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

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.