基因重組苜蓿夜蛾核多角體病毒對秋行軍蟲細胞株及斜紋夜蛾幼蟲殺蟲效力

將蘇力菌(Bacillus thuringiensis)δ－內毒素經修飾過之cryIC基因選殖至野生型苜蓿夜蛾核多角體病毒(Autographa californica NPV)基因組中，以間接免疫螢光法可測得毒素在被重組病毒感染的細胞之細胞膜上聚集，並可得知在不同感染後時間，毒素在細胞中之表現量亦有差異。以粒線體去氫酶測定細胞被病毒感染後細胞活性之變化，得知感染後48、72、及96小時，受重組病毒感染之細胞粒線體去氫酶活性均比野生型病毒感染者低，且有顯著性差異。利用trypan blue染色法，分別測定被野生型及重組病毒感染後不同時間，細胞存活比率之變化，結果就活細胞所佔比率而言，不論野生型或重組病毒感染，存活細胞比率均隨感染病毒後時間之增加而降低，但三株重組病毒感染組，其存活細胞率在感染後48小時降低至10%以下，而野生型病毒感染組則至感染後96小時，存活細胞比率仍佔70%左右。比較野生型病毒與重組病毒對四齡斜紋夜蛾幼蟲之毒性，結果供試之野生型及重組病毒對幼蟲之致病力並無顯著性差異。

關鍵字

重組桿狀病毒；蘇力菌； δ－內毒素基因；細胞毒性；斜紋夜蛾

並列摘要

The production of Bacillus thuringiensis (Bt) toxic protein in the genetically engineered Autographa californica nuclear polyhedrosis virus containing Bt δ-endotoxin gene was detected by indirect immunofluorescence. The Bt toxic protein proved to be aggregated around the cell membrane, revealing that the toxin is a membrane binding protein. More toxic protein was expressed as post-infection time progressed. The infected cell lysed 72h after infection and toxic protein was released into the medium. The viability of infected cells was assayed using trypan blue and mitochondrial dehydrogenase; the results reveal that the infectivity of recombinant virus was higher than wild type in infected cells. The cloning of modified cry IC gene to AcNPV not only improves the toxicity of recombinant virus to cell but also reduce the time of infection leading to cell death. The recombinant and wild type viruses were injected into haemocoel of 4th instar larvae of Spodoptera litura to test the insecticidal activity, and results showed larval mortality increased with time after infection, but there was no significant difference in killing time between wild type and recombinant virus against Spodoptera litura larvae.

並列關鍵字

Recombinant Baculovirus ； Bacillus thuringiensis ； δ-endotoxin ； Cytotoxicity ； Spodoptera litura

國際替代計量

基因重組苜蓿夜蛾核多角體病毒對秋行軍蟲細胞株及斜紋夜蛾幼蟲殺蟲效力

全文下載

主題瀏覽