作　　者： ; ; ; ; ; ;

机构地区： 华中农业大学生命科学技术学院农业微生物学国家重点实验室

出　　处： 《中国兽医学报》 2008年第5期479-484,共6页

摘　　要： 以致猪水肿病大肠杆菌鄂E株为模板,通过PCR的方法扩增大肠杆菌菌毛F18主要亚基FedF的全长及去信号肽亚基因FedFs,扩增片段分别为1000、900 bp。将纯化的扩增产物克隆到pMD18-T中,通过酶切鉴定和序列分析表明,含SacⅠ及HindⅢ酶切位点的基因全长为967 bp,鄂E株FedF基因编码区全长903 bp,编码301个氨基酸,碱基序列同标准株107/86的同源性为100%。与菌毛AF/R1相比,编码的蛋白质没有同源性。利用pGEX-KG分别构建表达载体,SDS-PAGE检测表明FedFc/pGEX-KG无表达带,FedFs/pGEX-KG则有约58 000的特异性表达带;West-ern blotting检测表明重组蛋白具有免疫原性。利用重组蛋白GST-FedF免疫新西兰兔所制备的抗血清,能抑制Ee株与刷状缘细胞的粘附。利用表达的蛋白建立了F18的ELISA检测方法,并检测790份临床送检血样,其中536份呈阳性,血清学阳性率为67.8%。结果表明,FedF是猪大肠杆菌病新型疫苗及F18+E.coli感染鉴别诊断的良好候选抗原,通过本试验建立的ELISA方法揭示国内的F18+E.coli感染严重,血清学阳性率高达67.8%。 The 967 bp nucleotide fragment including the whole encoding regions of FedF was amplified by PCR from Ee strain which was responsible for the oedema disease of piglets in Hubei province,while the 897 bp fragment excluded the signal peptide sequence. The 967 bp and 897 bp fragments were cloned into pMD18-T vector, respectively. The 967 nueleotides sequence including the whole encoding regions of FedF between site Sac Ⅰ and Hind Ⅲ as compared with the strain 107/86 reported by Imberechts H. Results of the comparative sequence analysis indicated that there was 100% of nueleotide homology between the isolated Ee strain and 107/86 strain. Comparsion of the FedF gene with the AF/R1 pilus,the levels of identity of the protein homology was zero. The FedF gene was then cloned into pGEX-KG, the recombinant plasmid pGEX-KG/FedFc and pGEX-KG/FedFs were constructed. The constructs were transformed into E. coli BL21 （DE3）. After induction by 1.0 mol PL IPTG,a recombinant protein of about 58 000 in size,designed as GST-FedF, was detected,which was present as inclusion bodies and reacted speeifically with swine antisera to the F18 fimbriae by Western blot. The anti-FedF sera from the rabbit of New Zealand after immunization by GST-FedF protein were able to inhibit F18＋ E. coli adhension to porcine brush borders. ELISA was preliminaryly established based on GST-FedF antigen to detect FedF antibody in the sera. The clinical application showed 536 of the 790 sera were positive. All results suggested that FedF may be not only a better vaccine candiate for swine colibacillosis but also a better candiate antigen used to differentiate the F18＋ E. coli infected pigs or not. The swine in our country were seriously infected by F18^＋ E. coli with the rate of the seropositive of 67.8%.

关 键 词： 大肠杆菌 基因 克隆 原核表达

领　　域： [生物学]