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Expression, Purification And Immunoreactivity Of Antigenic Epitopes Of Bovine Viral Diarrhea Virus (BVDV) Glycoprotein E2

Posted on:2018-12-03Degree:MasterType:Thesis
Country:ChinaCandidate:X F ShenFull Text:PDF
GTID:2370330512983637Subject:Biology, microbiology
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Bovine viral diarrhea/mucosal diseases caused by bovine viral diarrhea virus(BVDV)infection resulted in substantial economic losses for the cattle industry in the world.BVDV belongs to the genus estivirus within the family Flavivirdae.The genome of BVDV is a single positive-strand RNA which contains 5'-untranslated region(5'UTR),3'-untranslated region(3'UTR)and a single large open reading frame(ORF)encoding a polyprotein about 4000 amino acids.The polyprotein is co-and posttranslationally processed into at least 11 mature proteins by viral protease and cellular signal peptidases.These viral proteins include structural proteins C(Core),Erns,E1 and E2,and nonstructural proteins Npro,p7,NS2-3(NS2 and NS3),NS4A,NS4B,NS5A and NS5B.Among them,E2 glycoprotein is the major antigenic protein and responsible for eliciting neutralizing antibodies.Classical swine fever virus(CSFV)and border disease virus(BDV)also belong to genus Pestivirus within the family Flaviviridae.CSFV cause a highly contagious viral disease of pigs,classical swine fever.BVDV has serological cross-reactions with CSFV leading to great difficulties in differentiating disease diagnoses.Based on alignment of amino acid sequences,we designed antigenic epitopes of BVDV E2 corresponding to the conserved antigenic epitopes of CSFV E2 for expression and antigenic analyses.BVDV E2 gene was amplified by RT-PCR using viral genomic RNAs of BVDV NADL strain as a template.The truncated BVDV E2 mutants were amplified by PCR using BVDV E2 gene as a template and cloned into pET-32a to generate pET/BtE2333 and pET/BtE2243,respectively.Similarly,the truncated CSFV E2 gene was amplified by PCR cloned into pET-28a to generate pET/CtE2177.The five epitopes of BVDV E2,CKPEFSYAIAKDERIGQLGAEGLT,AEGLTTTWKEYSPGMK,FDGRKQ,TSFNMDTLA and TYRRSKPFPHRQGCITQKNLGE.were designed,respectively.Six tandem repeat sequence of each epitope with a linker GSGS was synthezed and cloned into pGEX-6p-1 vector to generate pGEX-6E1,pGEX-6E2,pGEX-6E3,pGEX-6E4 and pGEX-5E5,respectively.All recombinant plasmids were identified by sequencing.After transformation,the recombinnat proteins in E.coli were analyzed by SDS-PAGE and Western blot.Recombinant proteins were purified by Ni-NTA affinity chromatography.The specific antibodies directed to BVDV E2 and CSFV E2 were prepared by inoculating BALB/c mice using purified BVDV E2 or CSFV E2 as the antigen,respectively.Indirect immunofluorescence(IF)staining showed that the anti-BVDV E2 and anti-CSFV E2 antibodies were able to react specifically with PK15 cells infected with BVDV and CSFV,respectively.Indirect ELISA assay was developed using the purified BVDV or CSFV as the coating antigen for detection of E2 specific antibodies.Using ELISA assay with the five purified antigenic epitope proteins as the coating antigen,we analyzed the immunoreactiviy between antigenic epitopes of BVDV E2 and the anti-BVDV E2 and anti-CSFV E2 antibodies.The results demonstrated that the five antigenic epitopes reacted with both anti-BVDV E2 and anti-CSFV E2 antibodies.Our works contribute to the understanding of antigenicity of pestivirus E2.
Keywords/Search Tags:Bovine viral diarrhea virus, E2 glycoprotein, Prokaryotic expression, Antigenic epitope
PDF Full Text Request
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