The Study On The Characteristics Of Aeromonas Sobria Of Pelteobagrus Fulvidraco And The Cloning And Expression Of Its Hemolysin Gene

Aeromonas sobria,aslo called Aeromonas veronii bv.sobria,was one of the important pathogenicbacterias to endanger the fresh water breeding in Chinese.It could lead to hemorrhagic septicaemia of Fish,Reptile,Amphibia,and so on.And it was the important pathogenicbacteria of zoonosis,it could be raised the symptom of diarrhea,wound infection and septicemia.Research showed that the Aeromonas sobria was a kind of conditioned pathogens,and extensive indwell in vivo of Aquatorium,Soil and Aquatic animals.The major virulence factors were produced variety hemolysin and enterotoxin of pathogenicbacteria.They coused involute pathological changs of animal for toxins have variety biological activity.At present,we have not yet found the report about Aeromonas sobria of Pseudobagrus fulvidraco.This study indentified of the pathogenicbacteria what isolated from the vivo of the death Pelteobagrus fulvidraco by biochemistry and molecular biology indentification.At the same time,we carried out the study of biological characteristics,pathogenicity test and drug susceptibility test of pathogenicbacteria.Also cloning and expressing the gene flagment of hemolysin that was the primary pathogenic agents of isolation strain.1 Indentification of the isolation pathogenicbacterias of Pelteobagrus fulvidracoIn our study,we isolated two bacterial strains from vivo of the death Pelteobagrus fulvidraco. We obtained strains RC-07-KA and RC-07-XB by sublimated them with culture mediums.They were identified as Aeromonas sobria through morphological characters,cultural characters,physio-biochemical properties and the method of cloning system of bilchemistry identification of vibrio bacteria.At the same time,a 1431bp gerie fragment of 16S rRNA gene was amplified by PCR. The result showed that have nearly sibship that compared 16S rRNA gene sequence of isolation strain with others reported Aeromonas sobria in Genbank,and the homology of attain above 97%.2 The biological properties study of isolated strain of Aeromonas sobriaIn this study,Crucian and Pelteobagrus fulvidraco was used to pathogenicity test,and cultured the isolation strain RC-07-KA of Pelteobagrus fulvidraco with difference pHs and temperatures to filter the optimum growth conditions of pathogenicbacteria.And researched the sensibility of isolation strain with 20 antibiotics and 6 antiseptics.The results showed that the Aeromonas sobria have stronger pathogenicity with Crucian and Pelteobagrus fulvidraco.The minimum lethal dose of Crucian and Pelteobagrus fulvidraco was 1.7×10~7CFU and 1.95×10~6CFU.The optimum pH among 7.0 to 7.5 of pathogenicbacteria growth,and the optimum growth temperature was about 30℃.Drug susceptibility test showed that,among 20 antibiotics,Amikacin,Ciprofloxacin,Piperacillin, Piperacillin,Kalamycin,Selectrin,Gentamicin have the most jnhibitive effeet on the strain.And 6 antiseptics have obvious bacteriostatic action with pathogenicbacteria,in particolare Formaldehyde,Glutaraldehyde,and so on.3 Cloning and expressing the hemolysin gene fragment of isolation strain of Aeromonas sobriaAccording to the published hemolysin nucleotide sequence from human Aeromonas sobira in Gen-Bank,a pair of primers that contain BamHⅠand HindⅢenzyme sizes were designed and synthesized,which were localized in conservative regions of hemolysin gene.A 830bp gene fragment was amplified via PCR that was used the genome DNA of isolation strain of Pelteobagrus fulvidraco,then constructed the recombinant plasmid pMT-hly.Nucleotide sequence analysis indicated that the gene was 830bp in length and homology of nucleic acids between this gene and other similar gene in GenBank was 98%.The recombinant expression plasmid pMT-GEX-hly was constructed with pGEX-KG and transformed into E.coli BL21(DE3).Then the recombinant strain was obtained and SDS-PAGE analysis indicated that the hly gene was expressed about 57KD protein when the recombinant strains was induced by IPTG.This study could amplify the hemolysin gene fragment of Aeromonas sobira of Pelteobagrus fulvidraco,and highly active expressed the hemolysin protein,it showed that the human and Pelteobagrus fulvidraco Aeromonas sobira have highly homology.