Cloning, Expressing Of Tdh,Trh And Tlh Genes And Analyzing Of Hemolysis Of These Gene Deleted Mutants Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus has been listed one of the pathogenic vibrios endangering the net-cage cultured Pseudosciaena crocea, Fennropenaeus chinensis and shellfish in coastal areas of China. Several kinds of hemolysin produced by Vp are believed the major virulence factors. These are thenmostable direct hemolysin, TDH-related hemolysin and thermolabile hemolysin. In this study, tdh,trh and tlh three genes were cloned from the genome DNA of Vibrio parahaemolyticus and expressed in prokaryotic expression system. The purified proteins were activated and showed the hemolytic activity after renature by gradient dialysis. At the same time, The skeleton plasmid pMD18-T-neo of targeting vector was successfully constructed. The gene knockout studies were carried out separately. The three gene deleted mutants were identified by PCR and hemolysis were identified too. It indicated that knockout of single gene had no influence on hemolysis of VP. The follo wings are the details.(1) We cloned tdh,trh and tlh genes from the genome DNA of Vibrio parahaemolyticus by polymerase chain reation. The three genes were ligated into prokaryotic expression vector pET-28a(+), the recombinant plasmids were transformed into E. coli BL21 and induced to express the recombinant proteins by addition of IPTG. The recombinant proteins were expressed in fusion bodies and purified with Ni-IDA affinity chromatography. The three purified proteins showed expected MW in SDS-PAGE,27 kDa,27 kDa and 50 kDa, respectively, accordant with the calculated value. Western blotting results showed that recombinant proteins, TDH, TRH and TLH would reacted with rabbit antiserum immunitied by VP antigen. Because the fusion proteins were expressed as inclusion bodies, the three purified proteins were renatured by gradient dialysis. Except TLH in the addition of phmphatidyehdine, The renatured proteins were activated and showed the hemolytic activity.(2) To construct skeleton plasmid of targeting vector, the neomycin resistance gene (neor) including self-promoter was amplified by PCR with pEGFP-Nl plasmid DNA as the template. The amplified DNA fragment was inserted into cloning vector pMD18-T, and recombinant plasmid pMD18-T-neo was successfully constructed.(3) The long and short arms of targeting vector were cloned. Restriction enzyme cutting sites were introduced in the 5'flank of the primers of homologous long and short arms of tdh, trh and tlh genes. These homologous arms were amplified by PCR and inserted into vector pMD18-T simple, Then these DNA fragments were ligated into targeting vector in certain order. Two ligation methods were chosen. The first method:at first, skeleton plasmid pMD18-T-neo with single enzyme digestion separately, were treated with Alkaline Phosphatase (CIAP), then it was ligated with homologous short arm. In a similar way, homologous long arm was ligated, thus targeting vector was successfully constructed; The second method:big linear pMD18-T fragment was recovered after skeleton plasmid pMD18-T-neo was digested, then small linear neor gene. The two recovered fragments were ligated with homologous long and short arms, thus targeting vector was successfully constructed. The constructed targeting vector was linearized and electroporated into VP cells. Positive transformers were screened from the neor resistance plate. The tdh, trh and tlh gene deleted mutants were identified by PCR. Hemolysis of tdh, trh and tlh gene deleted mutants were separately identified on 5% rabbit blood agar plate. The results suggested that the there gene deleted mutants could all induce hemolysis. It indicated that knockout of single gene had no influence on hemolysis of VP. Whereas, Hemolysis of tlh gene deleted mutants was identified on 5% horse blood agar plate, it could not induce hemolysis. The result suggested that tlh gene was knockouted successfully.In this study, we cloned and expressed tdh,trh and tlh genes of VP, and prepared Polyclone antibody using purified proteins. The successful cloning and expressing of these genes made it possible to describe these genes' function under asingle factor level, and also provided technical support for developing an advanced gene engineering vaccine against VP. Moreover, tdh, trh and tlh gene deleted mutants were constructed by homologous recombination and their hemolysis were identified to discuss molecular mechanism of hemolysis of VP. At the same time, an effective method in which unknown functional genes in VP genome were researched was provided.