产肠毒素大肠杆菌K88ab/K88ad菌毛操纵子fae全基因的克隆、表达及生物学活性的初步研究

Cloning and expression of K88ab/K88ad fimbrial operons from enterotoxigenic Escherichia coli and their bioactivity

为研究K88ab/K88ad菌毛对细胞的黏附作用,本研究分别以产肠毒素E.coli(ETEC)K88ab C83901株和K88ad C83903株基因组DNA为模板,采用PCR技术扩增这两种K88菌毛操纵子fae基因(均约7.9 kb)。将其分别克隆于表达质粒pBR322中构建pBR-K88ab和pBR-K88ad重组质粒,并将其分别转化至不含任何菌毛的E.coli SE5000株中。该重组菌能够分别与鼠抗K88菌毛阳性血清和抗K88菌毛单克隆抗体(MAb)产生凝集反应;在电镜下观察到重组菌表面大量表达K88菌毛。采用热抽提法提取其体外表达的K88ab和K88ad菌毛,SDS-PAGE电泳检测结果显示,菌毛蛋白的分子量约为26 ku。玻板凝集试验和western blot结果表明:重组表达的K88ab及K88ad菌毛与K88+参考株菌毛均能够被抗K88菌毛阳性血清和MAb识别。以猪小肠上皮细胞系IPEC-J2为模型进行黏附和黏附抑制试验,结果表明表达K88菌毛的重组菌及K88+参考株均能够黏附于IPEC-J2上皮细胞表面;而且阳性血清和MAb能够有效抑制重组菌或K88+参考株对猪小肠上皮细胞系的黏附结合。

The fae operon with size of 7.9 kb, encoding the K88 fimbriae, was amplified by high fidelity long-PCR with the genomic DNA as the templates from K88ab and K88ad E.coli strains. The PCR products of K88ab and K88ad fimbriae genes were cloned into pBR322 to construct the pBR-K88ab and pBR-K88ad which were transformed into the E.coli SE5000, respectively. The expressed fimbriae were observed by transmissible electromicroscope, and the fimbriae K88ab and K88ad were purified from the recombinant E.coli, which were about 26 ku detected by SDS-PAGE. The results of agglutination assay and western blot showed that the rabbit sera and MAb against K88 fimbriae reacted positively with the K88ab and K88ad fimbriae from recombinant E.coli..In addition, piglet small intestine epithelial cell line IPEC-J2 was prepared and tested for the adherence assay. The results showed the recombinant E.coli expressed K88 fimbriae was able to adhere to IPEC-J2 cell in vitro as wild typeE.coli did. The anti-sera and MAb against fimbriae K88 was able to efficiently block the recombinant E.coli （expressing K88 fimbriae） and wild type E.coli adherence to IPEC-J2.