Preparation Of Oral Subunit Vaccine Of Recombinant Lactobacillus Casei Of Grass Carp Reovirus And Evaluation Of Immune Effect

Grass carp(Ctenopharyngodon idella)is one of the most important freshwater farmed fishes in my country.Grass carp hemorrhagic disease caused by grass carp reovirus(GCRV).Seriously endanger the development of grass carp breeding industry in our country.It is a viral disease that needs to be tackled.GCRV can be classified into type I,type II and type III according to genotype.Grass carp type Ⅱ reovirus is the main epidemic strain that causes grass carp hemorrhagic disease in my country,and its fatality rate is as high as 80%.Effective vaccines are urgently needed for prevention.The VP56 protein encoded by the S7 sequence is the fibrin on the outer capsid of GCRV,which has been proved to have good immunogenicity through experiments.Lactic acid bacteria(LAB)are common bacteria in the intestines of bony fishes and mammals,and are rated as green and safe probiotics.LAB can efficiently express foreign genes and load recombinant proteins into the outer membrane of bacteria to obtain better immunogenicity.Compared with other carrier strains,LAB has stronger acid-base salt tolerance,which makes it exert more stable immunity in the intestinal tract.The oral subunit vaccine prepared by mixing recombinant lactic acid bacteria and feed has a high survival rate in fish,can stably present antigen proteins,is convenient to use,has low cost,and has extremely high application potential.This research work is divided into two parts: the preparation of GCRV-VP56 oral subunit vaccine and the detection of the effect of live grass carp oral vaccine.1.Preparation of Lactobacillus casei p PG612-VP56/L.casei 393 vaccineIn this study,we selected the VP56 protein encoded by the S7 sequence in GCRV type Ⅱ as the antigen to prepare the subunit vaccine.According to the laboratory’s existing recombinant plasmid containing the full-length VP56 gene as a template,the full-length VP56 gene was amplified.Choose Bam H I as the restriction site and insert it into the expression vector p PG612.1 using the one-step cloning method to form the recombinant plasmid p PG612-VP56(p VP56).The p VP56 was electroporated into competent Lactobacillus casei to obtain recombinant Lactobacillus casei p PG612-VP56/L.casei 393(p VP56/L.casei 393).Optimize the expression conditions for the expression of VP56 protein,detect the expression of VP56 protein by SDS-PAGE and Western blotting experiments,and observe the position of VP56 protein expression in Lactobacillus casei using a fluorescence microscope.The results showed that the complete gene sequence of VP56 with a size of 1539 bp was successfully amplified,and successfully ligated into the p PG612.1 vector,and the recombinant plasmid p VP56 was successfully constructed.SDS-PAGE and Western blotting experiments showed that VP56 protein was expressed in the supernatant and pellet of p VP56/L.casei 393,and the protein expression in the pellet was more than that in the supernatant.A time gradient was set to induce p VP56/L.casei 393,and it was found that as the induction time increased,the band of interest gradually deepened.Indirect immunofluorescence analysis showed that VP56 protein was expressed in the outer membrane of Lactobacillus casei.So far,p VP56/L.casei 393,which can express VP56 protein,has been successfully prepared.The induced expression of p VP56/L.casei 393 was mixed with grass carp feed to prepare an oral vaccine for in vivo effect testing.2.Detection of the live immune effect of the vaccineIn order to explore the actual effect of the vaccine,we divided 400 grass carp into 8 groups,named L.c-p PG612 group,L.c-p PG612-VP56 group,L.casei 393 group,and PBS group.Each group of 50 grass carp,each group of tanks are independently cultured.On days 1,3,7,14,17,21,24,and 28,3 fish were randomly selected from each group for sampling and testing.Through specific antibody level detection,viral load detection,quantitative immune-related genes,in vivo protection rate analysis and other methods to obtain experimental data for each group,verify the feasibility of preparing the recombinant Lactobacillus casei p VP56/L.casei 393 into an oral vaccine.The results showed that after the oral subunit vaccine was used to immunize grass carp,compared with other groups,the L.c-p PG612-VP56 group produced high levels of Ig M.Serum C3 content,lysozyme and TSOD activity increased in L.c-p PG612-VP56 group.Taking various tissue samples at different time points for q RT-PCR detection before and after the challenge,the L.c-p PG612-VP56 group can effectively activate the immune genes IL-1β,IFN1,TNF-α and MHC-II.The viral load test results showed that after the GCRV challenge,the viral load of the Lc-p PG612-VP56 group was significantly lower than that of the other groups,and with the increase in the number of days after the challenge,there was no upward trend,indicating that the vaccine has an effect on GCRV.The proliferation of the virus in the tissue plays an inhibitory role.Through mortality statistics,it was found that the protection rate of the L.c-p PG612-VP56 group was significantly higher than that of other groups.According to the analysis of pathological sections,compared with other groups,the L.c-p PG612-VP56 group had the least degree of tissue lesions and the least degree of virus damage.Overall.After immunizing grass carp with the oral subunit vaccine of Lactobacillus casei,it activates the humoral immune response of the fish body,produces specific antibodies,up-regulates the activity of immune-related enzymes,and activates related immune pathways.As a probiotic,Lactobacillus casei alone also improves the immunity of the fish.After the challenge,the use of oral vaccines slows down the damage to the organs caused by the virus attack,and achieves the effect of increasing the survival rate of the fish.It is proved that the oral subunit vaccine of Lactobacillus casei has the function of preventing GCRV Ⅱ infection of grass carp.It provides a solution for the preparation and practical use of oral vaccines for aquatic animals.