Cloning And Expression Of FimA Gene Of Extraintestinal Pathogenic Escherichia Coli From Pig

In recent years, a new class of E. coli named Extraintestinal pathogenic Escherichia coli（ExPEC） has been reported. The strains colonized in the extraintestinal organizations of host,which are serious harmful to the host. ExPEC can cause extraintestinal organizations infectionin humans and animals, leading to meningitis, pticemia, urinary tract infections and respiratoryinfections of different hosts. Annually, the disease caused the huge economic losses to the globalpublic health sector, it has aroused wide attention all over the world.ExPEC is characterized by specific virulence factors （VFs） causing extraintestinalorganizations infections. Fimbria is protein structure which is one of VFs located in cell surface,associated with cell motility, biofilm formation, adhesion, release of pathogenic factors. It isconcerned as an important drug target by scholars. Therefore, the research of fimA geneencoding FimA protein subunits of typeâ… fimbria was of great significance.Lung, brain, heart and other tissues were collected from a pig farm in Jilin province, wheresome pigs are suffering from pneumonia. The isolated strain from lung was identified bydifferential medium and16S rDNA PCR identification. Mice virulence experiments, reversionanimal tests confirmed that the isolated strain had virulent, it could cause lung infectionsindependently, and resulted in death. Thus, the isolated was qualitatived as ExPEC, namedSLPE. The drug-resistance of SLPE to8kinds of antimicrobial drugs were determined bymicrodilution assay, results showed that SLPE has multiple resistance to drugs, only wassensitive to β-lactam drug. To further explore the causes of virulent and multidrug resistance,the whole genome of SLPE was sequencing analyzed,66genes associated with Fimbria and35resistance genes （efflux regulation gene MarC, MATE efflux pump genes, resistance genesoriginal mobile IS, tetracycline resistance gene tet, florfenicol resistance gene FloR etc.） wereselected out. The bioinformatics analysis of amino acid sequence encoded by ORF₈90gene,further proved this DNA fragments was fimA gene. Based on this, ORF₈90fimA gene wasamplified, with SLPE genome been as template, and cloned by T-A technique. The prokaryoticexpression plasmid pET-28a-fimA, pET-32a-fimA were constructed successfully. After inducedby IPTG, the expression protein of pET-32a-fimA-BL21was approximately38.7ku. Theexperimental results would offer the basis for preparing FimA antibodies, establishing a rapidand effective method for the detection of gene expression levels, and clarifying pathogenicmechanism of ExPEC by a certain extent.