Application Of ORFV Whole Genome Sequencing And B2L Protein

Orf is a kind of acute,high-contact human and beasts.This experiment performs all genome sequencing on the ORFV-Q separation strain and analyzes data such as NR annotations,GO annotations,KEGG annotations;the B2L gene sequence is used to design a pair of primers,with the extraction ORFV genome as a template to expand the destination fragment.Build a reorganized plasmid PET-30A-B2L;after induction expression and purification,B2L protein is obtained.Use B2L protein as a pack of antigens,establish an ORFV antibody indirect ELISA method,establish the best indirect Elisa method test conditions,and detect the critical value of 32 known ORFV negative sheep serum;The mouse is immunized,and the spleen cells of immune mice are fused with osteoma cells.According to the results of the whole genome sequencing,the genome size of the ORFV-Q strain is 127 160bp,and a total of 123 genes are predicted.The SDS-PAGE analysis shows that B2L protein is expressed in the form of precipitation,and the size after purification is about 48 kda.The purification B2L protein is used as an antigen with8μg/ml.The antibody is diluted in 1:10,the two resistance is diluted by 1:5000,and the substrate TMB color is 15 min.Elisa method,when OD₄₅₀≥0.372,the sample is positive;when OD₄₅₀≤0.318,the sample is negative and determines the critical value.After the purification of B2L protein is immune to mice,3 plants can stabilize anti-ORFV-positive hybrid tumor cell strains from 13 positive hybrid tumors cells.This study provides data support for subsequent ORFV genomic research through the entire genome sequencing and gene function annotation analysis of the ORFV-Q strain;uses a purified B2L protein to detect the indirect ELISA method of the ORFV antibody and prepare a single clone antibody to follow the subsequent Studies in the study of the ORFV detection kit,ORFV-related strains,and vaccine production provides direction.