Expression Of Porcine Epidemic Diarrhea Virus Antigen Gene In Tomata And Animal Immunoassay

Our country is a big agricultural country,pig breeding in country's agricultural production occupies a important position,related to the national economy and people's life.Porcine epizootic diarrhea(PED)is a type of contact intestinal infectious diseases seriously acting the healthy development ofpig industry,which is characterized by diarrhea,vomiting and dehydration.The most effective method for PED prevention and control is vaccination.The rapid development of molecular biology and plant genetic engineering technology further highlights the advantages of transgenic plant vaccine,such as low cost,higher safety and easier operation.Transgenic exogenous protein is relatively stable and can be used as oral vaccine or feed supplement.In this study,PED virus antigen gene was transferred into tomato genome by plant binary expression vector and agrobacterium-mediated method,to make the immunogenic gene expressed in tomato genome;then,an immunological test was conducted for mice and piglets.The study on the antibody level and immunogenicity provided strong theoretical evidence for the development and production of plant genetic vaccines for PED virus.The transgenic exogenous proteins are relatively stable in inheritance,and they can be taken as oral vaccine or feed additive;therefore,there is a broad prospect for development.In this study,the construction of plant expression vector,integrated expression of pathogen in tomato,and animal immunity experiment were mainly studied.The results are shown as follows:1)The plant biome expression vectors p BI121/S1 B and p BI121/S1 D of structural protein S1 gene of porcine epidemic diarrhea virus He NPEDV?01 were constructed.The genomic initiation codon of S1 B and S1 D antigen region was ATG,and S1 B encodes 720 nucleotides in total.A total of 1,140 nucleotides were encoded in S1 D,and the nucleotide homology between S1 B and S1 D gene sequences and the new He NPEDV?01 was 100% and 99.8%,respectively,through sequence alignment analysis of nucleotides.2)Construction of tomato expression system of transgenic plants.According to the preference of tomato codon,sequence optimization of exogenous target antigen genes S1 B and S1 D was conducted,the endoplasmic reticulum guided polypeptide sequence SEKDEL was introduced at the 5 'end of the fragment to improve the accumulation of tomato protein,and Kozak sequence was added at the 3' end to improve the translation efficiency.In this paper,using cauliflower Mosaic virus Ca MV 35 S as the promoter,p BI121/S1 B and p BI121/S1 D transgenic tomato expression vectors of porcine epidemic diarrhea virus S1 gene were successfully constructed.3)Optimization and detection of tomato transformation system of transgenic plants.The somatic cells of plants have a strong ability of regeneration and differentiation,and the regeneration produces buds and grows into complete plants.In this experiment,tomato transformation conditions were optimized on the basis of the existing studies.IAA0.1 mg /L and ZT1.0 mg /L were added to the MS medium,and agrobacterium agriculturum and cotyledon were infected for 3 to 5 minutes.The transformed tomato plants were examined by the enzyme-linked immunosorbent assay(ELISA)showed that the positive p BI121/S1 B Kana-resistant plants accounted for about 40%,and the success rate of p BI121/S1 D Kana-resistant plants was 25%.The study showed that after integrating the PED immunogenic genes S1 B and S1 D into tomato genome,the expressed proteins would have immunogenicity.4)Animal immunization experiments.The mice were immunized by tomato leaf protein extractcontaining the antigen gene,and 10 d after the first immunization,there were no PEDV antibodies;10 d after the second immunization,ELISA found the antibody level and the first peak;21 d after the third immunization,ELISA found that the antibody level was significantly increased,and the second peak appeared,where 21 d after immunization with p BI121/S1 B,the serum titer reached 1:64 at the highest and the immune protection rate for strong poison attack was 80%;21 d after immunization with p BI121/S1 D,the serum titer reached 1:128 at the highest,and the immune protection rate was 72%.5)Animal regression experiment.As shown in the experiment regarding the effect of transgenic tomato containing antigens on piglets,the transgenic edible tomato vaccine could resist the virus infection and improve the survival rate of the piglets.The piglet infection experiment based on different viruses showed that PRV,PRRS and TGE had the same effect on virus replication as PBS,while S1 B,S1D and PEDVvaccines had relatively low virus titer.After 21 d of infection,the spleen,liver,lung,and salivary gland progeny particles were respectively 1,000,2,000,4,000,and 15,000 times lower than those of the control group,while transgenic tomato vaccine immunity was respectively 100,200,800,and 1,000 times lower.Therefore,S1 B and S1 D of porcine epidemic diarrhea virus S gene can stimulate the body to produce immune response and resist virus infection in vivo.In summary,the transgenic tomato binary expression vector of S genes p BI121/S1 B and p BI121/S1 D of PED virus was successfully constructed,and the transgenic tomato plants were successfully transfected with agrobacterium tumefaciens GV3101 in this study.As shown in the experiment immunizing mice and piglets with transgenic tomato leaves containing antigens,the mice and piglets had good immunogenicity in vivo,which indicated that transgenic tomatoes could induce local immune response and systemic immune response in vivo,thus providing theoretical support forscale production of PEDV transgenic plants.