牛轮状病毒VP4基因与LTB基因的融合表达及免疫原性研究  

Construction,Expression and Immunogenicity of Fusion Gene VP4 Gene of Bovine Rotavirus and LTB Gene

作　　者：南晓伟[1,2] 杨少华[1] 高运东[1] 王长法[1] 杨宏军[1] 刘晓[1] 何洪彬[1] 申之义[2] 仲跻峰[1] 

机构地区：[1]山东省农业科学院奶牛研究中心,山东济南250100 [2]内蒙古农业大学,内蒙古呼和浩特市010018

出　　处：《西南农业学报》2010年第1期206-209,共4页Southwest China Journal of Agricultural Sciences

基　　金：山东省农科院青年基金项目(2007YQN018);山东省农科院产业开发项目(2007-013);山东省农科院"杰出人才"项目;现代农业产业技术体系项目(nycytx-0107)

摘　　要：PCR扩增轮状病毒VP4基因和大肠杆菌LTB基因编码区,将其克隆至pET32a载体上,构建pET32a-VP4-LTB质粒,转化大肠杆菌BL21(DE3),PCR、酶切鉴定后进一步测序,测序结果表明融合基因片段由2637 bp组成,为编码879个氨基酸残基的多肽。经IPTG诱导和SDS-PAGE检测发现表达产物分子量约为118.9 KD,以包涵体的形式存在。融合蛋白经镍离子螯合次氨基三乙酸(Ni-NTA)亲和层析纯化后免疫小鼠,结果所产生的抗体效价高于单独VP4蛋白的免疫结果但差异不显著(P>0.5),但与对照组相比差异显著。表明所表达的融合蛋白具有较好的免疫原性,为研制牛轮状病毒亚单位疫苗奠定了基础。The coding region of rotavirus VP4 gene and LTB gene was obtained by PCR amplification. The plasmid pET32a -VP4-LTB containing VP4-LTB fusion gene was constructed by cloning VP4 and LTB gene into pET32a, which was further confirmed by restriction enzyme digestion and nucleotide sequence. SDS-PAGE analysis demonstrated that the recombinant plasmid could express the VP4-LTB fusion protein in form of inclusion body , and its molecular weight was around 118.9 KD. The fusion protein of VP7-LTB and VP7 were purified by NiNTA. Vaccine were prepared and immune to mouse. The result showed that the antibody tilter of fusion protein VP4-LTB was higher than that of VP4, but the difference was not significant （ P 〉 0.5）, compared with control group there was significant difference （ P 〈 0.1 ）. The resuits demonstred that VP4-LTB fusion protein had a better immunogenicity and could act as a eandidate vaccine against bovine rotavirus infection.

关 键 词：牛轮状病毒 融合基因 原核表达 免疫原性 

分 类 号：S852.612[农业科学—基础兽医学] S852.659.4[农业科学—兽医学]