Preliminary Study On The Immunogenicity Of Recombinant Lactobacillus Expressing Porcine Epidemic Diarrhea Virus S1 Gene

Porcine epidemic diarrhea(PED)is an acute and contact infectious swine intestinal disease with watery diarrhea symptoms,which brings great harm to the pig industry.As it has no effective treatments currently,vaccination is the main control measure to prevent the occurrence of the disease.Lactobacillus is the most suitable living carrier for antigen presentation because of its acid resistance,bile salt resistance and safety.Studies showed that secretory antibodies(sIgA)produced by intestinal mucosal immunity were effective antibodies against PEDV infection.After oral immunization,sIgA antibodies could be detected in both intestinal fluid and serum.Piglets could obtain maternal sIgA from colostrum,resulting in passive immune protection against swine epidemic diarrhea.Therefore,it is important to choose a safe and non-toxic carrier system that can survive in the intestine and can express and deliver antigenic substances,to construct a vaccine that can effectively stimulate the mucosal immune system to produce mucosal immune response,which is of great significance for the prevention and control of porcine epidemic diarrhea.In this study,a recombinant expression plasmid carrying the S1 gene was constructed,and the S protein was successfully expressed in Lactobacillus and the immunogenicity of the recombinant Lactobacillus vaccine expressing the porcine epidemic diarrhea virus S1 gene was studied in mouse model.1 Construction of PEDV S1 gene recombinant plasmidReference laboratory PEDV EM-P strain has been determined sequence,and the hydrophobicity,homology and epitope of PEDV S1 protein were analyzed by bioinformatics software Protean,and the dominant antigen of size 307 aa was selected as the target protein.The cleavage sites Sal I and Eco R V were added to the 5'and 3'ends of the target gene,and codon optimization and gene synthesis were performed to construct the PUC57-S1 cloning plasmid.After double digestion of pVE5523 vector and pUC57-S1,the ligation was carried out to obtain a recombinant expression plasmid pVE5523-S1,which was electroporated and transformed into Lactobacillus plantarum to obtain recombinant Lactobacillus plantarum.PCR,SDS-PAGE and Western blotting were used to identify the recombinant Lactobacillus plantarum.The SDS-PAGE and Western blot results showed that the heterologous protein with a molecular mass of about 34 KD successfully expressed in Lactobacillus plantarum.The growth curve of the recombinant bacteria and the parental bacteria and the analysis of acid resistance and bile salt resistance showed that the transfer of the heterologous plasmid did not significantly affect the biological characteristics of the bacteria.The recombinant strain was passaged to the 5th generation,and its stability was studied.PCR analysis showed that the gene of the fifth generation could still be stably present in the recombinant strain.2 Preliminary study on the immunogenicity of recombinant bacteriaSafety experiments were carried out on MRS medium containing erythromycin,Lactobacillus plantarum containing plasmid pVE5523 and recombinant lactobacilli plantarum at a concentration of 10~9 CFU/mL and in a dose of 0.2 mL.The results showed that there were no significant clinical symptoms among the guinea pigs groups,indicating that the amount of this bacteria is safe for oral administration.MRS medium containing erythromycin,Lactobacillus plantarum containing expression plasmid pVE5523,Lactobacillus plantarum containing recombinant plasmid pVE5523-S1 were orally immunized three times for three consecutive days,immunization of guinea pigs with PED-TGE bivalent attenuated vaccine thigh muscle as a positive control group,three times of immunization.Feces were collected at 0d,7d,14d,24d,31d,41d,and 48d after immunization for sIgA detection,and cardiac blood was collected for ELISA antibody detection and neutralization test.The spleen cell proliferation test was performed on the spleen of guinea pigs on 48d.The experimental results showed that the vaccine could stimulate the body to produce secretory antibody sIgA and specific neutralizing antibodies,and also promoted the expression of IL-4 and IFN-?and the proliferation of spleen cells.