猪丹毒丝菌C43065株表面保护性抗原AN端保护区在大肠杆菌中的表达

目的：在大肠杆菌中表达猪丹毒丝菌C43065株表面保护性抗原A（SpaA）N端保护区（SpaA-N）,并检测其抗原性。方法：利用PCR方法从猪丹毒丝菌C43065株基因组中扩增出spaA基因片段,构建pMD18-spaA重组质粒并对插入片段进行测序;以pMD18-spaA重组质粒为模板,PCR扩增得到spaA-N基因片段,构建重组表达质粒pGEX-spaA-N,经序列测定证实正确后转化大肠杆菌BL21（DE3）,再经IPTG诱导表达GST-SpaA-N融合蛋白并纯化。结果：扩增得到的spaA基因长1881bp,编码由626个氨基酸残基构成的多肽;SDS-PAGE和Western印迹检测结果表明,诱导表达获得相对分子质量约64000的GST-SpaA-N融合蛋白,该融合蛋白能与相应抗体发生特异性反应。结论：获得了在大肠杆菌中可溶性表达的GST-SpaA-N融合蛋白,为进一步研究猪丹毒丝菌免疫保护性抗原奠定了基础。

Expression of N-Terminal Protective Domain of Surface Protective Antigen A from Erysipelothrix rhusiopathiae C43065 Strain in Escherichia coli

Objective：To express the N-terminal protective domain of surface protective antigen A（SpaA） of Erysipelothrix rhusiopathiae C43065 strain in E.coli,and to assay its antigenicity.Methods：The gene encoding SpaA was amplified by PCR with specific primers from genomic DNA of E.rhusiopathiae C43065 strain,and was cloned into the pMD18-T vector and then sequenced.The N-terminal protective domain of the spaA（spaA-N） gene was amplified by PCR from the recombinant plasmid pMD18-spaA,and was cloned into the prokaryotic expression vector pGEX-6p-2 to provide a recombinant plasmid pGEX-spaA-N.Under the IPTG induction,the recombinant fusion protein was produced by E.coli BL21（DE3） harboring the recombinant expression plasmid pGEX-spaA-N.Results：The sequence analyses showed that the open reading frame of spaA gene was 1 881 bp and encoded a peptide of 626 amino acid residues.SDS-PAGE analysis revealed a single fusion protein band with a molecular weight of 64 kD,and the Western blot results showed that the fusion protein can reacted to an antibody against the fusion protein GST-SpaA-N.Conclusion：GST-SpaA-N fusion protein is expressed and purified,which will contribute to further research.