| Goose yolk peritonitis, occurred mainly in the egg layer geese is an acute infectious disease that can lead to decreased performance and even death. The disease occurs mainly because of the gander genital infected with E. coli and then infect to the goose in the mating process, leading to inflammation of the fallopian tubes and the emergence of E. coli infection, resulting in egg yolk peritonitis. Egg laying goose because of the occurrence of the disease appear performance decline, never produced even death, resulting in huge economic losses to the aquaculture industry. OmpA is widely present in the outer membrane of Gram-negative enterobacteria. The part of N-terminal signal peptide sequence of ompA consist of21amino acids. OmpA, also known as heat-modifiable protein, is the factor that anti the damaging effects from the host. As the microporous protein OmpA can maintain the integrity of outer membrane structure and the integrity of the normal form of the bacteria. At the same time, it is also an important virulence factor of the bacteria and plays a key role in both the bacterial adhesion and the beginning of the process of invasion.1. Cloning and expression of ompA of Escherichia coli inducing salpingitis-peritonitis in layer geeseThe gene encoding protein ompA was amplified from the genomic DNA of E.coli G8107by PCR technique. The purified PCR product was digested with enzymes (EcoR I and Xho I), and then cloned into expression vector pET32a(+) with the same sticky ends, and then transformed into E.coli BL21(DE3). Plasmids containing the right insert were sequenced to confirm its identity, and then the supernatant of the bacterial lysates from cultures induced with IPTG were directly loaded onto SDS-PAGE. Sequence analysis showed that the full coding length of ompA was1041bp, which could encode347amino acid residues with a molecular mass of36kD. The SDS-PAGE analysis showed that the best expression was induced in5hours by28℃and1.0 mM IPTG, under which a relative molecular weight of54kD recombinant protein OmpA was produced. Western-blot results showed that the recombinant OmpA could interact with MAb against His-tagged protein. The ompA gene of E.coli was cloned and successfully expressed in E.coli, and the results may provide the foundation to further study the function of the protein and the pathogenesis of the pathogen.2. Preparation of monoclonal antibodies against the OmpA of Escherichia coli inducing salpingitis-peritonitis in layer geeseThe expression protein of the outer membrane protein A of Escherichia coli from Goose yolk peritonitis were used as immunogen,6-8week old BALB/C mice were immunized three times. Spleen cells were collected from BALB/C mice and fused with murine SP2/0myeloma cells. By using the indirect ELISA and limiting dilution methods, seven hybridoma cell lines secreting monoclonal antibodies were established after twice fusion. The hybridoma cell lines were named as OmpA-2H11,OmpA-lA3, OmpA-4A7, OmpA-4B8, OmpA-6A6, OmpA-8H10and OmpA-9D3. Western-blot results showed that the seven hybridoma cell lines could interact with the recombinant OmpA and did not interact with the His tagged protein. The subclass isotype of monoclonal antibodies were IgGl or IgG2a. The titers of the ascetic fluids of6A6and1A3were1:128000and1:32000respectively. The clinical gram-negative bacteria isolates were screened and it was found that the OmpA is not in all Gram-negative bacteria.3. The construction of the OmpA deletion mutants from the Escherichia coli inducing salpingitis-peritonitis in layer geese and some related function analysisBased on the principle of Red/ET homologous recombination, the wild type isolate G8107was selected as the prototype of ompA, and the gene mutant was constructed by using λ Red-mediated recombination system. According to the ompA gene and FRT-flanked PGK-gb2-neo cassette kanamycin resistant fragment, one pair of primers was designed to amplify the fragment containing50bp homologous arms respectively. The pRedET plasmid was first transformed into E.coli G8107by electro transformation. The recombinase was expressed and the fragment with homologous arms was transferred into bacteria. Then the homologous recombination would be generated. Based on the AompA mutant, some assays related to the biochemical characteristics and function, such as growth, biochemical characteristics, drug sensitivity and so on, were carried out. It was found that the mutant strain could not get an effective decomposition of arginine, ornithine,could decompose β-galactoside, and have smaller growth velocity compared with wild type. Susceptibility testing showed that the drug sensitivity of mutant strain to most antibiotics has been raised. Through mice injected vagina, we found that the gene deletion seriously affected the adhesion of bacteria colonized the site where was addicted easily before. The results showed that the drug sensitivity of mutant have raised, therefore, this study provided the basis for the development of new antimicrobial agents. |
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