论文部分内容阅读
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。
In order to screen the induced genes of AeromonasVirginia in fox in vivo, we infected Artemisia foxtii with Artemisia fonnula by artificial infection and mixed the sera of different stages of infection with Aeromonas sobria And Escherichia coli BL21 whole bacteria, both cell lysate and heat-denatured lysate were adsorbed, and detected by ELISA. At the same time, a recombinant plasmid expression library of p ET system of Aeromonas foxtii genomic was constructed. Then the serotypes of Adsorption-treated sera were used to screen the A. foxii genomic library for colony-specific in situ hybridization. The positive clones were sequenced and verified by RT-PCR. The results showed that in vivo induction of 8 Aeromonas sobria fox genes were GTP pyrophosphate kinase gene, transcription factor Mar R gene, hydrogenase cell membrane subunit gene, outer membrane transporter Ton B Gene, the type III secretion system asc V gene and three conserved hypothetical protein genes. The results laid the foundation for the screening of protective antigens of Aeromonas sobria, molecular pathogenesis and cross-species transmission mechanism.