Analysis of Biological Domains in Insecticidal Protein from B. Thuringiensis

苏云金芽孢杆菌变种以色列产杀虫晶体蛋白(ICP)--β-内毒素，能特异性杀死双翅目昆虫幼虫、蚊子和黑蝇。其毒性包括：1)双翅目昆虫杀虫；2)昆虫细胞毒性；3)昆虫特异性神经毒性；4)哺乳动物细胞毒性；5)哺乳动物溶血性；6)乳鼠毒性。ICP SENE编码128 kDa(ISRH3)和135 kDa(ISRH4)蛋白质，并根据核苷酸序列确定。ISRH3基因由3405个氨基酸组成，编码1135个氨基酸。在本研究中，我们试图确定ISRH3蛋白在活性上的活性区域。通过几种限制性内切酶的处理，构建了几个N端C端缺失的ISRH3突变体，并用蚊子幼虫和建立的昆虫细胞(TN-368)对这些基因的蛋白进行了检测。编码N端到第635位氨基酸的基因产生的蛋白质也具有杀虫剂…更多的铝和昆虫的细胞溶解活性。然而，从N端到第602位的多肽失去了这两种活性。613-1135氨基酸(C端)的蛋白质具有这两种活性，而632或641-C端的蛋白质则没有明确的活性结果。然后是716号的蛋白质。C端失去了活动。ISRH3蛋白缺失了中央部分第560-772位氨基酸，也失去了ITS。560-820的几个小肽中心部分失去了活性。这些结果表明，613-635号氨基酸的中心部分是一个结构域，另一个结构域(R或R‘)分别位于N端或C端。推测杀虫和昆虫细胞毒活性结构域需要两个结构域，即A结构域和R或R‘结构域，并认为杀虫活性是昆虫细胞毒活性。然而，ISRH3不具有哺乳动物细胞毒性、溶血活性和乳鼠毒性。抗ISRH蛋白抗体的表位识别第773-820号附近的氨基酸序列。较少

Bacillus thuringiensis var. Israelensis (Bti) produces insecticidal crystalline protein (ICP), delta-endotoxin, which specifically kills dipteran insect larvae, mosquito and black fly. The ICP have several toxicities, that is, 1) dipteran insecticidal, 2) insect cell toxic, 3) insect specific neurotoxic, 4) mammalian cell toxic, 5) mammalian hemolytic and 6) suckling mouse toxic activities. The ICP senes encode 128 kDa (ISRH3) and 135 kDa (ISRH4) proteins and determined as respects the nucleotide sequences. The ISRH3 gene is consisted of 3405 by encoding 1135 amino acid. Then in this project, we attempt to identify the active domain in ISRH3 protein on the activities.Several mutants of ISRH3 deleted from C of N terminal were constructed by treatment of several restriction enzymes and the proteins from the genes were assayed by the mosquito larvae and an established insect cell (TN-368 ). The protein produced by the gene encoding from N terminal to No. 635 amino acid has also insecticid … More al and insect cytolytic activities. However, the peptide from N terminal to No. 602 loses these two activities. The proteins of No. 613 - No. 1135 amino acid (C terminal) have the two activities, though the proteins of No. 632 or No. 641 - C terminal showed no clear results as to the activities. Then that protein of No. 716 - No. C terminal had lost the activities. The ISRH3 proteins deleted a central parts No. 560 - No. 772 amino acid had also lost its. The several small peptides central parts, No. 560 - No. 820 had lost the activities. From these results the central parts included No. 613 - No. 635 amino acid is one of domain and another domain (R or R') locate at N or C terminal, respectively. It was suggested that the domains for insecticidal and insect cell toxic activities required two domains, that is, A and another R or R' domains and also suggested the insecticidal activities was the insect cell toxic activity. However, it is ISRH3 does not have the mammalian cell toxic, hemolytic activities and the suckling mouse toxicity. The epitop of an antibody against ISRH proteins recognizes amino acid sequences around No. 773 - No. 820. Less