猪繁殖与呼吸综合征病毒M蛋白和SEA共表达核酸疫苗的构建及鉴定  

Construction and identification of DNA vaccine co-expressing M protein of porcine reproductive and respiratory syndrome virus and staphylococcalenterotoxin A

作　　者：韩贞珍[1] 金宁一[1] 沈国顺[1] 庄天中[1] 孙中锋[1] 李臻[1] 付延军[1] 李太元[2] 李红文[1] 郑敏[1] 

机构地区：[1]军事医学科学院军事兽医研究所,吉林长春130062 [2]延边大学农学院,吉林龙井133400

出　　处：《中国兽医学报》2008年第1期9-11,共3页Chinese Journal of Veterinary Science

基　　金：吉林省科技发展计划资助项目(20060554)

摘　　要：构建猪繁殖与呼吸综合征病毒(PRRSV)M蛋白和金黄色葡萄球菌肠毒素A(SEA)共表达的核酸疫苗,并研究其表达产物的抗原性。将采自长春患PRRS病猪的组织剪碎充分研磨,提取病毒RNA后,用RT-PCR的方法特异性扩增PRRSV长春株的ORF6基因(M蛋白)片段。将该片段定向插入到载体pKS-SEA上,双酶切获得片段SEA-ORF6,再将其克隆到真核表达载体pVAXI中,构建重组质粒pVAXI-SEA-ORF6,转化大肠杆菌。经酶切鉴定后,将构建的阳性重组质粒在BHK细胞中表达。通过间接免疫荧光方法初步检测所表达蛋白的抗原性,用Western blot分析表达蛋白的特异性。结果表明,所构建的重组质粒pVAXI-SEA-ORF6在BHK细胞中得到了表达,为进一步研究PRRSV基因工程疫苗奠定了基础。To construct DNA vaccine co-expressing M protein of porcine reproductive and respiratory syndrome virus （PRRSV） and staphylococcalenterotoxin A （SEA） ,and explore the antigenicity of expressed preduct,the tissues of porcine carrying PRRSV were cut and ground completely, and the total RNA was extracted. Then ORF6 gene fragment of Changchun strain was amplified by RT-PCR and inserted into vector pKS-SEA. Then the expression cassette SEA-ORF6 was acquired by double restriction endonuclease digestion, and cloned into eukaryotic expression vector pVAXI to construct recombinant plasmid pVAXI-SEA-ORF6, then transformed E. coli. After identified by restriction endonclease digestion, the recombinant plssmid pVAXI-SEA-ORF6 was transferred into BHK cells.And antigenicity of the expressed protein was identified by IFA and Western blot. The results showed that the expression vector pVAXI-SEA-ORF6 was successfully constructed ,and object gene was expressed in BHK cells,so it laid the foundation for further study on genetic engineering vaccine of PRRSV.

关 键 词：猪繁殖与呼吸综合征 表达 核酸疫苗 

分 类 号：S852.65[农业科学—基础兽医学]