狐源维氏气单胞菌体内诱导基因的筛选

Screening of in vivo-induced genes of Aeromonas veronii isolated from fox

为了筛选狐源维氏气单胞菌体内诱导基因,本研究将狐源维氏气单胞菌人工感染家兔,并取不同感染时期的血清混合,分别用体外培养的维氏气单胞菌和大肠杆菌BL21全菌、二者菌体裂解物以及热变性裂解物进行吸附,用ELISA进行检测;同时构建狐源维氏气单胞菌基因组的p ET系统重组质粒表达文库。再用经吸附处理的血清对狐源维氏气单胞菌基因组文库进行菌落原位免疫杂交筛选,将筛选的阳性克隆进行测序,并用RT-PCR进一步验证。结果显示,最终获得8个狐源维氏气单胞菌的体内诱导基因,分别为GTP焦磷酸激酶基因、转录调控基因Mar R基因、氢化酶细胞膜亚基基因、外膜受体转运蛋白Ton B基因、Ⅲ型分泌系统asc V基因以及3个保守假想蛋白基因。研究结果为维氏气单胞菌保护性抗原的筛选、分子致病机制及跨物种间传播机制奠定了基础。

To screen the in vi vo -induced genes of Aeromonas veronii （A. veronii ） isolated from fox, sera were collected from rabbits experimentally infected with A. veronii. And sera were adsorbed extensively against bacterial whole cells,cell lysates and heat-denatured cell lysates of in vitro grown A. veronii and E. coli BL21 （DE3）. Absorption effect was detected by ELISA. In addition,the genomic expression library of A. veronii was constructed,and then it was screened by the adsorbed sera using colony in situ hybridiza- tion. The positive clones were identified by sequencing and RT-PCR. The results showed that 8 in vivo-induced genes were obtained by screening the library,including putative GTP pyrophosphokinase gene,MarR family transcriptional regulator gene, hydrogenase 3 membrane subunit gene, outer membrane receptor-mediated trans- port energizer protein TonB gene, typeIII seeretionsystem ascV gene and three of them were conserved hypothet- ical genes. In conclusion, the results will be helpful to screen protective antigens and lay the foundation for the study about the pathogenesis and mechanism of cross-speciestransmission of A. veronii.