Construction And Immunoprotective Effect Evaluation Of The Recombinant Lactococcus Lactis Expressing Grass Carp Reovirus VP6 Of Genotype Ⅱ

Grass carp(Ctenopharyngodon idella)is the highest produced freshwater aquaculture fish in our country,which has an important economic status.Grass carp hemorrhagic disease(GCHD)caused by grass carp reovirus(GCRV)has a mortality rate of up to 80%and there is currently no effective treatment,which brings seriously economic challenges to grass carp farming industry.GCRV has divided into three genotypes,and the main is GCRV-II.Vaccination is the most effective way to protect grass carp against GCRV infection.In this study,we evaluated the physicochemical properties and probiotic ability of Lactobacillus in the mucosal system of healthy grass carp.At the same time,the standard strain of Lactobacillus was used as a vector to construct a recombinant Lactobacillus displaying GCRV VP6 protein on the surface.The protective effect and immune mechanism of the vaccine was evaluated by rare minnow which as laboratory vaccines evaluation model.The main contents and results are as follows:(1)Isolation and identification of Lactobacillus from grass carp and its preliminary study on aquatic disease prevention and control.In this study,8Lactobacillus strains were isolated from the intestines of the healthy grass carp.The results of biochemical reaction and 16S r RNA identification showed that they were all L.plantarum.And then,the isolate strains Y190430 has good fermentation performance and good antibiotic sensitivity.In addition,L.plantarum Y190430 has a good tolerance to environmental stresses such as acid,alkali,salt and temperature.Besides,it also has a better acid production rate,which has more ability than the standard strain to inhibit the growth of pathogenic bacterias.All results show that Lactobacillus have the potential to be used as an ecological control for aquatic diseases.(2)Construction and identification of Lactobacillus oral vaccine of GCRV VP6genotype II.We analyzed that the functional domains of GCRV VP6 by bioinformatics methods and selected the N-terminal gene of VP6 to construct a recombinant plasmid p NZ8148-VP6,which was transformed into L.lactis NZ9000 by electroporation to obtain a recombinant Lactobacillus,named p NZ8148-VP6/L.lactis We determined the optimal induction condition for the expression of recombinant Lactobacillus by SDS-PAGE,which was 10 ng/m L Nisin,and was cultured at 28°C for 4 h.We identified the expression and localization of recombinant Lactobacillus target protein by Western blot and indirect ELISA.The results showed that the fusion protein of about57.6 k Da was expressed on the cell surface,and the size of the protein was consistent with the theoretical value.The successful expression of VP6 protein laid the foundation for the recombinant Lactobacillus oral vaccine to resist GCRV infection in experimental animals.(3)The immunoprotective effect of Lactobacillus oral vaccine against GCRV VP6 genotype II.We selected rare minnow as the vaccine evaluation model,and the dose of 10μL 2×10~9CFU/m L/d per fish was continuously administered by oral for 3days,with an interval of 2 weeks,and the effect was evaluated by immunization twice.The blank group and the Lactobacillus group were set as controls.We collected the hindgut tissue at 3 h,6 h,12 h,24 h,48 h and 72 h after the first immunization for viable counts to determine whether the recombinant Lactobacillus could stay in the hindgut for a long time for antigen presentation.On 7 d,14 d,21 d and 28 d after vaccination,we collected the spleen,kidney and hindgut tissues and determined the expression levels of immune-related genes in each tissue by RT-q PCR.We collected blood serum at 3 w and 6 w after immunization and determined the level of specific antibody Ig M by ELISA.On the 42 d,we conduct a challenge of GCRV by intraperitoneal injection,and record and calculate the mortality of the fish every day for14 days.The results showed that the recombinant strain could stay in the hindgut of the rare minnow for at least 72 hours for antigen recognition and presentation.The survival rate of rare minnow in the immunized group after challenge was 46.7%,and the RPS was 42.9%.In addition,the expression level of specific antibody Ig M in the serum of the immune group was significantly higher than the blank group and the Lactobacillus group(p<0.05).At the same time,the results showed that oral p NZ8148-VP6/L.lactis can stimulate the innate immune response and adaptive immune response,and has a protective effect on the rare minnow against GCRV infection.In this study,Lactobacillus with antagonism to aquatic pathogens was isolated from the intestinal tract of healthy grass carp.We used the standard strain of Lactobacillus as the carrier and successfully constructed the recombinant Lactobacillus with the GCRV VP6 protein displayed on the surface.And we used rare minnow as an oral vaccine laboratory evaluation model to evaluate the immune protection effect and initially studied the mucosal immune mechanism of the recombinant Lactobacillus,which laid an important foundation for the subsequent development of GCRV-II probiotic oral vaccine.