猪丹毒丝菌C43311株spaA基因N端免疫保护区的克隆和表达

Cloning and expression of N-terminal protective domain of spaA gene from Erysipelothrix rhusiopathiae C43311

摘要:

采用PCR从猪丹毒丝菌C43311株基因组中扩增出编码表面保护性抗原A的spaA基因片段, 将其克隆到pMD18-T载体并对插入片段进行测序。用spaA基因N端免疫保护区(spaA-N)的特异引物从重组质粒pMD18-spaA中扩增得到spaA-N片段, 将其定向插入原核表达载体pGEX-6p-2中, 构建重组表达质粒pGEX-spaA-N, 转化大肠杆菌BL21(DE3), 在IPTG诱导下表达融合蛋白GST-SpaA-N, 用SDS-PAGE和Western印迹检测表达产物。测序结果表明spaA基因片段大小为1881bp, 与已报道的不同血清型菌株spaA基因的核苷酸序列比较, 核苷酸序列同源性在93％～99％之间。SDS-PAGE结果显示表达蛋白约为64kDa, 与预期的大小相近。Western印迹检测结果表明, 表达的融合蛋白GST-SpaA-N能与C43311株SpaA蛋白的抗血清发生特异性反应, 证明原核融合表达蛋白具有免疫反应性。

关键词:猪丹毒丝菌; spaA基因; 融合蛋白; 免疫反应性; 原核表达

Abstract:

The spaA gene was amplified by PCR from the genomic DNA of Erysipelothrix rhusiopathiae C43311 strain, and inserted into the pMD18-T vector and then sequenced. The N-terminal protective domain of the spaA gene was amplified by PCR from the recombinant plasmid pMD18-spaA, then cloned into the prokaryotic expression vector pGEX-6p-2 and expressed in E. coli BL21 (DE3) by IPTG induction. The expressed protein was identified by SDS-PAGE and Western blot. The sequence analyses showed that the coding region of the spaA gene of C43311 strain was 1881bp in length, and the nucleotide sequence homology of the spaA genes between the C43311 strain and the pre-viously reported different serotype strains of E.rhusiopathiae was 93 to 99%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 64kDa, and the Western blot results showed that the GST-SpaA-N fusion protein was recognized specifically by an antiserum against the SpaA protein of C43311 strain, suggesting that the fu-sion protein of GST-SpaA-N possessed high immunoreactivity.

Key words:Erysipelothrix rhusiopathiae; spaA gene; fusion protein; prokaryotic expression; immunoreactivity