【摘要】 目的:在体外克隆和表达猪肠产毒性大肠杆菌(ETEC)K88ac菌毛操纵子fae结构基因,并检测重组菌毛的相关生物学活性。方法:利用长PCR技术以猪ETECK88ac株C83902基因组DNA为模板扩增编码K88菌毛操纵子fae基因,克隆入表达质粒载体pBR322,构建和筛选重组质粒pBR322-fae,转化至不含任何菌毛的大肠杆菌EP株;电镜观察重组菌表面菌毛表达情况;用热抽提法提纯表达的重组菌毛;用纯化菌毛免疫小鼠制备高效价抗血清;用SDS-PAGE和Westernblot检测重组菌毛的抗原性,用细胞黏附和黏附抑制试验检测其生物学活性。结果和结论:在电镜下观察到重组菌表面大量表达K88ac菌毛,该重组菌与兔抗K88ac菌毛单因子阳性血清、鼠抗K88ac菌毛单克隆抗体均产生凝集反应;纯化菌毛经SDS-PAGE,结构单位菌毛呈单一的相对分子质量约26×103的蛋白条带;纯化菌毛免疫小鼠后可制备出高效价的鼠抗血清,玻板凝集试验和Westernblot结果表明体外表达的K88ac菌毛具有与K88ac野生菌毛相同的抗原性;猪小肠上皮细胞系黏附和黏附抑制实验结果表明重组EP菌和野生菌株一样具有较强的黏附猪小肠上皮细胞系的能力,而且提纯重组菌毛制备出的鼠抗血清能有效抑制上述重组菌或野生菌株对猪小肠上皮细胞系的黏附结合。更多还原

【Abstract】 Objective:To clone and express fae operon gene clusters from enterotoxigenic Escherichia coli(ETEC) K88ac fimbriae in vitro,and to study the bioactivity of recombinant fimbriae from fae gene clusters expression.Methods:The K88 fimbria operon gene clusters were amplified by long-PCR using the genomic DNA templates of K88ac E.coli C83902 and cloned into expressing vector pBR322.The recombinant plasmid with the inserts of fae gene clusters were constructed and screened.The above recombinant plasmid DNA was transformed into non-fimbria E.coli EP strains.The expressed fimbriae from the recombinant E.coli EP with pBR322-fae expression plasmid were observed by transmissible electromicroscope.The fimbriae from the recombinant E.coli EP was isolated and purified,the mouse sera with high titer of anti-K88ac fimbriae were detected after being immunized with the purified K88ac fimbriae.The combination assays of SDS-PAGE/Western blot with the agglutination assay/agglutination inhibition assay were used to evaluate the antigenicity and biologic activity from recombinant K88ac fimbriae.Results and Conclusion:The recombinant E.coli was revealed and confirmed by transmissible electromicroscope observation.The fimbriae were isolated and purified from the recombinant E.coli,and only a single major band of protein with size of approximately 26 kD was visualized in Coomassie blue-stained gels after SDS-PAGE.The mouse sera with high titer of anti-K88ac fimbriae were detected after being immunized with the purified K88ac fimbriae.The results of agglutination assay with Western blot combination showed that both the polyclonal antibodies(in rabbits and mice sera) and monoclonal antibody directed against K88ac fimbriae reacted positively with the K88ac fimbriae from recombinant E.coli.The recombinant E.coli expressing K88ac fimbriae could adhere well to IPEC-J2 cells in vitro as wild type E.coli expressing K88ac fimbriae.The inhibitory effect of the mice polyclonal antibody against recombinant K88ac fimbriae demonstrated that the anti-sera can efficiently inhibit IPEC-J2,adherence to the recombinant E.coli and wild type E.coli,which both express K88ac fimbriae.更多还原