红斑丹毒丝菌SpaA蛋白保护区域的免疫学检测

Immunological Testing of Protein Protective Domain SpaA of Erysipelothrix rhusiopathiae

为了确定菌体表面保护性抗原A(SpaA)的免疫保护区域,通过PCR从红斑丹毒丝菌C43065株基因组DNA中分别扩增出编码成熟SpaA及其N端342个氨基酸序列(SpaA-N)和C端160个氨基酸序列(SpaAC)的基因片段,将它们分别克隆到表达载体pET32a上,转化入大肠埃希菌BL21,IPTG诱导,亲和层析法纯化重组蛋白,并检测它们对小鼠的保护作用。SDS-PAGE结果显示重组SpaA、SpaA-N和SpaA-C以可溶性形式表达在大肠埃希菌BL21细胞质中并具有良好的免疫原性。保护试验结果表明重组SpaA、SpaA-N和NaOH提取抗原完全保护小鼠受强毒株C43065的致死性感染,但重组SpaA-C没有保护作用。结果表明SpaA-N可以作为预防猪丹毒的亚单位疫苗。

In order to determine immuno-protective domain of surface protective antigen A （SpaA） , the gene fragments encoding mature SpaA and its N-terminal 32 amino acids sequence （SpaA-N） and C-terminal 160 amino acids sequence （SpaA-C） were respectively amplified by PCR from genome DNA of E. rhusiopathiae strain CA3065 and respectively cloned them into expression vector pET32a and transferred into E. coli BL21 by IPTG induction. The recombinant proteins were purified by affinity chromatography, and tested their protective effect in mice. SDS-PAGE resuits showed that SpaA, SpaA-N, and SpaA-C were expressed in cytoplasm of E. coli BI21 in the soluble form and possessed fine immunogenieity. The protection experiment results showed that the recombinant proteins SpaA, SpaA- N, and NaOH-extraeted antigens completely protected the mice from lethal infection by strong virulent strain CA3065 ; however, the recombinant SpaA-C had no protective effect. The results of the study showed that SpaA-N could be used as sub-unit vaccine to prevent swine erysipelas.