Wu D, Johnson J J, Federici B A
Department of Entomology, University of California, Riverside 92521.
Mol Microbiol. 1994 Sep;13(6):965-72. doi: 10.1111/j.1365-2958.1994.tb00488.x.
The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp. israelensis and the PG-14 isolate of B. thuringiensis subsp. morrisoni is at least 20-fold greater than any of the four mosquitocidal proteins of which it is composed (CytA, CryIVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CryIVD toxins were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis cells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bioassays against first instars of Aedes aegypti. The LC50 for the CytA inclusion was 60 ng ml-1, whereas the LC50 for the CryIVD was 85 ng ml-1. In comparison, the LC50s for different combinations of CytA and CryIVD inclusions ranged from 12-15 ng ml-1, 4-5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild-type parasporal bodies of B. thuringiensis subspp. israelensis and morrisoni is due to synergism among three or four of their major proteins.
苏云金芽孢杆菌以色列亚种产生的伴孢晶体以及苏云金芽孢杆菌莫里森亚种PG - 14分离株对蚊虫幼虫的毒性，比其所含的四种杀蚊蛋白（CytA、CryIVA、B和D）中的任何一种至少高20倍。这种高毒性据推测是由于伴孢蛋白之间的协同相互作用。然而，这一点仍存在争议，因为由于用于纯化毒素和检测其对幼虫毒性的方法不同，单个蛋白（尤其是CytA蛋白）的比毒性报告值差异很大。为了解决纯度、比毒性和协同作用的问题，编码CytA和CryIVD毒素的单个基因被克隆，并使用穿梭载体pHT3101在无晶体的苏云金芽孢杆菌以色列亚种细胞中表达。纯化了CytA和CryIVD包涵体，并通过针对埃及伊蚊一龄幼虫的生物测定法单独测定了它们的毒性，以及以不同比例组合时的毒性。CytA包涵体的LC50为60 ng/ml，而CryIVD的LC50为85 ng/ml。相比之下，CytA和CryIVD包涵体不同组合的LC50范围为12 - 15 ng/ml，比单独任何一种蛋白的毒性高4 - 5倍，表明这两种蛋白之间存在明显的协同作用。这些结果表明，苏云金芽孢杆菌以色列亚种和莫里森亚种野生型伴孢晶体的高毒性是由于其三种或四种主要蛋白之间的协同作用。
