STUDY ON PHYSIOLOGICAL FUNCTION OF CHYMOTRYPSIN INHIBITORS IN BOMBYX MORI

家蚕胰凝乳蛋白酶抑制剂的生理功能研究

• 批准号: 11306005
• 负责人: FUJII Hiroshi
• 金额: $ 26.27万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11306005/
• 关键词: Bombyx mori.; chymotorupsin inhibitor; hemolymph; CI-8 receptor; NF-kB; 体液; CI-13i遺伝子ゲノム解析; CI-b1遺伝子ゲノム解析; Bomyxmon; CI-10の精製; CI-8結合蛋白質; 中腸組織; リガンドアッセイ法; 35k protease遺伝子; Bomyx mori; Bomyxmori; キモトリプシンインヒビター; CI-13遺伝子; CI-3cDNA塩基配列決定

(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.

(1)糜蛋白酶抑制物基因结构与功能分析：家蚕血淋巴中有16种糜蛋白酶抑制物(CI)。在酸性凝胶上电泳法检测到由ICT-H基因控制的CI-b1。CI-b1是一种碱性蛋白，经纯化后含有62个氨基酸残基。为了阐明CI-b1和CI-13基因的基因组结构，克隆了这两个基因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，发现了一致的TATA和CCAAT盒。还检测到了转录因子的其他结合侧，如核因子-kB、GATA、C/EBp、COUP-Tf/hNf-4、RoRα1和HOX3。AGT第293位缺失了CI-13基因93个碱基，缺失了93个碱基上的核因子-kB、GATA、C/EBP、基序。在CI-13内含子中，每个内含子上检测到一个反转录Bm1，而CI-b1在第一内含子上存在2个Bm1，在第二内含子上缺失Bm1。根据这一结果，我们推测CI-b1和CI-13在体内对生理功能的作用不同，尽管它们都对牛胰凝乳酶有抑制作用。(2)CI-13相互作用蛋白的检测：在幼虫体内注射糜蛋白酶后，CI-13与血淋巴中的CI-b1结合。将经甲醛处理的内毒素和大肠杆菌注入5龄幼虫第2天，在血淋巴中可诱导出大小分别为31 kDa和14 kDa的CI-b1结合蛋白，而不能诱导CI-13结合蛋白。(3)CI-13在鸡蛋中的分布：为了检测与CI-13相互作用的内源性蛋白水解酶，在天然凝胶上检测到与CI-13迁移相同的酶。我们正在努力建立一个检测系统来检测蛋白酶的活性，因为很难检测到每周的酶活性。(4)CI-8受体的纯化：从凝胶中分离CI-8受体，对其N端氨基酸序列进行分析。

项目成果

Ujita M., A.Kimura, D.Nishino, H.yokoyama, Y.Banno, H.Fujii, and A.Hara: "Silkworm Bombyx mori 30-kDa lipoprotein s specifically bind to glucose-containing carbohydrate."Biosci.Biotechnol.Biochem.. 66(10). 2264-2266 (2002)

Ujita M.、A.Kimura、D.Nishino、H.yokoyama、Y.Banno、H.Fujii 和 A.Hara：“家蚕 Bombyx mori 30-kDa 脂蛋白特异性结合含葡萄糖的碳水化合物。”Biosci.Biotechnol

Takashi Nakamura et al.: "Genetic of the "wild silkworm translucent" mutant (ows) discovered in the progenies after the cross between the domesticated silkworm, Bombyx mori and the sild mulberry silkworm Bombyx mandarina"Int J.Wild Silkworm and Silk. 6. 7

Takashi Nakamura 等人：“家蚕、家蚕和桑蚕 Bombyx mandarina 杂交后在后代中发现的“野蚕半透明”突变体（ows）的遗传”Int J.Wild Silkworm and Silk。

Yamamoto: "Isolation and characterization of prophenoloxidase isoforms from the silkworm, Bombyx mori(a80 strain)"J.Seric.Sci.Jpn.. 68・1. 65-72 (1999)

山本：“家蚕（Bombyx mori）（a80株）中酚氧化酶原异构体的分离和鉴定”J.Seric.Sci.Jpn.. 68・1（1999）。

姜永煌: "カイコ消化液35kプロテアーゼの精製とその性質"日本蚕糸学雑誌,. 69・1. 47-53 (2000)

康永皇：“蚕消化液35k蛋白酶的纯化及其特性”日本血清学杂志，69・1（2000）。

Ujita M et al.: "Silkworm Bombyx mori 30-kDa lipoproteins specifically bind to glucose-containinig carbohydrates"Bioscience, Biotechnology and Biochemistry. 66(10). 2264-2264 (2002)

Ujita M 等人：“蚕 Bombyx mori 30-kDa 脂蛋白特异性结合含葡萄糖碳水化合物”生物科学、生物技术和生物化学。