HIV-2 Gp105基因重组鸡痘病毒的构建及鉴定

目的　研制预防HIV -2的重组鸡痘病毒活载体疫苗。方法　将HIV -2Gp10 5基因插入鸡痘病毒转移载体pUTA2复合启动子的下游 ,构建鸡痘病毒转移载体pUTA2 Gp10 5。将该重组质粒与鸡痘病毒 (FPV) 2 82E4株共转染鸡胚成纤维细胞 (CEF) ,进行同源重组。通过 3次BrdU加压筛选 ,以PCR、RT PCR、IFA和Westernblot鉴定重组病毒。结果　从重组鸡痘病毒的基因组和总RNA中能扩增出大小为 6 5 3bp的目的基因 ;感染重组病毒的CEF细胞与特异性荧光抗体发生阳性反应 ;表达产物与人HIV- 2血清发生阳性反应 ,目的蛋白相对分子质量约为10 5 0 0 0。结论　成功获得 1株重组鸡痘病毒 ,可正确表达HIV 2Gp10 5基因。

Construction and Identification of Recombinant Fowlpox Virus Expressing HIV-2 Gp105 Protein

Objective To obtain a safe and effective recombinant fowlpox virus (FPV) as a vaccine against HIV-2.Methods Insert HIV-2 Gp105 gene downstream to the composite promoter of fowlpox virus transferring vector pUTA2.Co-transfect chick embryo fibroblast (CEF) with the constructed recombinant plasmid pUTA2/Gp105 and FPV 282E4 strain for homologous recombination.Recombinant virus was screened 3 times in presence of 5′-bromo-deoxyuridine (BrdU) and identified by PCR, RT-PCR,IFA and Western blot.Results The goal gene, at a length of 653 bp, was amplified from the genome and total RNA of recombinant fowlpox virus.The CEF transfected with recombinant fowlpox virus reacted with specific fluorescent antibody.The expressed protein, with a relative molecular weight of about 105 000, reacted with HIV-2 positive human serum.Conclusion A recombinant fowlpox virus strain for expression of HIV-2 Gp105 gene was successfully obtained.