Development Of Transmissible Gastroenteritis And Porcine Epidemic Diarrhea Bivalent Inactivated Vaccine And Evaluation Of Immunogenicity

Porcine transmissible gastroenteritis(TGE)and porcine epidemic diarrhea(PED)are acute and high contagious diseases,which are caused by the porcine transmissible gastroenteritis virus(TGEV)and porcine epidemic diarrhea virus(PEDV)respectively.Porcine transmissible gastroenteritis and porcine epidemic diarrhea showed symptoms of diarrhea,vomiting,dehydration,all of which can lead to death of piglets and infected adult swine body weight loss,reduced feed efficiency,increase the cost of human drugs,causing huge economic losses to the pig industry.Currently,vaccination is an effective method of controlling both diseases.To develop a vaccine for targeted the pandemic strain has practical significance for control this two kinds of disease.In this study,we developed a porcine transmissible gastrornteritis-porcine epidemic diarrhea bivalent inactivated vaccine successfully with TGEV A/E strains and PEDV A/D strain,and studied its immune effects.First,in order to study the proliferation dynamic of TGEV in the PK-15,the TGEV was inoculated onto PK-15 cells,and cell suspension and supernatant were obtained at Oh,811,16h,24h,32h,40h,48h,56h,64h,72h,80h.After inculation respectively,their titers were detected by the method of TCID50,each checkpoint set up three parallel controls.After finishing the data we plotted TGEV dynamic curves in PK-15 cells and analyzed comparatively.The results showed that:The proliferation reached a peak at 48h after TGEV inoculated into PK-15(TCTD50 for 10-6.83/mL),and the peak can sustain to 56h after detoxification(TCID50 for 10-6.5/mL).From the test we can see that the virus can be harvested in 48-64 hour when TGEVwas proliferated on the PK-15.Second,TGEV A/E strains and PEDV A/D strains were used as bivalent inactivated vaccine strains and subcultured on PK-15 cells and VERO cells respectively,amplified high titers virus solution,prepared oil emulsion inactivated vaccine,add the prokaryotic expression of PEDV pandemic strain S genetic before 1077 bases of protein expression SA.Then vaccine physical traits detection,sterility testing,safety inspection and animal immunity test were conducted.Through the trace serum neutralization test and ELISA test get the antibody levels of the experimental pigs after immune.Then process the data by SPSS 17.0.Results showed that the vaccine is qualified;No side effects in mice and pigs security;Will TGE-PED duplex inactivated seedlings points high,medium and low dose immune pigs found between high,middle dose group antibody levels close to,the data of two groups are significantly higher than the low dose group and significant difference(P<0.05),to determine the best immune dose for 2ml/head.The antibody level of TGEV reached the highest after Secondary immune four weeks,neutralizing antibody detection test results and ELISA test results respectively were 1:319 andl:1280,the antibody level of PEDV reached the highest after Secondary immune three weeks,neutralizing antibody detection test results and ELISA test results respectively were 1:512 and 1:1600,have the same antibody level with the TGEV single vaccine(1:319 andl:1280)and the PEDV single vaccine(1:512 and 1:1600),no significant difference(P>0.05),but were higher than the antibody level of commodity vaccine.SA proteins can be induced the low antibody level of PEDV,but the group of bivalent inactivated vaccine add SA protein have the higher ELISA antibody level than the TGE-PED bivalent inactivated vaccine group.The trace serum neutralization test and ELISA test have the same ebb and flow curve of antibody level.Both showed the higher antibody levels of TGEV and PEDV can sustainable to three months after the second immunization.The above results suggested,this TGE-PED bivalent inactivated vaccine got the good effect,provided an alternative vaccine to prevention and control these two kinds of disease.