Efficient Expression And Identification Of Vibrio Vulnificus Cytolysin Genes In Escherichia Coli

Objective:Vibrio vulnificus is an estuarine gram-negative,halophilic bacterium that is naturally present in coastal waters and the gulf of the water and seabed sediments.Vibrio vulnificus is a new food poisoning,its occurrence and the consumption of raw or cooked shellfish processing at the end of oysters,and can be through the damaged skin or gastrointestinal mucous membrane into the body and infection.The infection caused by this bacterium has attracted special interest because of its powerful rapid development.It can be extended the eventual development of the septic shock diseases,multi-functional organ failure, and then died.Mortality from this infection is more than 70%and death may occur within 1 to 2 days after the first signs of illness.A variety of endotoxins and exotoxins,including a cytolytic hemolysin,lipopolysaccharide,polysaccharide capsules,an elastolytic protease,and aphospholipase A2,have been implicated as virulence factors for this organism.Although a definite role of cytolysin in bacetrial infection is controversial,Vibrio vulnificus cytolysin is still one of the prime candidates in the pathogenesis of disease.To construct expression vector of Vibrio vulnificus cytolysin genes so that we can expression and identification them in the E.colis,which can provide a basis for further study.Method:In this paper,the cloning and expressing of the cytolysin of Vibrio vulnificus were discussed.A pair of prmiers were designed according to the GeneBank published nucleotide sequence of we.The action site of BamHâ… was added to 5' terminal of upper primer and Hindâ…¢to the lower one.Vvc gene was cloned by polymerase chain reaction. This result was tested by 1%agarose electrophoresis,and the length of polymerase chain reaction production accorded with the anticipation.With the cleavage of BamHâ… and Hindâ…¢,the target fragment was inserted oriently into pET-32a(+)prokaryotic expression vector. After enzyme restriction and sequence analysis,the nucleotide data had been further analyzed. The recombinant plasmid was transformed into E.coli BL21(DE3).After 1.0 mmol/L IPTG induce expressed 6 hours,we used SDS-PAGE to analyze and mouse-anti-vv antibody to have Western blot identification.Results:The nucleotide sequences of the cleavage of enzyme restriction was matched with the NCBI GeneBank reported.It analysis showed that this sequences were composed of 1311bp encoding mature peptide of 437 amino acids.SDS-PAGE analysis suggested that the VVC protein had efficient expressed,the molecular mass of fusion protein was 71kDa.Gray scanning software suggested that fusion protein was about account of 32%of total protein in VVC.Antibody against three Vibrios was got through acquisition mice serum.Then these serums was diluted with 1:200.The western blot result suggested that the prokaryotic expression VVC fusion protein can particularly combined with Vibrio vulnificus anti-serum.Conclusion:We not only successful gained the vvc genes,but also constructed a recombinant expression plasmid,pET32a(+)-vvc plasmid,which was efficiently expressed in E.coli BL21(DE3).Expressed VVC protein can be used for understanding its biological activity,preparing its diagnostic kits and laying the foundation on its pathogenic mechanism.