仙人掌桿菌及蘇力菌營養期殺蟲蛋白基因之篩選與表現

Screening and Expression of the Vegetative Insecticidal Protein (vip) Gene from Bacillus cereus and Bacillus thuringiensis

陳怡均(Yi-Chun Chen)

指導教授 ： 高穗生 ； 劉炳嵐

朝陽科技大學/理工學院/應用化學系/碩士(2010年)

摘要

本研究利用聚合?鏈鎖反應(PCR)從四株仙人掌桿菌中篩選出BCRC 12811 (經cry gene typing重新鑑定為蘇力菌)及蘇力菌HD-1具有營養期殺蟲蛋白基因vip3，其PCR產物大小約為2.4 kb。將vip3基因與載體pET29b接合構築成表現質體pET29b-vip3-12811及pET29b-vip3-HD1，並分別以轉形方式表現於Escherichia coli BL21宿主中。兩株重組菌株利用IPTG誘導，可分別表現出Vip3-12811及Vip3-HD1兩個蛋白質，分子量大小約為88.5 kDa，由核?酸序列轉譯後可以推衍出789個胺基酸，經由MS/MS分析，部分胺基酸片段與先前由核?酸序列推衍之胺基酸序列相吻合。生物檢定結果顯示，轉殖株純化之Vip3-12811毒蛋白對小菜蛾、擬尺蠖及玉米穗蟲1-3齡幼蟲的LC50分別為：91、152、220 μg/mL及256、358、459 μg/mL與279、376、481 μg/mL。而轉殖株純化的Vip3-HD1毒蛋白對小菜蛾、擬尺蠖及玉米穗蟲1-3齡幼蟲的LC50分別為：82、135、188 μg/mL及240、332、415 μg/mL與248、350、432 μg/mL。

關鍵字

蘇力菌 ； 仙人掌桿菌 ； 生物檢定 ； 營養期殺蟲蛋白

並列摘要

Four Bacillus cereus and Bacillus thuringiensis kurstaki HD-1 isolates were screened by polymerase chain reaction (PCR) to detect the presence of vegetative insecticidal protein genes. The vip3 gene was amplified from B. cereus 12811 (reidentified as B. thuringiensis by cry gene typing) and B. thuringiensis HD-1 respectively by PCR using vip28F and vip26R primers. The vip3 gene was cloned into vector pET29b to form construct recombinant expression plasmid pET29b-vip3-12811 and pET29b-vip3-HD1. By transformation, the recombinant expression plasmids were cloned into Escherichia coli BL21 respectively to form recombinant strains of pET29b-vip3-12811 and pET29b-vip3-HD1. The two recombinant strains were induced by IPTG to produce a protein of approximately 88.5 kDa. Moreover, by MS/MS analysis, the nucleotide sequence was determined to predict a protein of 789 amino acid residues. The LC50 of purified recombinant protein (Vip3-12811) against 1st~3rd instar Plutella xylostella, Trichoplusia ni, and Helicoverpa armigera were 91, 152, 220 μg/mL, 279.18, 375.90, 480.93 μg/mL and 256, 358, 459 μg/mL and 279, 376, 481 μg/mL respectively. The LC50 of purified recombinant protein (Vip3-HD1) against 1st~3rd instar Plutella xylostella, Trichoplusia ni, and Helicoverpa armigera were 82, 135, 188 μg/mL, 240, 332, 415 μg/mL and 248, 350, 432 μg/mL respectively.

並列關鍵字

Bacillus thuringiensis ； Bacillus cereus ； vegetative insecticidal protein (VIP) ； bioassay

參考文獻

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