頁籤選單縮合
| 題 名 | 豬瘟病毒E2蛋白之表現與純化=Expression and Purification of the Glycoprotein E2 of Hog Cholera Virus in Pichia Pastoris |
|---|---|
| 作 者 | 劉堂輝; | 書刊名 | 臺糖畜產 |
| 卷 期 | 4:3 1998.12[民87.12] |
| 頁 次 | 頁51-60 |
| 分類號 | 437.657 |
| 關鍵詞 | 豬瘟病毒; E2封套蛋白; 重組Pichia pastoris; 基因表現; HisTrap冄親合性管柱純化; HisTrap冄 affinity chromatography; Hog cholera virus; Pichia pastoris recombinants; Gene expression; Envelope protein; |
| 語 文 | 中文(Chinese) |
| 中文摘要 | E2蛋白可激發豬隻產生中和抗體預防豬瘟。因此本試驗在於探討豬瘟病毒封套蛋 白 E2 於含此基因之重組酵母菌 Pichia pastoris 之生產及其純化。 於其 3' 端不含轉譯 為穿越膜區蛋白的 E2 基因以 PCR 方法從豬瘟病毒 P97 毒株增幅、選殖後再以同質重組方 式將其嵌入 P. pastoris 的染色體上。此重組 P. pastoris 唯有於具緩衝能力之培養基中 培養才能表現 E2 融合蛋白。Casamino acids 則具有穩定 E2 蛋白的效果。 以甲醇誘導後 12 小時其培養液中開始出現 E2 蛋白並積聚其中， 至 72 小時開始出現降解之 E2 蛋白。 此分泌至培養液中之 E2 融合蛋白約 51.4 kDa。 以 0.5% 甲醇量誘導 P.pastoris 而產生 E2 蛋白最為適當且誘導時培養細胞數愈多產量亦多。甲醇誘導培養 72h 後酵母菌 OD 值為 15， 其培養液中之 E2 融合蛋白可達 6.Oug/ml。 分泌至培養液之 E2 融合蛋白可利用 HisTrap �薿M組純化之。本試驗結果顯示含 E2 基因之重組 P.pastoris 於特定培養條件下 可分泌高量之完整 E2 融合蛋白並可以 HisTrap �蟫辿X性管柱純化。 |
| 英文摘要 | Vaccination of pigs with E2, an envelope glycoprotein of hog cholera virus elicits neutralization antibody and protects pigs from hog cholera. The production and purification of the protein from a recombinant Pichia pastoris were investigated. The recombinants that contained the E2 gene inserted into their chromosomes were constructed. E2 gene without its 3' transmembrane region amplified by PCR from a P97 strain in Taiwan was cloned into a Pichia expression vector, transformed into Pichia cells, and finally integrated into Pichia genomic DNA by homologous recombination. The recombinants produced E2 fusion protein only in a buffered medium, and the addition of casamino acids into the medium increased the stability of the protein. The secreted fusion protein with a size of 51.4 kDa was first detected at 12 h after methanoi induction, steadily increased thereafter, and began to degrade at 72 h. Methanoi with a concentration of 0.5% was suitable for induction. Pichia recombinants with higher cell number produced more E2. E2 accumulated in the medium to about 6.0 ug/ml at 72 h after induction of the recombinants (OD ≒ =15). Secreted E2 was purified from the medium by HisTrap �� affinity chromatography. The results show that E2 protein can be secreted in high amounts from Pichia recombinants in special conditions and purified by affinity chromatography. |
本系統中英文摘要資訊取自各篇刊載內容。