Analysis of Biological Domains in Insecticidal Protein from B. Thuringiensis

苏云金芽孢杆菌（Bacillus thuringiensis var. Israelensis, Bti）产生杀虫结晶蛋白（ICP）、三角洲内毒素，对双翅目昆虫幼虫、蚊子和黑蝇具有特异性杀伤作用。ICP具有几种毒性，即1)杀虫双翅虫，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)哺乳动物溶血毒性和6)哺乳小鼠毒性。ICP基因编码128 kDa （ISRH3）和135 kDa （ISRH4）蛋白，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。然后在本项目中，我们尝试鉴定ISRH3蛋白的活性结构域上的活性。利用几种限制性内切酶处理，构建了N端C缺失的ISRH3基因突变体，并利用蚊子幼虫和已建立的昆虫细胞TN-368检测了突变体的蛋白表达。该基因编码的N端至635号氨基酸所产生的蛋白也具有杀虫和昆虫的活性。然而，从N端到602号的肽失去了这两个活性。613 - 1135号氨基酸（C端）的蛋白具有这两种活性，而632和641 - C端的蛋白没有明显的活性。那么716号的蛋白质。C终端失去了活动。ISRH3蛋白缺失了一个中心部分，编号560 - 772的氨基酸也丢失了。560 ~ 820号中间的几个小肽部分失去了活性。从这些结果可以看出，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。表明杀虫活性和昆虫细胞毒性活性的结构域需要两个结构域，即A和另一个R或R'结构域，并表明杀虫活性是昆虫细胞毒性活性。然而，ISRH3不具有哺乳动物细胞毒性、溶血活性和哺乳小鼠毒性。抗ISRH蛋白抗体的表位识别773 - 820号附近的氨基酸序列。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