Expression And Preliminary Application Of Baculovirus Of S Gene A Site Of Porcine Transmissible Gastroenteritis Virus

Porcine transmissible gastroenteritis virus(TGEV)is an enteric coronavirus that causes high morbidity and high mortality in pigs worldwide,invading the intestine and respiratory tract.The main symptoms are vomiting,severe diarrhea,and dehydration.In order to control the occurrence and prevalence of the disease,it must have corresponding diagnostic techniques and vaccine immunization.S protein is a protective antigen of TGEV.It has four main antigen sites,A,B,C,and D.The A site is located on the surface of TGEV.Inducing factors,and play a key role,the deletion of this site can cause S protein can not produce neutralizing antibodies,so this site is the main object of research.In this study,for the TGEV S gene A site(abbreviated as Sa,the same below),a recombinant baculovirus expression vector was constructed and the fusion protein rBacmid-TGEV-Sa was successfully expressed,and a monoclonal antibody against rBacmid-TGEV-Sa fusion protein was prepared,the fusion protein was used as the coating antigen to establish the ELISA diagnostic method of TGEV antibody.The specific research contents are as follows1 Construction and identification of recombinant baculovirus expressing S Gene A Site of porcine transmissible gastroenteritis virusIn this study,a strain of porcine infectious gastroenteritis TZ-10-2016 saved in the laboratory was used as a template,and a pair of specific pairs designed and synthesized based on the sequence of the A gene site of the porcine transmissible gastroenteritis virus S gene in the study of Wang Xian and others Primers,the Sa gene sequence was amplified by RT-PCR,the TGEV-Sa gene was cloned into the pFast-bac-HTA baculovirus expression vector,transposed in DH10Bac competent cells,screened by blue and white spots and three times.After purification,a positive recombinant shuttle vector plasmid was finally obtained,and then the plasmid was transfected in Sf9 insect cells mediated by liposome LipofectamineTM 3000 to obtain recombinant baculovirus rBacmid-TGEV-Sa.IFA identification showed that the recombinant baculovirus fusion protein can be specifically recognized as cytoplasmic fluorescence by anti-porcine infectious gastroenteritis virus antibody.The results of SDS-PAGE and Western-blot showed that about 28kDa of soluble recombinant protein was obtained.It is specifically recognized by 6×His monoclonal antibody and porcine polyclonal sera and has good reactogenicity,which lays the foundation for antibody screening and establishment of serological detection methods.2 Preparation of Monoclonal Antibody of S Gene A Site of Porcine Infectious Gastroenteritis VirusThe immunogen used in this study was the recombinant baculovirus rBacmid-TGEV-Sa fusion protein prepared in Chapter 1.The 6-week-old Balb/c mice were immunized.After cell fusion,indirect immunofluorescence(IFA)was used for screening.After three fusions,five hybridoma cells capable of secreting monoclonal antibody(Mab)against TGEV were successfully obtained,named Mab-TGEV-2B 1,Mab-TGEV-2D1,Mab-T GEV-4E3,Mab-TGEV-5E3,Mab-TGEV-5G2.The subtype of these Mabs were identified Mab-TGEV-2B1,Mab-TGEV-2D1,Mab-TGEV-5G2 subtype as IgGl,and the other two subtypes as IgM.The monoclonal antibodies obtained after ELISA and Western-blot verification only recognize the TGEV virus antigen and do not cross-react with PEDV and PoRV.The ascites of Mab-TGEV-2B1 and Mab-TGEV-2D1 monoclonal antibodies was purified by Protein G column,which laid the foundation for the subsequent establishment of TGEV detection method.3 Establishment of an indirect ELISA method for detecting antibodies to porcine infectious gastroenteritis virusIn this study,the supernatant of recombinant baculovirus rBacmid-TGEV-Sa cell virus suspension after ultrasonic disruption was used as the coating antigen.Through screening the various reaction conditions of ELISA,the P/N value method was used to determine the antigen concentration.?g/mL,coated overnight at 4?,blocked with 5%goat serum for 2 h,diluted 1:200 in serum,incubated at 37? for 60 min in water bath,and incubated with enzyme-labeled secondary antibody(HRP-sheep anti-mouse)for 60 min at 37?,Color development for 15 min,as the final working conditions.22 negative sera of pigs were tested to determine the positive cut-off value was 0.476,and the negative cut-off value was 0.422,which was determined to be suspicious.The specificity,sensitivity,and repeatability tests confirmed that the ELISA method established in this study had good specificity,no cross-reactivity with other viruses,and good sensitivity.The coefficients of variation within and between batches were less than 10%.The established indirect ELISA method was compared with the commercial ELISA test kit and IFA method.The 96 pig sera submitted for clinical testing were tested.The results showed that the total coincidence rate was 92.7%.It shows that the ELISA method established in this study can be applied to the detection of clinical TGEV infection,and it also lays a certain material foundation for the further development of TGEV commercial diagnostic kits.