STUDY ON PHYSIOLOGICAL FUNCTION OF CHYMOTRYPSIN INHIBITORS IN BOMBYX MORI

（1）胰凝乳蛋白酶抑制剂基因结构与功能分析：B血淋巴中有16种胰凝乳蛋白酶抑制剂（chymotrypsin inhibitor，CI）。森。在酸性凝胶电泳上检测到受Ict-H基因控制的CI-b1。CI-b1是一种碱性蛋白，经纯化后由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，克隆并测定了它们的核苷酸序列。它们由3个外显子和2个内含子组成。在它们的5 '侧翼区，鉴定了共有TATA和CCAAT盒。还检测到其他转录因子如NF-κ B、加塔、C/EBP、COUP-TF/HNF-4、ROR α1和HOX 3的结合侧。CI-13基因第293位AGT缺失93个碱基，因此缺失NF-κ B、加塔、C/EBP等基序。在CI-13内含子中，每个内含子上检测到一个反转座子Bm 1，而CI-b1在第一内含子上存在2个Bm 1，在第二内含子上缺少Bm 1。根据以上结果，我们推测CI-13和CI-b1虽然都具有抑制牛胰凝乳蛋白酶的作用，但在体内发挥的生理功能不同。(2)CI-13相互作用蛋白的检测：注射胰凝乳蛋白酶后，血淋巴中CI-13与CI-b1结合。LPS和E.将甲醛处理的大肠杆菌注射到5龄2日龄幼虫体内<th>，在血淋巴中诱导出31 kDa和14 kDa的CI-b1结合蛋白，而未诱导出CI-13结合蛋白。(3)CI-13在蛋中的分布：为了检测蛋中与CI-13相互作用的内源性蛋白酶，在CI-13的相同迁移下在天然凝胶上检测蛋白酶。由于蛋白酶活性很弱，很难检测到蛋白酶的活性，因此我们正在努力建立一种检测蛋白酶活性的检测系统。(4)Cl-8受体的纯化：从凝胶中分离Cl-8受体，进行N-末端氨基酸序列分析。

(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.