猪葡萄球菌脱落毒素ExhC基因的克隆及原核表达

Cloning and Prokaryotic Expression of Exfoliative Toxin ExhC Gene of Staphylococcus hyicus

为研究猪葡萄球菌表皮脱毒素Exh C基因的分子特征及其原核表达产物的免疫学活性,利用PCR技术从猪葡萄球菌广东增城分离株(GDZC株)扩增出表皮脱落毒素Exh C基因,测序与序列比较分析显示,其编码区全长为837bp,编码279个氨基酸,与国内外猪葡萄球菌和松鼠葡萄球菌表皮脱落毒素Exh C基因的核苷酸同源性、氨基酸同源性均为100%。将Exh C基因克隆于pET-28a(+)表达载体中,经测序正确后,重组质粒转入大肠埃希菌BL21(DE3)进行诱导表达,表达产物经SDS-PAGE鉴定和Western blot分析。表达的融合蛋白主要以包涵体形式存在,其分子质量大小约为32ku,与预期大小一致。Western blot分析表明,Exh C重组蛋白能与猪葡萄球菌GDZC株的阳性血清发生特异性反应,说明表达的GDZC株Exh C重组蛋白具有良好的免疫学活性。

This research was intended to study the molecular characteristics and reactogenicity of prokary-otic expression products of exfoliative toxin Exh C gene in S.hyicus.The Exh C gene was amplified in GDZC strain of S.hyicus which came from Zengcheng of Guangdong province by PCR.Sequence analysis indicated that the coding region has 837 bp,to code 279 amino acid.The nucleotide homology and amino acid homology were 100% both with Exh C gene of S.hyicus and S.sciuri which came from abroad.The recombinant expression plasmid pET-28a （＋）-Exh C was shifted to E.coli BL21（DE3）for inducible ex-pression.After the SDS-PAGE,fusion protein was as inclusion body,and its molecular mass was 32 ku as the expected.Western blot indicated that recombinant protein of Exh C can response specifically with posi-tive serum of GDZC strain,and indicated that it has good immunologic activity.