共表达HIV-1 gag-gp120与白细胞介素6重组鸡痘病毒的筛选

Selection of recombinant fowlpox virus coexpressing HIV-1 gag-gp120 and IL-6

目的构建共表达中国株HIV1gaggp120与白细胞介素6(IL6)的重组鸡痘病毒(FPV)。方法分别将HIV1gaggp120基因和IL6基因插入到FPV表达质粒pUTAL复合启动子和单一启动子下游,构建重组FPV表达质粒pUTAGEIL6。利用脂质体法将重组质粒和FPV282E4株共转染鸡胚成纤维细胞,经BUdR加压筛选,重组病毒分别用SDSPAGE和WesternBolt进行鉴定,观察病毒样粒子的形成和重组病毒在哺乳动物细胞中的表达,并分析其免疫原性。结果阳性重组病毒基因组点膜处有显色斑点,WesternBolt结果显示重组病毒表达了gaggp120嵌合蛋白和IL6,在病毒感染细胞中有反转录病毒样粒子形成,且重组病毒可在哺乳动物细胞中表达目的蛋白。小鼠免疫指标检测表明该重组病毒具有很好的免疫原性。结论成功构建了共表达中国株HIV1gaggp120与IL6的重组FPV,为HIV-1基因工程活载体疫苗和巨分子颗粒化疫苗的制备奠定了基础。

Objective To construct the recombinant fowlpox virus （rFPV） coexpressing HIV-1 gag-gp120 and hIL-6. Methods The recombinant expressing plasmid pUTA-GE-IL6 was successfully constructed by inserting gag-gp120 gene and hIL-6 gene into the downstream of the combined promoter ATI- pT. 5 and p7.5 tandem promoter respectively. After transfect;ng the plasmid into chicken embryonic fibroblast （CEF） cells preinfected with FPV 282E4 strain and selecting the recombinant virus under the pressure of BUdR. The recombinant virus was analyzed by nucleic acid probe hybridization and immunoblotting. In addition, the formation of vlrus-like particle and the expression of interested proteins in the recombinant virus-infected p815 cells were observed, and the immunogenicity of the recombinant virus was also analyzed. Results There was colorable dot for the positive recombinant virus, immunoblotting analysis showed that the recombinant virus could expressed both gag-gp120 and IL-6. Virus-like particles （VLP） were formed in virus-infeeted cells, and the interested proteins could be expressed in mammalian cells infected by the recombinant virus. The immunity index from the immunized mice showed that the recombinant virus had good immunogenieity. Conclusion The recombinant fowlpox virus coexpressing gag- gp120 and IL-6 was successfully constructed, which may provide basis for the preparation of live vector genetic engineering vaccine and maeromolecule particle vaccine against HIV-1.