Cloning And Expression Of VscD Gene And Study On The Function Of VscB Gene In Type ? Secretion System Of Vibrio Alginolyticus

Vibrio alginolyticus is one of the major pathogens of marine aquatic animals and Vibriosis,it has caused serious damage to the aquaculture industry in the world.Therefore,effective control of Vibrio alginolyticus is of importance.The type III secretion system of Vibrio alginolyticus is closely related to its pathogenicity as a virulence factor.Vsc B protein and Vsc D protein are important components of type III secretion system,and it is speculated that they may play an important role in the process of invading the host.vsc B and vsc D genes of V.alginolyticus T3 SS were studied and vsc B and vsc D genes were cloned.The results showed that the vsc B gene has a length of 429 bp and encodes 142 amino acids with a theoretical molecular weight of 16.4 k Da and a p I value of 5.48.The vsc D gene is 1302 bp in length and encodes 433 amino acids with a molecular weight of47.9 k Da and a theoretical p I value of 4.98.The amino acid sequence homology comparison results showed that the Vsc B of vibrio alginolyticus had the highest similarity with that of Vibrio parahaemolyticus,reaching 91%.And the Vsc D of vibrio alginolyticus had the highest similarity with that of Vibrio diabolicus,reaching 97%.Vsc B and Vsc D proteins were analyzed by Western-blot,respectively.The results showed that the two protein was successfully expressed in Escherichia coli.Overlap PCR and homologous recombination technique were used to construct the target gene vsc B unmarked gene in-frame deletion strain and its complementary strain in combination with chloramphenicol(Cm)forward screening method and susceptibility of vmt gene to L-arabinose.The results showed that the growth curve,the extracellular enzyme activities and the biofilm formation ability of Vibrio alginolyticus did not significantly changed compared with the wild strain and the regenerative strain in the absence of vsc B gene.The test of bacterial swimming showed that the swimming ability of vsc B-missing strain was significantly lower than that of the wild strain.The toxicity of the deletion mutant strain was 23.2 times lower than that of the wild strain.But the toxicity of the complementary strain restored to the original level.The results showed that vsc B gene is related to the pathogenicity of Vibrio alginolyticus.The Epinephelus was immunized with the mutant strain ?vsc B as immunogen by intraperitoneal injection.After a month of immunization,the wild strains of Vibrio alginolyticus were used to attack the groupers.It was found that the immune resistance ability of the grouper was significantly enhanced,and the relative immune protection rate reached 77.6%.The results show that the mutant strain ?vsc B can be used as a live attenuated vaccine against Vibrio alginolyticus in fish aquaculture.