STUDY ON PHYSIOLOGICAL FUNCTION OF CHYMOTRYPSIN INHIBITORS IN BOMBYX MORI

(1)凝乳胰蛋白酶抑制剂基因结构与功能分析：家蚕血淋巴中存在16种凝乳胰蛋白酶抑制剂（CI）。由Ict-H基因控制的CI-b1在酸性凝胶上进行电泳检测。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，我们克隆了它们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。在他们的5'侧翼区域，共识TATA和CCAAT盒子被确定。转录因子的其他结合侧如NF-kB、GATA、C/EBP、COUP-TF/HNF-4、ROR α1和HOX3也被检测到。CI-13基因在第293个AGT上缺失了93个碱基，因此缺失了93个碱基上的NF- kB、GATA、C/EBP等motif。在CI-13内含子中，每个内含子上检测到一个逆转录子Bm1，而CI-b1在第一个内含子上存在2个Bm1，在第二个内含子上缺乏Bm1。综上所述，我们推测CI-b1和CI-13在体内的生理功能上有不同的作用，尽管它们都能抑制牛糜凝胰蛋白酶。(2) CI-13相互作用蛋白的检测：注入幼虫体内的Chymotorypsin在血淋巴中与CI-13和CI-b1结合。将LPS和经甲醛处理的大肠杆菌注射到5^< >龄幼虫的第2天，在血淋巴中诱导出了CI-b1结合蛋白，分别为31kDa和14kDa，而CI-13结合蛋白未被诱导。(3)鸡蛋中CI-13的分布：为了检测鸡蛋中与CI-13相互作用的内源性蛋白酶，在与CI-13相同的迁移过程中，在天然凝胶上检测蛋白酶。我们正在研制一种检测蛋白酶活性的检测系统，因为很难检测到蛋白酶在一周内的活性。(4) CI-8受体的纯化：从凝胶中分离CI-8受体，分析n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.