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目的构建并探讨人类免疫缺陷病毒(HIV)-2 gag重组鸡痘病毒(FPV)在小鼠体内的免疫应答,为开发HIV-2基因重组活载体疫苗提供实验依据。方法将HIV-2结构基因gag插入到转移载体pUTA2复合启动子ATI-P7．5×20下游．构建出鸡痘病毒重组转移质粒pUTA2-gag;经转染和BrdU加压筛选,以基因组聚合酶链反应(PCR)和Western blot法鉴定重组病毒;将获得的重组病毒大量制备并纯化后,肌内注射免疫Balb/c小鼠,酶联免疫吸附实验(ELISA)检测小鼠血清HIV-2抗体,流式细胞仪测定CD4~+、CD8~+T淋巴细胞亚类数量,乳酸脱氢酶(LDH)释放法检测脾特异性细胞毒性T淋巴细胞(CTL)杀伤活性。结果构建出重组鸡痘病毒转移质粒pUTA2-gag;从重组病毒的基因组中可扩增出大小为766bp的目的基因,其表达产物可与人HIV-2阳性血清发生反应,目的蛋白的相对分子质量约为55000;重组病毒免疫组小鼠的血清出现HIV-2抗体．脾T细胞亚类的数量增加,并产生针对HIV-2靶细胞的特异性CTL杀伤活性。结论获得一株重组鸡痘病毒,该病毒可稳定表达HIV-2结构蛋白Gag,并能诱导小鼠产生特异性细胞和体液免疫应答。
Objective To construct and investigate the immune response of human immunodeficiency virus (HIV) -2 gag recombinant fowlpox virus (FPV) in mice and provide experimental basis for developing HIV-2 recombinant live vector vaccine. Methods HIV-2 structural gene gag was inserted into the downstream vector ATI-P7.5 × 20 of pUTA2 recombination promoter. Recombinant plasmid pUTA2-gag was constructed and transfected and BrdU-pressed. The recombinant virus was identified by polymerase chain reaction (PCR) and Western blot. The obtained recombinant virus was prepared and purified in large quantities. Serum HIV-2 antibody was detected by enzyme-linked immunosorbent assay (ELISA), the number of CD4 ~ +, CD8 ~ + T lymphocyte subsets and the activity of lactate dehydrogenase LDH release assay was used to detect the cytotoxic T lymphocyte (CTL) killing activity. Results The recombinant plasmid pUTA2-gag was constructed. The target gene of 766bp was amplified from the genome of the recombinant virus, and its expression product reacted with human HIV-2 positive serum. The relative molecular mass of the target protein About 55000; HIV-2 antibody appeared in serum of recombinant virus-immunized mice. The number of splenic T-cell subsets is increased and produces specific CTL-killing activity against HIV-2 target cells. Conclusion A recombinant fowlpox virus was obtained. The virus can stably express Gag, a HIV-2 structural protein, and induce specific cellular and humoral immune responses in mice.