| Due to Vibrio alginolyticus is responsible for significant economic losses in the fish industry,it is urgent to develop a vaccine against Vibrio alginolyticus.Lactic acid bacteria as probiotics can improve aquatic environment,promote intestinal microbial balance and resist to disease.According to the advantage of probiotics and vaccine in this study,Lactic acid bacteria was isolated from the intestine of healthy grouper,and cloning the Vsco gene of Vibrio alginolyticus into the Lactic acid bacteria as a recombinant strain by molecular biology techniques,we studied the biological characteristics,safety and immune protection of the recombinant lactic acid bacteria.The main results and conclusions are as follows:1.The total of 3 lactic acid strains,name as ZJS01?ZJS02 and ZJS03,were isolated from grouper(Pearl Gentiana).Both physiological and biochemical methods and molecular biological techniques were used to determine that isolates ZJS01-03 were Lactococcus lactis.Safety assays revealed the Lactococcus lactis was not virulent for grouper.The results of antibiotic susceptibility showed that all strains were extremely susceptible to penicillins,tetracyclines,macrolides,clindamycin,chloramphenicol and cephalosporins and Resistance to aminosaccharides,quinolones,compound neotamine,polycandida B and aztreonam.Six pathogenic bacterias were used to measure the bacteriostatic activity of the three strains by agar diffusion method.The results showed the three strains hadn't antibacterial effect on Escherichia coli,but had significant effect on Aeromonas hydrophila,Vibrio alginnolyficus,V.parahemolyticus and V.harveyi,with the antibacterial circle diameter was greater than 21 mm on V.alginnolyficus and V.parahemolyticus.2.By the molecular biology techniques,the recombinant strain(Lc-pMG36e-Vsco)was constructed through cloning the Vsco gene into the E.coli/ Lactococcus lactis shuttle plasmid pMG36 e,name as Lc-pMG36e-Vsco.In order to verify the expression of Vsco gene in Lactococcus lactis by Western blotting,the results showed that18 kDa protein was expressed and had good reaction-ogenicity.the plasmid stability was evaluated,resules showed that the plasmid pMG36e-Vsco had good stability after culturing 45 generations continuously.3.In order to measure whether recombinant strain could induce Intestinal mucosa and systemic immune response,recombinant Lactobacillus Lc-pMG36e-Vsco was coated with granular bait and fed to Pearl gentian grouper.To evaluate immune responses,specific IgM,activity of relevant enzymes(ACP,SOD and lysozyme)were determined by ELISA in serum,showed that recombinant strain had high level of IgM in serum.And the activity of ACP?SOD and lysozyme were up-regulated.To measure the epression level of IL-1??IL-10?TNF-? and MHC-1? in relative tissue by Rt-Q PCR.The result indicated that recombinant strains had significantly improved the expression level of IL-1??IL-10?TNF-? and MHC-1? had significantly improved in the spleed and kidney for recombinant strain(P<0.05).Which showed constructive strain could improve immune responses,disease protection,and stimulate immunerelated gene expression.4.When the immunized grouper were challenged with V.alginolyticus HY9901,the elative percentage survival(RPS)of wild strain was 55% and recombinant strain was 68%.The result indicated that Both recombinant strains and wild strains could alleviate the infection of pathogenic bacteria and had certain protective effect.The immune effect of recombinant strains was higher than wild strains.In summary,constructive Lactococcus lactis,which could express Vsco gene of V.alginolyticus HY9901.The level of IgM and relative enzyme,the Relevant cytokines had significantly improve by immunized with constructive Lactococcus lactis.As the result,constructive Lactococcus lactis have capacity to improve immune response in grouper(Pearl Gentiana),and It provides the foundation for the development of recombinant lactic acid bacteria by oral vaccine against Vibrio alginolyticus. |
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