表达猪繁殖与呼吸综合征病毒GP5蛋白减毒鼠伤寒沙门氏菌疫苗株的构建及动物免疫试验

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.

Construction of the Attenuated Salmonella typhimurium Vaccine Strains Expressing PRRSV GP5 Antigen and Animal Immunity Test

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...