Truncated Expression Of B2L Protein And ORFV Establishment Of ELISA Method

Orf also known as Contagious Ecthyma,a contagious disease caused by Orf virus(ORFV)in the genus Parapoxvirus of the poxvirus family.The Protean function in DNAstar software was used to analyze the sequence of the membrane protein B2L of the ORFV published in GenBank,and the highly hydrophilic dominant epitope region was screened.The full length of the B2L gene was named B2L-1 and the height was selected.The hydrophilic dominant epitope region is named B2L-2.The B2L-1 and B2L-2 coding genes were optimized for E.coli bias codon,and the HIS-tag and B2L-1 and B2L-2 genes were respectively ligated to the pET32a(+)vector by gene synthesis.The expression vector was transformed into BL21(DE3)competent cells for prokaryotic expression,and the expression conditions were optimized,followed by purification by Ni-NTA Agarose affinity purification column.The B2L-1 and B2L-2 proteins were separately immunized to obtain positive serum,and the purified recombinant fusion protein was used as a coating antigen to establish an indirect ELISA method for detecting the antibodies of the ORFV,and the ELISA conditions were optimized.The results of SDS-PAGE showed that the recombinant fusion protein B2L-1 and truncated B2L-2 protein with good solubility and high expression efficiency were successfully obtained.Western Bolting results showed that B2L-1 and B2L-2 proteins could specifically bind to Anti-His Mouse mAb;agar diffusion test results showed that the expressed product could react with rabbit serum immunized with ORFV,indicating that B2L-1 and B2L-2 proteins have good reactogenicity;the optimal condition for the established ELISA method is B2L-1:the coating amount is 55ng/mL,the optimal dilution of serum is 1:200;the optimal coating is 5%skim milk;the best dilution of the second antibody is 1:5000;the optimal color development time is 20min;B2L-2:the coating amount is 56.5ng/mL,the optimal dilution of serum is 1:400;the optimal coating is 5%skim milk;the best dilution of the second antibody is 1:5000;the best color development time It is 20min.The detection of 40 sheep serum collected from Inner Mongolia was compared with the results of 40 neutral serum neutralization tests.The results showed that the detection rate of the indirect ELISA method was 100%and 97.5%.The established indirect ELISA method for detecting antibodies against ORFV is expected to provide a rapid and simple means for the detection of Orf.