猪丹毒丝菌C43150株表面保护性抗原A的免疫功能区分析

【目的】确定丹毒丝菌新疆分离株C43150表面保护性抗原A(SpaA)中起免疫保护作用的主要区段。【方法】PCR扩增SpaA氨基端(spaA-N)和羧基端(spaA-C)基因片段,测序鉴定正确后,克隆至pGEX4T-1原核表达载体,以IPTG诱导表达重组蛋白r-SpaA-N及r-SpaA-C,表达产物经亲和层析柱纯化后,免疫小鼠制备抗血清,Western Blot及ELISA检测重组蛋白的免疫反应性,最后分别从主动和被动免疫两方面分析r-SpaA-N及r-SpaA-C对小鼠的免疫保护活性。【结果】Western Blot结果表明抗r-SpaA-N及r-SpaA-C小鼠血清均能与天然的SpaA在64 kDa处特异性结合,ELISA检测也表明重组表达和纯化的融合蛋白都具有免疫原性,但小鼠免疫保护实验显示只有r-SpaA-N对攻毒小鼠保护作用显著(P<0.01)。【结论】丹毒丝菌新疆分离株C43150表面保护性蛋白A起主要保护功能的核心区段为氨基端,且是由抗体介导的免疫保护。

Identification of the Major Domain in Surface Protective Antigen (SpaA) Responsible for Immunological Protection from Erysipelothrix rhusiopathiae C43150

【Objective】To confirm the major domain of surface protective antigen A(SpaA) from Erysipelothrix rhusiopathiae Xinjiang isolated strain C43150 which is responsible for protective immunity.【Method】The coding sequences of the N-terminal domain and C-terminal domain of SpaA(SpaA-N & SpaA-C) were obtained by PCR from the genomic DNA of C43150 and cloned into prokaryotic expression vector pGEX4T-1.After expression in E.coli BL2l by IPTG induction,the recombinant fusion protein of expected size(r-SpaA-N & r-SpaA-C) were obtained and purified for preparing the specific antibodies which were used in ELISA and Western Blot detection.And then the protection effects on different groups of immunized mice against the invasive infection from E.rhusiopathiae C43150 were evaluated by active immune and passive immune experiments.【Result】The ELISA assay showed high anti-rSpaA-N(1:102400)and anti-rSpaA-C(1:51200)antiserum were raised in mice,indicating both proteins have immunogenicity.Furthermore,the native spaA proteins extracted from E.rhusiopathiae C43150 were bound both by anti-r SpaAN and anti-r SpaA-C serum at the band of 64 kDa in western blot.However,the protection experiment showed that only r-SpaA-N have the effective protection against infection of E.rhusiopathiae C43150(P<0.01).【Conclusion】The results indicated that SpaA-N maybe the major protective domain of SpaA from E.rhusiopathiae C43150.