Barusrux Sahawat, Sramala Issara, Katzenmeier Gerd, Bunyaratvej Ahnond, Panyim Sakol, Angsuthanasombat Chanan
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom, 73170 Thailand.
J Biochem Mol Biol. 2003 May 31;36(3):294-8. doi: 10.5483/bmbrep.2003.36.3.294.
The pathological effect of the Bacillus thuringiensis Cry delta- endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protoxin. However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.
苏云金芽孢杆菌Cryδ-内毒素对敏感昆虫幼虫的病理作用对中肠上皮细胞造成了广泛损害。在本研究中，设计了一种体外试验，用于评估在大肠杆菌中表达的克隆Cry4B杀蚊幼虫蛋白的杀虫效力。通过对从5日龄埃及伊蚊幼虫解剖得到的中肠进行细胞活力测定来确定毒性。在用毒素蛋白孵育后，通过监测490nm处生物还原的甲臜产物的量，以光度法测定存活上皮细胞的数量。结果表明，65kDa的胰蛋白酶激活型Cry4B毒素的毒性效力约比130kDa的Cry4B原毒素高3.5倍。然而，无生物活性的Cry4B突变体（R158A）和鳞翅目特异性Cry1Aa毒素经胰蛋白酶处理后的产物在蚊幼虫中肠上相对没有体外活性。此处呈现的体外细胞毒性研究证实了生物测定中获得的数据。
