Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13411
標題: 傳染性化膿性皮膚炎病毒主要封套蛋白之表現與純化
Expression and Purification of the Major Envelope Protein of Orf Virus
作者: 歐啟明
Ou, Chi-Ming
關鍵字: contagiou pustular dermatitis
傳染性化膿性皮膚炎病毒
orf
B2L
phospholipase
liposome
磷脂酶原核表現
微脂粒
出版社: 獸醫學系暨研究所
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摘要: 中文摘要 傳染性化膿性皮膚炎(contagiou pustular dermatitis)又叫做Orf,可發生在全世界的山羊與綿羊,尤其是在年幼動物。Orf會在嘴唇、口鼻周圍以及口腔黏膜產生增生性的病變。Orf病毒屬於副痘病毒屬,痘病毒科。病毒顆粒為卵圓形,基因體為線狀的雙股DNA。Orf病毒的B2L基因可以產生高度免疫原性的主要封套蛋白,且與vaccina virus 的封套蛋白p37有42%的相似性。本實驗中我們以PCR的方式增幅出B2L全長基因,並將其轉接至原核表現載體pET24a。將建構好的重組質體轉型至E. coli BL21(DE3)中,再以IPTG誘導表現B2L重組蛋白。表現出的重組蛋白以西方墨點法與質譜儀鑑定確定為orf病毒的B2L蛋白。接著,以親和性樹脂管柱純化出B2L蛋白。以軟體分析B2L蛋白序列,可以預測到其序列的C端附近具有磷脂酶phospholipase D(PLD)的保留區,可能具有PLD的活性。由於vaccina virus的封套蛋白 p37 具有磷脂酶的活性,因此,我們推測B2L蛋白亦具有磷脂酶的功能。同時,我們更進一步地建構出四個C端缺損型質體以表現較短的B2L C端缺損蛋白,若B2L蛋白具有活性,希望藉此觀察缺損蛋白是否失去磷脂酶的活性;此外,因為B2L蛋白具有高度免疫原性,因此希望觀察是否可以藉此提高表現量。結果只有四個C端缺損質體中,只有兩個C端缺損蛋白有表現出,且表現量較全長為低。而在磷脂酶功能分析中,我們無法偵測出重組蛋白B2L的磷脂酶酵素活性。
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.
URI: http://hdl.handle.net/11455/13411
其他識別: U0005-2407200714015900
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2407200714015900
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