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標題: 牛傳染性鼻氣管炎封套醣蛋白gI基因之選殖
Cloning of Infectious Bovine Rhinotracheitis Virus (Yunling Stra in )Envelope Glycoprotein gI Gene
作者: 謝耀清
Hsieh, yao-ching.
關鍵字: infectious bovine rhinotracheitis
glycoprotein gI
出版社: 獸醫學系
摘要: 牛傳染性鼻氣管炎病毒屬於阿爾發庖疹病毒亞科是牛傳染性鼻氣管炎的致 病原牛傳染性鼻氣管炎病毒至少合成五種醣蛋白,其中四種醣蛋白被研究 的較透徹,分別被命名為醣蛋白gI,gII,gIII及gIV;在這四種醣蛋白之中, 醣蛋白gI,gIII及gIV可誘發宿主細胞產生中和抗體是當為次單原疫苗的理 想材料.先前我們實驗室已選殖到牛傳染性鼻氣管炎病毒(雲林株)封套醣 蛋白 gIII及gIV基因.為了往後對牛傳染性鼻氣管炎病毒之研究及次單原 疫苗之製造,分離封套醣蛋白gI基因是相當重要的.本實驗首先分析外國 Cooper株牛傳染性鼻氣管炎病毒的基因體圖譜,發現若將牛傳染性鼻氣管 炎病毒基因體以限制酵素HindIII及BamHI同時切割,可得到含有gI基因大 小約12kd的 HindIII/BamHI DAN片段.因此我參考Cooper株將牛傳軟性鼻 氣管炎病毒 (雲林株)以限制酵素HindIII/BamHI切割,以pBR322質體為選 殖載體來選殖含gI基因大小約12kd之HindIII/BamHI片段.並根據Cooper株 gI基因核甘酸序列設計PCR引子,以進行聚合酵素連鎖反應來對選殖株做初 部篩選.為了將來gI基因表現載體構築及定序的方便性,特將gI基因選殖 成3.8kd的DNA片段並以 pBSII殖體連接.最後定序約300dp來證明我已選殖 到gI基因.
Infectious bovine rhinotracheitis virus (IBRV),an alphaherpesvir us, is a causative agent of infectious bovine rhinotracheitis (IBR) in cattle. The IBRV synthesizes at least seven viral enve lope glycoproteins and four of them have been characterized and named gI,gII.gIII and gIV.Among the four glycoproteins,gI,gIII and gIV are the major target antigens involved in virus neutra lization, therefore, they are used as the subunit vaccine against the infection of IBRV.Previously our labortary has cloned IBRV (Yunlin strain)glycoprotein gIII and gIV genes. For the purpose of future study on genetic structure and development of sudunit vaccine of IBRV it is essential to proceed the mole cular cloningof IBRV glycoprotein gI gene. In this experiment, A 12kb BamH I/Hind III fragment of IBRV (Yunlin strain) DNA was cloned and identified A clone pBR322A contained gI gene ident ified by polymerase chain reaction (PCR) of which the primers were desigend according to IBRV Cooper strain gI gene sequence. A Hpa I/Kpn I fragment of pBR322A DNA was sudcloned and identi fied. Asubclone pGI-01 of 3.8kd contained gI gene was obtained by restriction mapping and sequencing. The sequences with 300 base pair long have been sequenced and included translation start site AUG. The sequence region was 100%homologous to IBRV Cooper strain. The results suggested that IBRV (Yunlin strain) glycoprotein gI gene has been cloned.
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