Isolation Of Orf Virus In Chongqing And Prokaryotic Expression Of F1L Gene

Orf virus is the prototype species of the Para-poxvirus genus of the Poxviridae family with capsule membrane.The Orf V genome consists of linear double-stranded DNA.Research at home and abroad had proved that the virus’ variability was strong.In order to obtain local pathogenic strains and lay the foundation of controlling and preventing the virus host expanding,the following study had been carried out in this experiment.1. Isolation and identification of Orf virus(OrfV-CQ) Chongqing strainA strain Orf virus was isolated from the scab skin of infected goat by MDBK cell lines, named the OrfV-CQ. The experimental results indicated that the bland passages of generations 1 to 3 were undertaken until the CPE could be seen on generation 4 in 48 h. CPE characterized by cells becoming round,aggregation, fusion and falling off. Lots of mature and immature virions were observed under electron microscopy. The F1 L gene of OrfV-CQ was amplified by PCR method using specific primers(708bp) and shared a nucleotide identity of 97%~99%. The value of TCID50 of generation 4 was 10-4.25/0.1 mL. Animal regression test showed similar characteristics with the natural occurrence2. Bioinformatic analyzing of F1 L gene of OrfV-CQBy using bioinformatics softwares(DNAStar, SignalP 4.1,et al)and methods,the molecular characteristics of F1 L gene was been analysed and the protein’s biological functions encoding by F1 L gene was predicted.The results indicated the length of F1 L gene is 1023 bp, encoding 340 amino acids. The F1 L gene of OrfV-CQ strain shared a nucleotide identity of 93.9%~98.4% with twenty strains, and an amino acid identity of 95.0% with Shanxi strain.Prediction of the secondary structure of F1 L indicated that the β helix and random coil took a higher percentage.The F1 L protein was supposed contain 13 potential antigen and no signal peptidefound. These results provide a theoretical basis for immunologic diagnosis and further research of nucleic acid vaccine of OrfV.3. Prokaryotic expression of F1 L gene of Orf V-CQThe F1 L gene of OrfV-CQ was amplified by PCR and built into pET-32 a for expression in E.coli.The results indicated the length of F1 L gene is1023 bp.SDS-PAGE analysis and Western Blotting showed the target protein was highly expressed at 5h in E.coli. as inclusion bodies at the level of 57.4 KDa and had good reactivity.Conclusions: A virus in the scab samples collected from suspected Orfv infection goat was isolated, named Orf V-CQ.The molecular characteristics of F1 L gene showed the sequence was conserved, but there were base deletion and mutation existing. The F1 L gene protein was supposed contain lots of potential antigen epitopes.The recombinant plasmid was constructed and the target protein was highly expressed in E.coli.