Construction Of Phage Display Libraries Of E2 Gene Of Classic Swine Fever Virus And Study On Epitopes Of E2 Protein

Classical swine fever (CSF) is a highly contagious infectious disease caused by the agent of classical swine fever virus (CSFV). The E2 glycoprotein is one of the structural proteins of CSFV which is known to induce neutrailzing antibodies in swine, so this protein becomes target protein for diagnosis reagent and new type vaccine. Many researchers pay more attention to that if the E2 protein has relation to the differences of immunogenicity and pathogenicity between C-strain and wild strains. It has been proved that there are two epitopes on the E2 protein and their sequences are TAVSPTTLR and YYEP. In addition, there is one mimic epitope which sequence is TTWKEYSH.The E2 genes of Shimen strain (SM) and hog cholera lapinized virus (HCLV) strain were chosen to be amplified by RT-nPCR. Then the obtained products were digested randomly by DNase I . The small fragments which length was between 20bp and 125bp was collected and cloned into T7seclect-415b vector. So the E2 gene phage display peptides libraries of Shimen strain (SM libraries) and of HCLV strain (HCLV libraries) were constructed. The titers of SM libraries and HCLV libraries are 6.6×10~5pfu and 3.2×10~5pfu respectively, which meet the demand of contraction of libraries.CSFV positive serum and seven MAbs which are named with 1B34-37-9, 4B30-3-1, F105, F108, M56, M11 and 1E2 were used to screen the epitopes in SM-E2 libraries and HCLV-E2 libraries by biopanning and phage in situ hybridization. After analyzing the sequences of selected phage recombinants, a sequence which was highly homologous with TAVSPTTLR was selected by the MAbs and CSFV positive serum in SM-E2 libraries and/or HCLV-E2 libraries. A sequence which was highly homologous with YYEP was selected by CSFV positive serum in SM-E2 libraries and a sequence which was highly homologous with TTWKEYSH was selected by CSFV positive serum from HCLV -E2 libraries. These results prove that these three sequences are dominant epitopes on E2 protein. In addition, a sequence which was highly homologous with GGQ( V)VK(887aa～891aa) of E2 amino sequence was selected by MAb 4B30-3-1 from both of the two libraries. A sequence which were highly homologous with PDGLPHY(903 aa～909aa) was selected by M56 in HCLV-E2 libraries. These two unpublicized sequences were consistent with predicted epitopes by computer.So they might be the potential epitopes of E2 protein. But further researches should be done on it.SM-E2 libraries and HCLV-E2 libraries were constructed as specific phage display libraries of CSFV. The libraries contain information of E2 gene more directly and completely than pahge diplay random peptides libraries. These two libraries provide a solid base for the study of epitopes of CSFV E2 protein.