Immunoprotection Of The DNA Vaccine Of Transmissible Gastroenterits Virus S1 Gene In Combination With Porcine Interleukin-12 In Swine

Transmissible gastroenteritis virus(TGEV)can cause acute highly infectious enteritic disease transmissible gastroenteritis(TGE).Vomiting and severe diarrhoea and dehydration are the main symptoms of the infected animals,and often mixed with other diseases.Pigs of various breeds and ages are susceptible,newborn piglets are especially serious.Mortality rate gradually decreases with increasing age.Spike protein of the TGEV is protuberant on the surface of the virus,and it mediates the adhering to host receptors and membrane fusion,the S protein could be divided into S1 and S2 regions,it is a major target for neutralizing antibodies.Since 1990s,TGEV caused serious economic losses worldwide.Some vaccine preparation technologies have been developed and commercialized.At present,China mainly applied a trivalent attenuated vaccine of TGEV,porcine rotavirus and porcine epidemic diarrhea virus,to combat the infection of TGEV in pig industry,however,it had the disadvantage of virus dissemination,virulence reversion,etc.It is particularly urgent to develop new kind TGEV vaccine with higher effenciency,and now the new genetic engineering vaccines have been the highlighted poine in the related research field.Interleukin-12(IL-12)is an important multifunctional immunoregulatory cytokine.IL-12 can induce IFN-gamma production and induce CD4~+T cells to differentiate into Th1(T helper cell)type cells.IL-12 has many important biological functions,which are related to early nonspecific innate immunity and subsequent acquired immunity of specificity antigen.IL-12 has been regarded as an important candidate acting as immune adjuvant.This study investigated the distribution of TGEV infection in 60 pig farms in Fushun,Liaoning Province.Three hundreds samples of piglets with diarrhea symptoms were collected.The infection of TGEV was investigated by RT-PCR,and the S gene of the key gene of TGEV was analyzed by bioinformatics.Through molecular biology technology,pVAX1 vector was used to construct TGEV Purdue 46 strain S1 gene and pig IL-12(pIL-12)gene eukaryotic expression plasmid pVAX1-TGEV-S1 and pVAX1-pIL-12-(P40)-(p35)respectively.Two plasmids were transfected into BHK cells by liposome transfection technology,and their protein expression was detected by immunofluorescence assay.Twenty 7-day-old piglets were randomly divided into five groups:pIL-12+TGEV-S1 group,TGEV-S1 group,pIL-12 group,pVAX1 group and blank group.From 7 days of age,they were immunized with recombinant eukaryotic expression plasmids pVAX1-TGEV-S1 and pVAX1-pIL-12-(p40)-(p35).The immunization interval was 14 days.Blood samples were collected every 7 days.Pigs were killed 5 days after challenge with TGEV Purdue 46and blood and histological samples were collected.Samples were analyzed with lymphocyte proliferation test,flow cytometry,enzyme-linked immunosorbent assay,virus neutralization test,tissue frozen section and immunofluorescence assay and HE staining.Cellular immunity,humoral immunity and immune protection were measured in piglets of different groups.The RT-PCR results showed that three positive samples were detected in 300 samples from 60pig farms in Fushun City,Liaoning Province.By cloning and sequencing of S gene,structural analysis,sequence homology analysis,phylogenetic tree analysis and protein structure analysis,compared with the S gene of 17 other reference strains,five nucleotide mutations were found and resulted in four amino acid changes.The change of amino acid at the site leads to the change of secondary structure of protein,which has the highest homology and affinity with S gene of WH1and Almazan strains.It belongs to Purdue strain and is similar to the prevalent strains.The eukaryotic expression plasmid pVAX1-TGEV-S1 and pVAX1-pIL-12-(P40)-(p35)of TGEV Purdue46 strain S1 gene and pig IL-12 gene were constructed.After transfection of two plasmids into BHK cells,the expressed protein could be detected by its specific polyclonal antibody by the immunofluorescence assay.In animal immune protection test,the immune groups of TGEV-S1 group and pIL-12+TGEV-S1 recombinant expression plasmid increased in different degrees in different immune indicator detection,compared with blank group and pVAX1 group,there were significant differences,especially in pIL-12+TGEV-S1 group.In lymphocyte proliferation test,detection of specific IgG antibody to TGEV S1 and TGEV neutralizing antibody test,TGEV-S1 group and pIL-12+TGEV-S1 group increased significantly,especially in pIL-12+TGEV-S1 group,which was significantly different from that of TGEV-S1 group(p<0.05),and significantly different from blank group,pVAX1 group and pIL-12 group(p<0.01).CD4~+T lymphocyte detection,CD8~+T lymphocyte detection,IFN-gamma cytokine detection,cytotoxic T lymphocyte detection,pIL-12 group,TGEV-S1 group and pIL-12+TGEV-S1 group were significantly increased,especially in pIL-12+TGEV-S1 group,compared with blank group and pVAX1 group,the difference was very significant(p<0.01).Immunofluorescence detection and histopathological examination showed that there was a significant difference between the TGEV-S1 immunized group and the control group,in which the adjuvant and TGEV-S1 combined immunization group had fewer viruses and less tissue damage.It is suggested that pIL-12 as an immune adjuvant has an enhanced effect on immune response of piglets,and can enhance the immune protection effect of pVAX1-TGEV-S1 eukaryotic expression plasmid.In this study,the recombinant eukaryotic expression plasmid of TGEV S1 gene and pIL-12gene was used to immunize piglets for the first time.The results showed that the combination of adjuvant pIL-12 and TGEV S1 gene could obviously promote the cellular immune response and play a role in the humoral immune response,thereby improving the host immune level againsts viral infection.It provides a theoretical basis for the further study of the immune function and mechanism of the new nucleic acid vaccine and cytokine immune adjuvant.