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中文摘要：
【目的】在体外克隆和表达猪产肠毒素大肠杆菌（ETEC）K99菌毛操纵子fan结构基因,并检测重组菌毛的相关生物学活性。【方法】以猪源分离的表达K99菌毛ETEC C83907株制取模板,成功PCR扩增出编码K99菌毛的fan操纵子,约5.7 kb。将fan操纵子克隆入表达质粒载体pBR322,筛选出含正确阳性质粒的重组菌。进一步将上述的重组质粒DNA转化至不含任何菌毛的大肠杆菌SE5000株,同时将空载体pBR322质粒转化入SE5000构建阴性对照菌株。【结果】该重组菌能与鼠抗K99菌毛单克隆抗体发生明显的凝集反应,与新生仔猪小肠上皮细胞刷状缘BBV分子有强烈凝集反应。电镜观察到上述重组菌表面大量表达K99菌毛,用热抽提法提纯其表达的K99菌毛,并经SDS-PAGE电泳和考马斯亮蓝染色,可以得到分子量约为18.5kDa的主要蛋白条带。纯化菌毛免疫小鼠后制备出高效价的鼠抗血清,能与携带K99菌毛的C83907、C83914、C83260野生株发生强烈的凝集反应,而与携带其他菌毛的ETEC不反应。玻板凝集试验和Westernblot结果表明：体外表达的K99菌毛具有和野生K99菌毛相同的抗原性。用表达K99菌毛的重组菌进行HeLa细胞体外黏附试验和黏附抑制实验,结果表明：重组菌和野生菌株一样具有较强的粘附性,而且用重组菌毛制备的鼠抗血清能有效地抑制上述重组菌或野生菌株对细胞系的黏附结合。【讨论】本研究为进一步研究K99菌毛生物学作用建立了良好的实验平台。
英文摘要：
[Objective]To clone and express fan operon gene clusters of K99 fimbriae in enterotoxigenic Escherichia coli（ETEC） in vitro,and study the activity of the recombinant E.coli expressing K99 fimbriae.[Methods]K99 fimbriae gene clusters were amplified by long-PCR method,using the genomic DNA of K99-fimbriae E.coli C8307 as the DNA template.The 5.7Kb PCR products were inserted into expressing vector pBR322 with restriction endonuclease,then positive clones were screened.The positive recombinant plasmid was transformed into non-fimbriae E.coli SE5000 strains,and pBR322 plasmid was also transformed into SE5000 for negative control strain.[Results]The recombination E.coli expressing K99 fimbriae was tested with agglutination assay,using monoclonal antibody serum and brush border vesicles from the piglet small intestinal epithelia cells.The expressed fimbriae on the surface of the recombinant E.coli SE5000 were observed by transmissible electromicroscope.Heat extraction method was employed to isolate and purify K99 fimbriae,which was exerted SDS-PAGE,and 18.5 kDa protein band was detected.The mouse sera produced from recombinant fimbriae was used to test K99-fimbriae strains C83907,C83914,C83260 with positive agglutination results,while negative results were found with E.coli contain other kinds of fimbriae.The assays of SDS-PAGE,Western blot,agglutination assay were used to evaluate antigenicity and biologic activity between C83907 and recombinant strain.Adhesion test with HeLa cell line demonstrated the recombinant strain and wild type have the similar adherence ability,and this adhesion can be inhibited with mouse serum containing polyclonal antibody against recombinant K99 fimbriae.[Conclusion]This study has laid a good foundation for further study on bioactivity of K99.
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