Expression and Purification of the Major Envelope Protein of Orf Virus

• Main Authors: Chi-Ming Ou, 歐啟明
• Other Authors: Min-Liang Wong
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/50383988977441905067

碩士 === 中興大學 === 獸醫學系暨研究所 === 95 === Abstract Orf is also known as contagious ecthyma, contagiou pustular dermatitis, Scabby mouth and sore mouth. Orf occurs worldwide in sheep and goats, especially in young animal. The disease is characterized by proliferative lesion on the lips, around the nostrils and in the oral mucosa. Orf virus is a member of the Parapoxvirus genus of Poxviridae. The virus particles are ovoid and the viral genome is a linear double-stranded DNA. The viral B2L gene encodes for the highly immunogenic major envelope protein , which is the homologue of vaccinia virus envelope protein antigen p37 . We amplified the B2L gene by polymerase chain reaction (PCR) and cloned the gene into the expression vector pET24a. The plasmid was transformed into the E. coli. strain BL21(DE3) and the expression of recombinant B2L protein was induced by IPTG. The recombinant B2L protein was identified with western blotting and with mass spectrometry analysis. Then, the B2L protein was purified by affinity chromatography. Moreover, we made four truncated constructs to express shorter B2L proteins; although two of these deletion mutants were also expressed, they showed lower level of production. The vaccinia virus envelope protein p37 has phospholipase activity; therefore it has been proposed that the B2L protein has phospholipase function. However, we were not able to detect the phospholipase activity of our purified recombinant protein.