肠炎沙门氏菌SEFA基因表达和间接ELISA检测方法的初步建立

为了给临床检测肠炎沙门氏菌的感染和血清学调查提供一种切实可行的方法,本研究根据肠炎沙门氏菌SEF14菌毛操纵子亚单位sefA基因序列设计一对引物,利用PCR技术从国内标准株CMCC(B)50336中扩增sefA基因,并按预定的阅读框插入表达载体pET22b+中,获得重组质粒pET-sefA,限制性内切酶结合琼脂糖凝胶电泳分析和序列测定结果表明,该序列大小为498bp,与已发表的sefA结构编码序列完全一致。重组质粒pETsefA转化大肠杆菌E.coli BL21(DE3)并能获得高效诱导表达,通过对菌体裂解上清液SDS-PAGE和western blot分析鉴定,该重组菌可以表达大小为15.2ku的可溶性重组蛋白rSEFA。纯化的rSEFA免疫小鼠所得高免血清以及标准株CMCC(B)50336感染小鼠后所获阳性血清,均能识别标准株肠炎沙门氏菌SEF14菌毛蛋白以及纯化的重组蛋白rSEFA,结果表明明体外表达的rSEFA蛋白有较好的免疫原性和反应原性。而基于rSEFA介导的间接ELISA有较好的特异性,对肠炎沙门氏菌特异性抗体检测有潜在的应用前景。

Expression of subunit sefA of S. enteritidis SEF14 fimbriae and establishment of an indirect ELISA

The subunit sefA gene of SEF14 fimbriae from Salmonella enteritidis was amplified by PCR using a pair of primers derived from reference strain CMCC(B)50336 genomic DNA. The PCR products was cloned into the expression vector pET22b+ and then transformed to E. coli BL21 (DE3). A soluble recombinant SEFA protein (rSEFA) of 15.2 ku was expressed in BL21 cells and confirmed by SDS-PAGE and westem blot analysis. Antibody against rSEFA could recognize the purified SEF14 fimbriae from reference strain CMCC(B)50336 and rSEFA, respectively. An indirect ELISA assay was established by using rSEFA protein as coating antigen for the antibody detection against Salmonella enteritidis.