禽病原性大肠杆菌1型菌毛主要亚单位结构基因的克隆、测序与表达

Cloning,Sequencing and Expression of FimA of Type 1 Pili on Avian Escherichia Coli Isolates

从禽源大肠杆菌037(O78)、166(O78)、120(O18)分离株和猪源大肠杆菌107/86分离株分别提取基因组DNA,并以此为聚合酶链反应(Polymerasechainreaction,PCR)的模板,扩增上述分离株的1型菌毛主要亚单位结构基因pilA,通过其编码的主要菌毛亚单位FimA蛋白氨基酸的序列比较发现:3个禽源株间FimA的同源性为94 3%至99 0%;禽源株和猪源株间FimA的同源性为89 6%至91 1%。通过对重组大肠杆菌的菌体裂解物的SDS PAGE电泳分析及Westernblot分析,禽源大肠杆菌O78037株、O18120株出现了一致的强反应,O78166株反应较弱,而猪源大肠杆菌107/86株反应最弱。这些结果表明:禽源大肠杆菌与猪源大肠杆菌1型菌毛间存在抗原多样性,这种多样性甚至出现在禽病原性大肠杆菌同一血清型的2个不同分离株之间,如O78037株O78166株之间,尽管其FimA氨基酸的同源性很高,为99 0%。

In order to determine the molecular basis for the antigenic diversity of type 1 pili on avian Escherichia coli,the genomic DNA samples of avian Escherichia coli isolates 037(O78),166(O78),120(O18) and porcine Escherichia coli isolate 107/86 were extracted respectively,and used as templates for polymerase chain reaction (PCR) to amplify the pilA gene encoding the major subunit protein of type 1 pili.The specific amplicons about 570bp in length were cloned into T vector,and 4 positive recombinant plasmids were obtained by RE digestion analysis.The nucleotide sequences of pilA genes from 4 Escherichia coli isolates were determined.By comparison,the homology of amino acid sequences of FimA proteins encoded by pilA of those isolates was 943% to 990% among 3 avian isolates,and 896% to 911% between avian isolate and porcine isolate.The pilA genes removed from T vector were cloned into the plasmid vector pGEX6p1 at the site downstream of the 3'terminus of glutathione Stransferase (GST) gene.Recombinant expression plasmids were constructed and transferred into E.Coli B21.After inducing with IPTG,recombinant FimA was expressed as fusion protein linked with the GST.The results of SDSPAGE and Western blot assay demonstrated that the FimA protein expressed by the recombinant expression plasmids specifically reacted with antiserum made with type 1 pili from avian Escherichia coli isolate 1794.Reaction with FimA expressed by pilA gene from avian isolate 037 and isolated 120 was strong,and that with FimA from avian isolate 166 was weak,and that with FimA from porcine isolate 107/86 was the weakest in Western bolt assay.These results suggested that antigenic diversities of type 1 pili exist not only between avian Escherichia coli isolates and procine Escherichia coli isolate,but also exist between different avian Escherichia coli isolates beloneged to the same O serotype,e.g.isolate O78 037 isolate and O78 166 isolate.