Development Of A System For Detection Of Pathogenic Edwardsiella Tarda By PCR Assay And Cloning And Expression Of The Virulence Gene MukF

7 virulence genes( orfA,citC,fimA,gadB,katB,mukF,esrB) were identified in overseas isolation strains of pathogenic Edwardsiella tarda by southern hybridization. The primers of orfA gene couldn't be designed for its sequence had been not published .Six pairs of primers were designed according to the other six published nucleotide sequences. PCR was developed to detect the distribution of above virulence genes in domestic isolation strains in order to perform the detection system of pathogenic E.tarda. The result indicated that citC,fimA,gadB,mukF genes were detected in domestic isolation strains of pathogenic E.tarda, and katB and esrB genes were not. The same result also appeared with the primers of katB and esrB genes according to the correlative references .The other four pairs of primers were specific in other familiar pathogenic bacterium detection. In the sensitivity assay, the sensitivity of gadB gene reached 100fg/μL of concentration of template DNA and others reached 10pg/μL by simple PCR. It was the same result in the sensitivity assay between simple PCR and duplex PCR. And the sensitivity of gadB/fimA was a little better than that of citC/mukF. There was different in the virulence genes distribution between oversea and domestic isolation strains. GadB gene and mukF gene presented in all domestic isolation strains ,which detected in the test.The positive rates of fimA and citC genes were 8/13 and 7/13 respectively. The results indicated fimA/gadB was used to detected the pathogenic Edwardsiella tarda.The mukF gene was considered as the primary virulence gene,playing a important role in killing function.It is valid to prevent and cure the Edwardsisellosis by cloning and expression the mukF gene to develop vaccine.A pair of primers with restriction enzyme cutting sites were designed according to the published nucleotide sequence (AY078510) .With the specific primers, a target fragement about 540bp was amplified from domestic isolation strain. The target fragement was inserted oriently into pblue vector.By using sequence analysis , there were 87.8% homology between the cloned gene of the domestic strain and the standard strain.The nucleotide sequence was predicted to encode a 180 amino acid protein .There were 12 amino acid sites changed .which distributing center of the sequence equably. The alignment of the amino acid sequence was 93.3%. The recombinant plasmid was transformed into E.coli BL21.The fusion protein was expressed under the IPTG inducing condition.exhibited a protein band with 29KD in size on SDS-PAGE gel, which almost matched to the predicted molecular weight of pET-mukF fusion.The results indicated the fusion protein was high-expressed under with 0.05mmol/L IPTG by 5h .The successful cloning and expression the mukF gene made it possible to study its fusion under a single factor level.and provided technical support for developing an advanced gene vaccine against Edwardsisellosis.