Cloning, Expression Of B2L Gene Of ORFV Shaanxi Strain And Establishment Of Indirect Elisa For Detection Of Seral Antibodies

Orf is a highly contagious infectious disease caused by ORFV,which is globally distributed.The lambs are more sensitive,and the disease often causes great economic losses.The study used the polymerase chain reaction(PCR)to amplify the B2 L gene ORFV Shaanxi strain,and analyze its sequence.The prokaryotic expression of recombinant protein was made,and purified recombinant protein was used as antigen to establish an indirect ELISA for the detection of the antibody levels after the optimization of various conditions.The following results were obtained.1.The B2 L gene of ORFV Shaanxi strain was amplified,the target gene fragment length is 1137 BP.The gene sequence was analyzed,and the similarities were compared with other 11 strains of virus B2 L protein in Gen Bank.The similarities of nucleotide sequence and amino acid sequence were between 95.9%~97.4% and 97.8%~99%,respectively.The target gene was successfully connected to the prokaryotic expression vector pET-28 a,and the recombinant plasmid pET-28a-B2 L was constructed.In the prokaryotic expression system of Escherichia coli,the recombinant plasmid pET-28a-B2 L was expressed after induction,and 42 kD B2 L recombinant protein was obtained.2.Using purified B2 L recombinant protein as antigen,the indirect ELISA method for detecting ORFV seral antibodies was established by optimizing various conditions.The optimal reaction conditions were as following: Coated antigen is 600 ng,the dilutions of serum and enzyme-labeled antibody ware 1: 200 and 1: 5000;the blocking time of PBST containing 5% milk powder is 1.5 h;the incubation time of serum and enzyme-labeled antibody are 1 h and 0.5 h;the developing time of TMB is 10 min.The method was specific,sensitive and reproducible,and 344 clinical seral samples were detected for ORFV antibody,96.8% of positive rate was got.