ISOLATION AND ANALYSIS OF RECEPTOR FOR INSECTICIDAL PROTEIN, delta-ENDOTOXIN

苏云金芽孢杆菌产生的三角洲内毒素被称为结晶蛋白体（CPB）。该CPB还被用作微生物农药。CPB特异性毒性的一个决定因素似乎是中肠上皮细胞刷状边界膜（BBM）中的受体。在本项目中，我们尝试从家蚕中肠BBM中分离并分析{CryIA(a)}受体。从苏云金芽孢杆菌中克隆CryIA (a)基因的大肠杆菌中分离到杀虫蛋白CryIA (a)。为了分离CryIA (a)受体，将刷状边界膜泡（BBMV）用2%胆酸溶解，吸附在CryIA (a)固定化Tresyl Toyopearl亲和柱上。亲和柱洗脱液涂于DEAE-Toyopearl上。deae柱测定了纯化受体与^<125>I-CryIA (a)的结合活性，并检测了^<125>I-CryIA (a)对BBMV或溶解BBMV的阻断活性。通过与^<125>I-CryIA的结合活性测定了受体蛋白在PAGE上的电泳图(a)。在^<125>I的自射线图上检测到主带180 kDa和次带170 kDa。在所有纯化过程中使用蛋白酶抑制剂去除170 kDa条带。通过^<125>I-CryIA (a)与BBMV或溶解BBMV的竞争实验，证实了唯一的180 kDa蛋白是受体蛋白。180kda蛋白既不是亮氨酸氨基肽酶也不是碱性磷酸酶。经凝胶过滤柱层析测定，天然受体蛋白分子量约为600 kDa。这些结果表明，天然受体蛋白是由3个180 kDa蛋白组装而成的。180kda蛋白的n端被一些残基阻断，然后用赖氨酸肽酶处理180kda蛋白，随后用高效液相色谱法从酶解液中分离出6个寡肽。寡肽的n端氨基酸序列由蛋白测序仪（Shimazu PSQ-1）测定。180kda蛋白是一种糖基蛋白。少

The delta-endotoxin produced by Bacillus thuringiensis was known as a crystalline protein body (CPB). This CPB was also used as microbial pesticide. It seem that a factor to determine the specific toxicity of CPB is a receptor in brush border membrane (BBM) of mid-gut epithelial cells. Then in this project, we attempt to isolation and analysis of the receptor from BBM of the silkworm mid-gut for {CryIA(a)}.An insecticidal protein, CryIA (a) was isolated from E.coli carrying CryIA (a) gene cloned from B.thuringiensis var.aizawai. In order to isolate the receptor for CryIA (a), the brush border membrane vesicle (BBMV) was solubilized by 2% cholic acid, and was adsorbed on the CryIA (a) -immobilized Tresyl Toyopearl affinity column. The eluate from the affinity column was applied on a DEAE-Toyopearl. The binding activities with ^<125>I-CryIA (a) of the purified receptor by the DEAE-column were determined and blocking activities of ^<125>I-CryIA (a) against BBMV or solubilized BBMV were al … More so detected. An electropherogram of the receptor protein on PAGE was determined by the binding activity with ^<125>I-CryIA (a). A main 180 kDa and minor 170 kDa bands were detected on the autoradiogram of ^<125>I.The 170 kDa band was removed by using of proteinase inhibitors in all purification processes. It was confirmed that the only 180 kDa protein was a receptor protein by the competition experiments of ^<125>I-CryIA (a) and BBMV or the solubilized BBMV.The 180 kDa protein was not a leucine aminopeptidase or an alkaline phosphatase. It was determined that the molecular weight of the native receptor protein was about 600 kDa by a gel filtration column chromatography. These results suggested that the native receptor protein was assemble by three 180 kDa protein. The N-terminal of 180 kDa protein was blocked by some residues, then the 180 kDa protein treated by a lysylendopeptidase and subsequently 6 oligopeptides were isolated from the digested solution by a HPLC.The N-terminal amino acid sequences of the oligopeptides were determined by a protein sequencer (Shimazu PSQ-1). The 180 kDa protein was a kind of glycosyl protein. Less