【摘要】 为筛选表达量高且免疫原性强的红斑丹毒丝菌表面保护性抗原A(SpaA)的抗原片段，本研究根据该菌SpaA的结构和功能区将其设计为6个子片段和1个全长片段，采用PCR技术从红斑丹毒丝菌HG-1菌株中扩增出SpaA的7个抗原基因片段，利用原核表达系统克隆并表达后采用SDS-PAGE和western blot检测表达及纯化情况。结果显示，获得7个重组蛋白，分别命名为rSpaA1～rSpaA7，其表达量占菌体总蛋白的17.44%～25.38%，其中rSpaA1、rSpaA2、r SpaA5以包涵体形式表达，r SpaA3以可溶性蛋白形式表达，而rSpaA4、rSpaA6、rSpaA7两种表达形式同时存在。纯化后，rSpaA1～rSpaA7的纯度介于84.22%～93.72%，浓度为0.89 mg/mL～3.25 mg/mL。Western blot结果显示，7个重组蛋白均显示出良好的反应原性，其中rSpaA1、rSpaA3、rSpaA4的反应原性更强。本研究实现了对红斑丹毒丝菌SpaA的分段及其全长的表达和纯化，为SpaA保护性抗原片段的筛选及猪丹毒亚单位疫苗的研发奠定基础。更多还原

【Abstract】 In order to select the SpaA antigen fragments with high expression and strong immunogenicity, according to the structure and functional region of surface protective antigen A from Erysipelothrix rhusiopathiae, it was designed into 6 subfragments and 1 full-length fragment, 7 antigen fragments of SpaA were amplified from Erysipelothrix rhusiopathiae HG-1 strain by PCR, and then they were cloned and expressed by prokaryotic expression system. The recombinant proteins were purified by affinity chromatography, then detected by SDS-PAGE and western blot. The results showed that all seven recombinant proteins were successfully expressed, which named as rSpaA1-rSpaA7, and the proportion of its expression in total bacterial protein were between17.44%-25.38% respectively, of which rSpaA1, r SpaA2, and r SpaA5 were expressed as inclusion bodies, rSpaA3 was expressed as a soluble protein, while rSpaA4, rSpaA6, and rSpaA7 were presented in both forms. After purification with His-tagged protein purification kit, the purities of rSpaA1-rSpaA7 were between 84.22%-93.72%, and the concentrations were between 0.89 mg/mL-3.25 mg/mL. All 7 recombinant proteins showed good reactogenicity by western blot, among of which rSpaA1, rSpaA3, and rSpaA4 had stronger responses. This study successfully achieved the expression and purification of SpaA and its six segments, laying the foundation for the selection of SpaA protective antigen fragments and the development of swine erysipelas subunit vaccines.更多还原