Expression Of The Glycoprotein E2 Of Classical Swine Fever Virus And Preparation Of Monoclonal Antibodies Against E2 Protein

Classical swine fever(CSF) is a highly contagious,acute and multi-systemic hemorrhagic viral disease of pigs,which causes by classical swine fever virus(CSFV),a member of the genus Pestivirus of the family Flaviviridae.The envelope glycoprotein E2 is the major antigenic protein of CSFV and contains A,B,C and D domains which are highly conserved among CSFV strains.Antibodies directed against E2 strongly neutralize virus and protect pigs from infection by CSFV.E2 is the most variable structural protein among the three viral structural proteins and always used as the target of genetically engineering vaccine and differentiation diagnosis.In this study,the recombinant E2 protein was expressed in E.coli BL21-condonplus-RIL and SF-9 insect cells respectively and immunized Balb/C mice and developed three monoclonal antibodies against E2 protein of CSFV.It will be useful to set up a rapid clinical diagnosis of CSFV in the future.1.Expression of E2 protein of CSFV in E.coli and SF-9 insect cellsCharacters of E2 protein of CSFV were analyzed according to its published protein sequences.Based on its characters,antigen domains ABCD and AD were selected and amplified by two pares of primers.The full length of E2 gene was amplified by the reverse transcription potymerase chain reaction(RT-PCR) from total RNA isolated from CSFV infected PK-15 cells and cloned into vector pMD18-T.ABCD and AD antigen domains were amplified by polymerase chain reaction(PCR) using E2 gene as template and sub-cloned into vector pMD18-T.The lengths of AD/ABCD antigen domains were 300bp and 645bp,which encoded 100 and 215 amino acids,respectively.Prokaryotic expression vector pKGE2ABCD and pKGE2AD were generated by inserting ABCD and AD antigen regions into pGEX-KG and fusion with GST.The recombinant plasmids were transformed into E.coli BL21-condonplus-RIL and induced by IPTG.SDS-PAGE results showed that the fusion protein of GST-E2AD and GST-E2 were highly expressed in E.coli,and molecular weight of the fusion proteins were approximately 50 kDa and 36 kDa and could react with CSFV antisera.These results showed that both of fusion proteins could be used as the immunization antigen.Baculovirus expression system is very useful to express foreign viral proteins because proteins expressed in SF-9 insect cells can be modified post-translation and kept them naive bio-characters.In this study,baculovirus Bac to Bac expression system was used.E2 gene of CSFV was cloned into BamHⅠand HindⅢsites of pFastbac-HT vector with 6 His tag.The recombinant plasmids were isolated and transformed into E.coli DH10-bac.The recombinant bacmid was extracted and identified by PCR,named bacmid-E2.Bacmid-E2 was transfected into SF-9 insect cells and generated recombinant baculovirus expressing CSFV E2 protein.Western blot and Immunofluorescence staining assay(IFA) were used to detect recombinant glycoprotein E2 in infected insect cells using monoclonal antibodies against His-tag.Immunofluorescence staining showed that SF-9 insect cells infected with the recombinant baculovirus could react specifically with anti-His tag.However,the level of target protein expression was much lower and not easy to detect it by western blotting,and could not be used for further reseach.2.Preparation of Monoclonal Antibodies against the envelope glycoprotein E2 of CSFVBalb/c mice were immunized with the purified recombinant E2 proteins of CSFV to stimulate the production of antibodies against the target antigen.Mice were euthanized when antibodies against E2 antigen of CSFV reached quite high levels.Mice's spleen cells were separated and fused with myelomas cells under the action of PEG-4000 solution to obtain hybridoma.Positive hybridoma clones were screened by indirect Enzyme Linked Immunosorbent Assay(I-ELISA),and a limiting-dilution method was performed to sub-clone the positive clones.After three rounds of sub-cloning,three McAbs against the E2 protein of CSFV were obtained.4H5 and 1C7 are McAbs against ABCD antigen domain,while 4H4 is McAb against AD antigen domain.The three hybridoma cell strains were named 4H5,1C7 and 4H4 and belonged to IgG2a,IgG2b and IgG1 subtype.The ELISA titers of culture supernatant were 0.4×10~3,0.8×10~3 and 0.4×10~3 and ascites were 0.2×10~6,0.5×10~5 and 0.4×10~6 respectively. Immunofluorescence staining indicated that all three McAbs could react with CSFV antigen,and 4H5 and 4H4 were much better than 1C7.Western blot showed that 4H5 and 4H4 McAbs reacted very well with CSFV.