構築含蘇力菌內毒素基因之重組昆蟲桿狀病毒

Construction of Recombinant Baculovirus Containing the Bacillus thuringiensis δ-endotoxin Gene

胡耀中(Yaochung Hu) ； 羅竹芳(Chu-Fan Lo) ； 石正人(Cheng-Jen Shih)

《中華昆蟲》 14卷4期 (1994/12) Pp. iii+445-461

https://doi.org/10.6660/TESFE.1994040

摘要

將蘇力菌（Bacillus thuringiensis aizawai 7.29）內毒素基因剪接入昆蟲桿狀病毒傳送載體，本試驗共構築三種傳送載體，分別為pIBL-T7、pIBL-UW1及pIBL-1393 。上述三種載體均經限制酶切割，以確定剪接方向正確；利用DNA定序，確定選殖區之核酸序列完整；並以PCR方法，確定δ內毒素基因接入無誤後，利用此三種含蘇力菌δ內毒素基因之昆蟲桿狀病毒傳送載體DNA，分別與野生型病毒DNA進行共同轉染，將蘇力菌δ內毒素基因選殖至野生型病毒基因組中。產生之重組病毒利用終點稀釋法純法。利用聚合酶聯鎖反應及南方浸漬轉印等方法鑑定重組病毒，分別定名為 Acendo-T7、Acendo-UW1及Acendo-1393。抽取經重組病毒感染之細胞RNA並用dot-blot雜合法測試，可測得重組病毒轉錄之δ內毒素mRNA。利用western blot可檢測到受重組病毒感染之細胞有δ內毒素蛋白質的表現。綜合以上之結果分析，本試驗所得之重組病毒確實嵌入蘇力菌δ內毒素基因，且試驗所構築之重組病毒在細胞內可表現δ內毒素蛋白。

關鍵字

桿狀病毒表現載體 ； 核多角體病毒 ； 蘇力菌 ； δ內毒素基因 ； 重組病毒

並列摘要

The δ-endotoxin gene from Bacillus thuringiensis aizawai 7.29 specifically active against the tobacco cutworm Spodoptera spp. has been cloned to baculovirus trasfer vector. Three transfer vectors were also constructed, ie.,1. PIBL-T7: The characters used baculovirus polyhedrin promoter to drive foregin gene and the insert δ-endotoxin gene was not truncated. 2. PIBL-UW1: This transfer vector using P10 promoter to drive δ-endotoxin gene, so the tentative recombinant virus would be expected to be a polyhedra positive virus and provide a good persistance for the recombinant virus. In addition, the 5' terminal was truncated to remove excess nucleotides between promoter and translation start site, which may reduce the influences on gene expression. 3. pIBL1393: The cloning vector provides multiple cloning sites, and is convenient for gene cloning. The insert gene will be driven by polyhedrin promoter. The δ-endotoxin gene used for cloning was synthesized by polymerase chain reaction, which would be expected to be a high expression of the δ-endotoxin due to the exactlly cloning the coding region. All of the above three transfer vectors were co-transfected with wild type DNA from Autographa californica nuclear polyhedrosis virus (AcNPV) to generate genetically engineered recombinant viruses. These recombinant viruses were purified by limiting dilution. Polymerase chain reaction and Southern blot were used to confirm that δ-endotoxin was exactly inserterd into the genome of AcNPV. Three recombinant viruses, ie, Acendo-T7, Acendo-UW1 and Acendo-1393, were obtained during this experiment. The δ-endotoxin gene expression was detected by northern hybridization and western blot. Those result indicated that all of three recombinant viruses produced δ-endotoxin when infected insect cell.

並列關鍵字

Baculovirus transfer vector ； nuclear polyhedrosis virus ； Bacillus thuringiensis ； δ-endotoxin gene ； recombinant virus