水泡性口炎病毒M基因的克隆表达及多克隆抗体的制备  

Cloning and expression of M gene of vesicular stomatitis virus and its preparation of polyclonal antibody

作　　者：夏振红 兰云刚[1] 贺文琦[1] 潘伟[1] 同娟珍 赵魁[1] 高丰[1] 宋德光[1] 

机构地区：[1]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医学报》2016年第5期772-777,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31572473)

摘　　要：为制备水泡性口炎病毒(vesicular stomatitis virus,VSV)重组M蛋白的鼠源多克隆抗体,本研究将扩增的VSV-M基因插入到pET-28a载体中,构建了pET-28a-VSV-M原核表达载体,将其转化至大肠杆菌表达菌株E.coli BL21(DE3)后,用IPTG进行诱导表达;SDS-PAGE和Western blot检测结果发现重组蛋白主要以包涵体形式存在,相对分子质量约为31 000。之后应用His GraviTrap 10prepacked gravity flow columns纯化重组M蛋白,并将其免疫小鼠,制备VSV重组M蛋白的多克隆抗体;间接ELISA方法检测其抗体效价为1∶12 800。将该抗体应用于VSV的检测,在稀释倍数为1∶800时仍可检测到清晰的条带,且大小与预期的一致,证实了其与病毒的特异性结合。结果表明,本研究成功构建了VSV-M蛋白的原核表达载体,将表达纯化后的VSV-M蛋白免疫小鼠成功制备了VSV-M蛋白的多克隆抗体,这为后期检测VSV-M基因的表达分布、功能特性及开发诊断试剂和疫苗的研制奠定了基础。For the preparation of vesicular stomatitis virus recombinant M protein source of rat polyclonal antibody,the amplified production of M gene was firstly obtained using RT-PCR and cloning it to the pET-28 aexpression vector named pET-28a-VSV-M in this study.Then convert it to the strains of E.coli expression and induced expression by IPTG.Testing by SDS-PAGE and Western Blot found that the recombinant protein mainly exists in the form of inclusion body.The relative molecular mass is about 31 000.The recombinant M protein was purified using His GraviTrap 10 prepacked gravity flow columns and immunity in mice to prepare the polyclonal antibody of recombinant M protein.Indirect ELISA to detect the antibody titer is 1：12 800.The antibody was applied to test VSV virus,in diluted multiples 1∶800,clear bands still can be detected.Proved that its can be combined with the virus specificity.The results show that successfully constructed prokaryotic expression vector of M protein,expressed and purified M protein in Escherichia coli types.After immune the mice by the purified M protein successfully prepared the polyclonal antibody of M protein.Laid a foundation for later detection of M gene eukaryotic expression product and development of diagnostic reagents and the research of vaccines.

关 键 词：水泡性口炎病毒 VSV-M 原核表达 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]