Prokaryotic Expression And Purification Of ORFV B2L Gene

In an effort to obtain the B2L protein of ORFV,a pair of specific primers were designed and synthesized based on the published nucleotide acid sequence of ORFV strain ORFV/ShanXi/2011/China.The primers were used to amplify the B2L fragment of ORFV by using PCR.The PCR product was cloned into PMD18-T vector to form recombinant PMD18-T-B2L.Using bioinformatic software and methods,we predicted and analyzed the secondary structure,tertiary structure,B-cell preponderant epitope,conserved domain,transmembrane domain and signal peptide of the B2L gene.The res?Lts showed that the length of B2L gene was 1137bp,encoding 379 amino acids.The B2L gene of ORFV/QD/2015 strain shared 96.8%-99.2%of amino acid identities and 96.8%-99.7%of nucleotide identities,respectively,with the B2L gene of other 12 ORPV reference strains.The res?Lts of phylogenetic analysis indicated that there was a close relationship between ORFV/QD/2015 strain and ORFV/ShaanXi/2015/China strain.The prediction of secondary structure of B2L protein indicated that alpha-helix and ?-sheet were dominant.The B2L protein was supposed to contain 7 potential antigen epitopes.However,no transmembrane domain or signal peptide was identified.The tertiary structure of B2L protein was curved spiral structure.The B2L fragment of interest was digested and subcloned into Prokaryotic Expressing Vector PET-32a(+)and verified by sequencing.The constructed recombinant PET-32a-B2L was transformed into E.coli strain BL21(DE3)and the expression of B2L protein was induced by IPTG.The B2L protein expressed was analyzed by SDS-PAGE.Subsequently,the B2L protein was purified by affinity chromatograph and was identified by Western Blot.The res?Lts showed that the B2L protein was successf?Lly expressed with the expected size.Our data provided basis for the following research on the development of approach for detection of ORFV.