STUDY ON PHYSIOLOGICAL FUNCTION OF CHYMOTRYPSIN INHIBITORS IN BOMBYX MORI

(1)糜蛋白酶抑制剂基因结构和功能分析：家蚕血淋巴中存在16种糜蛋白酶抑制剂(CI)。 CI-b1由Ict-H基因控制，通过酸性凝胶电泳检测。 CI-b1是一种碱性蛋白质，经过纯化，由62个氨基酸残基组成。为了阐明 CI-b1 和 CI-13 基因的基因组结构，对它们进行了克隆并确定了它们的核苷酸序列。它们由 3 个外显子组成，间隔 2 个内含子。在其 5' 侧翼区域，鉴定出了 TATA 和 CCAAT 共有框。还检测到转录因子的其他结合面，例如 NF-kB、GATA、C/EBP、COUP-TF/HNF-4、ROR α1 和 HOX3。而CI-13基因在第293位AGT处缺失了93个碱基，因此93个碱基上的NF-kB、GATA、C/EBP、基序缺失。在CI-13内含子中，每个内含子上检测到一个反座子Bm1，但CI-b1在第一个内含子上存在2个Bm1，而在第二个内含子上缺乏Bm1。根据结果​​，我们推测CI-b1和CI-13虽然都抑制牛胰凝乳蛋白酶，但对体内生理功能发挥不同的作用。 (2)与CI-13相互作用的蛋白质的检测：注射到幼虫体内的糜蛋白酶在血淋巴中与CI-13和CI-b1结合。将用甲醛处理的LPS和大肠杆菌注射到第2天的5龄幼虫中，在血淋巴中诱导出CI-b1结合蛋白(31kDa和14kDa)，但没有诱导CI-13结合蛋白。 (3)CI-13在鸡蛋中的分布：为了检测鸡蛋中与CI-13相互作用的内源蛋白酶，在天然凝胶上检测与CI-13相同迁移的蛋白酶。我们正在努力建立一个检测系统来检测蛋白酶活性，因为很难检测非常弱的蛋白酶活性。 (4)CI-8受体的纯化：从凝胶中分离CI-8受体以分析N-末端氨基酸序列。

(1)Analysis of chymotorypsin inhibitor gene structure and function : There are 16 kinds of chymotrypsin inhibitor (CI) in hemolymph of B. mori. CI-b1, which is controlled by Ict-H gene, was detected on acidic gel by electrophoresis. CI-b1 was a basic protein and was purified and consisted of 62 amino acid residues. To elucidate genomic structure of CI-b1 and CI-13 genes, they were cloned and determined their nucleotide sequences. They consisted of 3 exones spaced by 2 introns. In their 5'-flanking region, consensus TATA and CCAAT boxes were identified. Other binding sides for transcription factors such as NF-kB, GATA, C/EBP, COUP-TF/HNF-4, ROR α1, AND HOX3 were also detected. And CI-13 gene were deleted 93 bases at 293th form AGT, therefore NF- kB, GATA, C/EBP, motif on 93 bases were absent. In CI-13 introns, one retroposon Bm1 was detected on each intron, but CI-b1 existed 2 Bm1 on first intoron and was lack of Bm1 on second intron. On the results, we suppose that CI-b1 and CI-13 plays different role on physiological function in vivo, although they both inhibit bovine chymotrypsin. (2) Detection of proteins interacting CI-13 : Chymotorypsin injected into larva was combined CI-13 and CI-b1 in hemolymph. The LPS and E. coli treated with formaldehyde were injected into day 2 of the 5^<th> instar larvae, CI-b1 binding proteins, which were 31kDa and 14kDa, were induced in hemolymph, but CI-13 binding protein did not. (3) Distribution of CI-13 in egg : To detect endogenous proteases interacting with CI-13 in egg, The protease was detected on native gel at the same migration of CI-13. We are working to make a assay system to detect the protease activity because it is difficult to detect protease activity for the very week activity. (4) Purification of CI-8 receptor : CI-8 receptor isolated from gel was carried out to analyze N-terminal amino sequence.