Development Of IFA And Subunit Vaccine Against Classical Swine Fever Virus Basing On Its E2 Protein

Classical swine fever was an acute,serious,highly contagious infectious diseases caused by classical swine fever virus(CFSV).It still outbreak in our country,and causes a serious loss to the pig industry.C strain is world wide used attenuated vaccine strain.Plenty of data show that the C strain vaccine has a good immune efficacy,high safety,and don’t cause pathological damage for the piglets,pregnant sows,and pigs even in immunosuppression.However,the antibodyinduced by this kind of live attenuated vaccine is difficult to distinguish from that of the infected pigs,which hinder the control and clean-up program against classical swine fever.In order to develop a DIVA vaccine,the current researches mainly focus on the major protective antigen.Membrane glycoprotein E2 located in the external surface of the virus particles,and acrossed a membrane area,is the main structural protein of CSFV,whcich is associate with virulence and host range.Therefore,E2 is the major target proterin for development of CSFV vaccines and diagnostic reagents.In this study,E2 protein of CFSV was expressed using prokaryotic expression system monoclonal antibody of E2 protein was developed,the indirect immunofluorescence assay was established,and the subunit vaccine of CSFV was evaluated.1 Expression of the main antigen of E2 protein of CSFVThe RNA of CSFV was extracted by Trizol,and reversetranscripted to cDNA.Then,E2 gene was amplified by PCR and clones into the cloning vector of pMD18T to construct pMD18T-CSFV-E2’.The E2 gene was further ligated to theprokaryotic expression vector of pET28a to construct pET28-CSFV-E2’.After analysis of the rare condon(the restriction site of Sac I located the site of(2672bp),the 2433～2672bp of E2 gene was optimized,synthesized and cloned into pTOPO to consruct pTOPO-Simple-E2.Finally,the E2’ gene of pET28a-CSFV-E2’ was replaced with optimized E2 gene to construct pET28-CSFV-E2.After optimizing the induction condition,the E2 protein was expressed successfully.The E2 protein was purified by chromatography column of Ni-NTA,and the concentration of the protein was about 3.48 mg/ml.Its antigenicity was confirmed by Western blot analysis.2 Establishment of immuofluorescenceassay against CSFVBALB/c mice were immuned with the purified E2 protein,the spleen cells from immunized miceand myeloma cells of SP2/0 were fused by PEG4000.Through repeated screening,we finally got two strains of monoclonal antibody,2B1 and 3B4,which were against E2 protein of CSFV and all belonged to IgGl subtypes.The immunofluorescence assay(IFA)against CSFV was established using the mAb as the first antibody,the FITC-sheep anti-mouse serum as the second antibody,102TCID50/ml of CSFV,and 1:100 times dilution of McAb 2B1.The IFA could detect CFSV only,not for BVDV and PCV2,indicating the IFA have a high specificity.Three batches of classical swine fever vaccine were test by the established IFA,each batch of vaccine was repeated 3 times.Results showed that there is less variation between different bataches,indicating the repeatability of IFA.To find the relationship between the TCIDso/ml detected by the established IFA and rabbit body heat reaction,20 batches of classical swine fever vaccine were tested.The results showed that if the vaccines contained 1×105RID/ml determined by rabbit body heat reaction,the titers of vaccines determind by IF:A were 103 6TCID50/ml～104.25 TCID50/ml.if the vaccines contained 5×105 RID/ml determined by rabbit body heat reaction,the titers of vaccines determind by IFA were 104.5 TCID50/ml～106.5 TCID50/ml.Therefore,if the titers of vaccines should contained 5×105 RID or above,the titers of vaccines should not be less than 104.5 TCID50/ml.3 The preliminary evaluation of E2 subunit vaccineThe E2 protein was emulsified with the adjuvant to prepare the subunit vaccine.They were all qualified by testing about physical properties and aseptic detection.One lot of the subunit vaccine was used to evaluate the safety in piglets.The result showed that there was no apparent adverse reactions caused by vaccination.The piglets were divided randomly into three groups to compare the immune efficacy of the prepared vaccine.After 14 days for twice immunization with live vaccine or subunit vaccine,the piglets was challenged with "shimen" classical swine fever virus.The results showed that pigs of either immune groupshad good appetite,no temperature change,no classical swine fever related clinical symptoms and pathological damage.Both E2 subunit vaccine and live attenuated vaccine provided 100%protection against challenge.In contrast,the pigs in control group began to die in 9 days after challenge,and the diseased pigs showed typical classical swine fever symptoms of sepsis.The pig sera were detected for antibody by using ELISA test kit.The antibodies of pigs in both vaccine groups turned positive at 14 days and continued to rise until 21 days.The antibodies of pigs in live vaccine group rose fasterin prime vaccination than that of subunit vaccine group,and the antibodies of pigs in subunit vaccine group rose faster in boost vaccination than that of live vaccine group.The antibodies pigsin control group were kept negative.The viralloadingsin the lung lymph node,inguinal lymph node and tonsil were higher than that of the other two immune group,indicating that the prepared subunit vaccine of E2 protein.can be used asvaccinecandidate.