STUDY ON PHYSIOLOGICAL FUNCTION OF CHYMOTRYPSIN INHIBITORS IN BOMBYX MORI

点击翻译按钮获取中文摘要

(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.