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

苏云金芽孢杆菌产生的δ-内毒素被称为晶体蛋白体（CPB）。该CPB也可用作微生物农药。中肠上皮细胞刷状缘膜（BBM）上的受体可能是决定CPB特异性毒性的一个因素。本研究从苏云金杆菌蓝泽变种（B. thuringiensisvar. aizawai）中克隆了CryIA（a）基因，并从大肠杆菌中分离得到了一种杀虫蛋白CryIA（a）。为了分离CryIA（a）的受体，用2%胆酸溶解刷状缘膜囊泡（BBMV），并将其吸附在固定化CryIA（a）的Tresyl Toyopolysin亲和柱上。将来自亲和柱的洗脱液施加到DEAE-Toyopolymer上。用<125>DEAE柱测定了纯化的受体与~（131）I-CryIA（a）的结合活性，并测定了~（131）<125>I-CryIA（a）对BBMV或可溶性BBMV的阻断活性。 ...更多信息 如此检测。通过与^ I-CryIA（a）的结合活性确定受体蛋白在PAGE上的电泳图<125>。放射自显影图上检测到主要的180 kDa和次要的170 kDa条带<125>。在所有纯化过程中使用蛋白酶抑制剂去除170 kDa条带。通过与BBMV或溶解的BBMV的竞争实验，证实了仅有的180 kDa蛋白是受体蛋白<125>，该180 kDa蛋白不是亮氨酸氨肽酶或碱性磷酸酶。通过凝胶过滤柱层析确定天然受体蛋白的分子量为约600 kDa。这些结果表明，天然受体蛋白是由三个180 kDa的蛋白组装而成的。将180 kDa蛋白的N-末端封闭，然后用赖氨酰内肽酶处理180 kDa蛋白，用高效液相色谱法从酶解液中分离得到6条寡肽，用Shimazu PSQ-1蛋白质测序仪测定了N-末端氨基酸序列。180 kDa蛋白为糖基蛋白。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