[目的]通过原核表达系统表达小鼠细小病毒(Minute virus of mice，MVM)的非结构蛋白1(nonstructural protein 1，NS1)，并制备其多克隆抗体，为MVM NS1蛋白生物学功能研究及相关检测方法的建立提供研究材料。[方法]通过同源重组将MVM NS1基因与质粒pET-N-His-C-His进行连接构建重组质粒，将其转化大肠杆菌BL21(DE3)感受态细胞进行诱导表达，通过不同诱导条件筛选并纯化出无需复性的可溶性蛋白。将纯化后的重组蛋白NS1免疫新西兰大白兔制备多克隆抗体，通过间接ELISA法测定抗体效价。采用Western blotting和间接免疫荧光试验(IFA)鉴定重组蛋白的表达及免疫原性。[结果]SDS-PAGE分析显示，诱导表达的重组蛋白大小为76 ku，在诱导剂(IPTG)浓度为0。6 mmol/L、诱导温度为28 ℃条件下表达效果最佳。间接ELISA结果显示，制备的多克隆抗体效价为1∶32 000。Western blotting和IFA检测结果显示，制备的NS1多克隆抗体可与NS1蛋白及MVM特异性结合。[结论]本研究成功表达并纯化获得MVM NS1重组蛋白，并制备了免疫原性良好的多克隆抗体，为进一步研究NS1蛋白生物学功能及其在MVM复制感染机制中的作用、MVM临床诊断方法的建立奠定了基础。
Prokaryotic Expression,Preparations of Polyclonal Antibodies and Immunogenicity Evaluation of NS1 Protein of Minute Virus of Mice
[Objective]The objective of this experiment was to express nonstructural protein 1(NS1)of Minute virus of mice(MVM)through prokaryotic expression system and prepare polyclonal antibody,which provided research materials for the study of biological function of MVM NS1 protein and the establishment of related detection methods.[Method]MVM NS1 gene was linked to plasmid pET-N-His-C-His through homologous recombination to construct recombinant plasmid,which was transformed into Escherichia coli BL21(DE3)competent cells for induced expression.Soluble proteins without renaturation were screened and purified by different induction conditions.The purified recombinant protein NS1 was immunized with New Zealand White rabbits and polyclonal antibodies were prepared.The titer of the antibodies was determined by indirect ELISA.Western blotting and indirect immunofluorescence assay(IFA)were used to determine the expression and immunogenicity of the recombinant protein.[Result]SDS-PAGE analysis showed that the recombinant protein was 76 ku in size,and the optimal expression was achieved under the conditions of IPTG concentration of 0.6 mmol/L and induction temperature of 28 ℃.Indirect ELISA results showed that the titer of the prepared polyclonal antibody was 1∶32 000.Western blotting and IFA detection results showed that the prepared NS1 polyclonal antibody could bind specifically to NS1 protein and MVM.[Conclusion]This study successfully expressed and purified MVM NS1 protein,and successfully prepared polyclonal antibody with good immunogenicity,which laid a foundation for further study of the biological function of NS1 protein and its role in the mechanism of MVM replication infection,and the establishment of clinical diagnostic methods for MVM.
Minute virus of micenonstructural protein 1polyclonal antibodyimmunogenicity
万方数据
维普
