重组病毒感染细胞表面有绿色荧光物质 ,表达产物的Westernblot分析表明重组病毒可表达gp12 0和hIL 6蛋白。重组病毒可刺激小鼠产生特异性体液免疫应答。结论 成功构建了共表达中国株HIV 1gp12 0与hIL 6的重组鸡痘病毒 ,为研制HIV 1基因工程活载体疫苗提供有益的资料。

Objective To construct the recombinant fowlpox virus (rFPV) coexpressing of HIV-1 CN gp120 and hIL-6. Methods pUTA-GP-IL6 was constructed by inserting HIV-1 gp120 and hIL-6 gene into the downstream of combined promotor ATI-P7.5 and P7.5 tandem promotor of fowlpox virus expression vector pUTAL. The recombinant plasmid and FPV 282E4 strain were cotransfected chicken embryo fibroblast (CEF) via liposome. The recombinant virus was identified by PCR, indirect immunofluorescent assay (IFA) and Western blot after three times of pressure selection with BUdR,and the mice immunization experiment was performed. Results 1.4kb fragment could be amplified in the genome of rFPV, and the green fluorescence existed on the celluar surface infected by rFPV. Western blot analysis of expressed products showed that rFPV expressed gp120 and hIL-6 protein. rFPV could elicit the specific humoral immune response. Conclusion The rFPV coexrpessing HIV-1 gp120 and hIL-6 has been successfully constructed which lays the basis on the preparation of live vector genetic engineering vaccine against HIV-1.