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称为结晶蛋白体(CPB)。该CPB还可用作微生物农药。提示CPB特异性毒性的一个决定因素可能是中肠上皮细胞刷状缘膜上的受体。在本课题中，我们试图从家蚕中肠的BBM中分离并分析其受体{CryIA(A)}。从从苏云金杆菌aizwai中克隆的携带CryIA(A)基因的大肠杆菌中分离到一种杀虫蛋白CryIA(A)。为了分离CryIA(A)的受体，用2%的胆酸增溶刷状缘膜囊泡(BBMV)，并将其吸附在CryIA(A)固定的Tresyl Toyota opearl亲和柱上。将来自亲和层析柱的洗脱液涂抹在DEAE-Toyota opearl上。用DEAE柱测定了纯化受体与β-CryIA(A)的结合活性，发现其对甜菜花叶病毒和溶解的甜菜花叶病毒的阻断活性均为…。检测到的情况更是如此。PAGE上受体蛋白的电泳图是通过与~(125)&GT；I-CryIA(A)的结合活性确定的。在~(125)GT；I的放射自显影上检测到主要的180 kDa条带和次要的170 kDa条带，其中170 kDa条带在所有纯化过程中均被用酶抑制剂去除。通过与BBMV或BBMV的竞争实验，证实仅有的180 kDa蛋白是受体蛋白，不是亮氨酸氨基肽酶，也不是碱性磷酸酶。凝胶过滤柱层析法测定天然受体蛋白的相对分子质量约为600 kDa。这些结果表明，天然受体蛋白是由3个180 kDa的蛋白组装而成。将180 kDa蛋白的N端用一定量的残基封闭后，用高效液相色谱仪(HPLC)从消化液中分离得到6个寡肽，并用岛津PSQ-1蛋白质测序仪测定其N端氨基酸序列。180 kDa蛋白是一种糖基蛋白。较少

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