Expression Of Orf Virus B2L And F1L Gene And Preparation Of Polyclonal Antibody

Orf, also know as ovine contagious ecthyma, is caused by Orf virus. ORFV is Parapoxvirus, which belong to Chordopoxvirinae family and mainly infect the skin and mucous membranes. ORFV ordinarily infect sheep and goat, affect animal husbandry economy and human health. Orf is control by vaccination, but there were some differences in ORFV strain in different regions, and it can't protection of goat. Thus, the study of the ORFV in the region is important significance.The goat blister skin samples were collected from Hainan, the B2L, V1R and F1L genes was cloned and analyzed by genetic phylogenetic tree. The results showed that it had a close relationship with that of Fujian and Northwest strain.B2L and F1L gene fragment was amplification by PCR, and inserted into the prepared vector pET28a (+) to construct the recombinant expression plasmid,pET28a-B2L and pET28a-F1L. The expressed protein was purified by Ni-NTA His-Bind Resin and identified by SDS-PAGE and Western Blot. The results showed that the recombinant plasmids pET28a-B2L and pET28a-F1L was successfully constructed and the fusion protein with His tag was successfully expressed. SDS-PAGE results showed that the molecular mass of His-B2L and His-F1L was approximate 47 KDa and 44 KDa respectively.B2L and F1L gene fragment was amplification by PCR, and construct the recombinant expression plasmid pEGFP-N1-B2L and pEGFP-N1-F1L. The recombinant plasmid was transfected into goat testicular cells and the expression of EGFP-B2L and EGFP-F1L fusion protein were detected by fluorescence microscope,qRT-PCR and Western Blot. The result showed that the eukaryotic expression plasmids pEGFP-N1-B2L and pEGFP-N1-F1L was constructed successfully, and EGFP-B2L and EGFP-F1L were expressed, which lay the foundation for the research of B2L and F1L protein.The purified fusion protein was used to immune New Zealand white rabbit by hypodermatic multi-point injection to prepare rabbit anti His-B2L and His-F1L polyclonal antibody. Indirect ELISA method was used to determine the polyclonal antibody titer. The results showed that the polyclonal antibody titer was more than 1:12800, and which has strong specificity.