Abstract: In order to clone and express 121 gene of ORFV Anhui strain and analyze the gene bioinformatics. In this study, the 121 gene was PCR amplified, with the ORFV AH-F10 strain as the template. The amplified product was cloned into pGEX-6P-1 prokaryotic expression vector, and the recombinant plasmid pGEX-6P-1- 121 was constructed and identified by sequencing. Then, the correctly identified recombinant plasmid was transformed into E.coli Rosetta competent cells, and was induced by IPTG. And optimizing the induction conditions of the recombinant protein to determine the optimal conditions for the expression of ORFV 121 recombinant protein. The results of SDS-PAGE and Western-blot showed that the size of the recombinant protein was about 60 ku, which was highly expressed in Rosetta and mainly in the form of inclusion body with good reaction genicity. After inclusion body protein was purified to obtain the target protein, emulsified with Freund's adjuvant to inject subcutaneously into BALB/c female mice aged 6 weeks. The mice were booster immunized once every 14 d, and serum was collected after 4 times, which was specifically identified by Western-blot. The results showed the antibody serum could specifically react with ORFV 121 protein of positive prokaryotic expression, indicating the successful preparation of multi-antiserum. In addition, the physicochemical properties, signal peptides, transmembrane domains, phosphorylation sites, secondary structures and B-cell epitopes of the target gene coding proteins were predicted by bioinformatics software. The results of bioinformatics indicated that the isoelectric point of the protein was 8.86, which was an unstable hydrophilic protein. It was predicted that there were 50 possible phosphorylation sites, no signal peptide and transmembrane domain. In the secondary structure, the random coil accounted for 62.25%, the α-helix, β-turn and extended chain accounted for 27.81%, 2.98% and 6.95%, respectively. Eight potential dominant epitopes of B cells were predicted and analyzed. Inconclusion, the ORFV 121 protein of AH-F10 strain was successfully expressed and its biological characteristics were predicted. It laid a foundation for further research on the structure and function of the protein and the pathogenesis of ORFV.

Key words: Orf virus, 121 gene, Prokaryotic expression, Bioinformatics

CLC Number: 

• S855.3