本试验旨在构建表达猪繁殖与呼吸综合征病毒(PRRSV) GP5蛋白的口服重组减毒鼠伤寒沙门氏菌活载体疫苗株.PCR克隆除去信号肽序列的PRRSV ORF5基因,将其插入到表达载体pYA3341中,构建重组质粒pYA3341-ORF5.将重组质粒电转入鼠伤寒沙门氏菌疫苗株X4550(缺失Asd、Cya、Crp基因),获得重组疫苗菌株X4550(pYA3341-ORF5).鉴定重组菌GP5蛋白的表达；测定重组菌定居特性及安全性；检测免疫小鼠血清抗体；流式细胞仪检测重组菌对小鼠T淋巴细胞CD4+和CD8+亚群的影响；最后进行免疫猪血清抗体检测.结果表明,酶切鉴定证实重组质粒构建成功；Western blot证实重组菌表达的GP5蛋白能与PRRSV阳性血清特异性结合；重组菌在体内可较稳定地定居于小鼠的肠系膜淋巴结和脾脏中,并在其中表达出GP5蛋白；小鼠口服试验证实重组菌无毒性作用；重组菌口服免疫小鼠可以产生抗GP5蛋白抗体且抗体具有中和活性；重组菌株能不同程度地使CD4+、CD4+/CD8+升高,而使CD8+下降,表明重组菌对细胞免疫功能具有调节作用；淋巴细胞增殖试验表明,重组菌能诱发小鼠产生较强的细胞免疫应答；重组菌口服免疫猪可以产生抗GP5蛋白抗体.本试验成功构建了能稳定表达PRRSV GP5蛋白的口服减毒鼠伤寒沙门氏菌疫苗株,为研究PRRSV口服基因工程疫苗奠定基础.%To construct a recombinant attenuated Salmonella typhimurium oral live vector strain expressing the antigen of PRRSV GP5,the deleting N-terminal signal peptide sequence of PRRSV ORF5 gene was cloned and inserted into expression vector Pya3341. The recombinant plasmid Pya3341-ORF5 was electro-transformed into delta cya, delta crp, delta asd attenuated S. typhimurium vaccine strains X4550 to construct the live vaccine strains X4550 (Pya3341-ORF5). The recombinant strain of X4550 (Pya3341-ORF5) was identified, and its immunogenicity of expression protein, settlement and safety were evaluated. The recombinant strain of X4550 (Pya3341-ORF5) immunity was evaluated by detecting antibody in immunized mouse model. Flow cytometry were performed to analyze the effect of recombinant strain to CD4+ and CD8+ T lymphocyte subsets of mice. Lymphocyte proliferative responses were tested. At last, the recombinant strain of X4550 (Pya3341-ORF5) immunity was evaluated in swine model to detect antibody. The results showed that the recombinant plasmid has been constructed successfully. SDS-PAGE and Western blot could detect the GP5 protein band in S. typhimurium X4550 (Pya3341-ORF5) and expressed GP5 protein could be recognized by the positive serum of PRRSV. The re-combinant strain X4550 (Pya3341-ORF5) could colonize and persist stably in mesenteric lymph-nodes and spleens of mice. Its safety and reliability were verified by mouse experiment. The re-combinant strains were inoculated to mice and swine by oral way and the serum GP5 antibody could be detected by ELISA and neutralization test. Through statistical analysis, the recombinant strain could increase CD4+ and CD4+/CD8+, but decreased CD8+ compared with the control group. This indicates that the recombinant strain has regulatory role for the cellular immune function. Lymphocyte proliferative responses results indicated that recombinant strain also induced an obvious cellular immune response. A recombinant oral live vaccine S. typhimurium strain X4550 (Pya3341-ORF5) expressing PRRSV GP5 protein was constructed successfully. This live vaccine could be a basis to oral live vector vaccine for PRRSV infection.
展开▼
