菲牛蛭胰蛋白酶抑制剂Bdenlin-HM的分离纯化及功能研究
发布时间：2019-01-30 07:56
【摘要】：背景和目的胰蛋白酶抑制剂(trypsin inhibitor,TI)为丝氨酸蛋白酶抑制剂家族的成员之一,普遍存在于自然界的动植物及微生物中,能参与调解体内的多重生命活动过程。其中关于胰蛋白酶抑制剂的抗癌作用,是近年来的研究热点。吸血蛭类在医疗领域的应用历史悠久,我国药用蛭最早见于《神农本草经》。随着现代药理学研究的进展,水蛭的抗肿瘤作用得到了大量实验研究证实,水蛭提取物以及水蛭中提取的小分子蛋白质水蛭素显示出直接抑制肿瘤生长和增殖,诱导肿瘤细胞凋亡,抑制肿瘤血管生成等多方面的抗肿瘤作用。迄今为止,对菲牛蛭及其提取物的研究仍主要集中于抗凝活性、降血脂作用、抗栓和溶栓作用等方面,在抗肿瘤方面的作用少有报道。本研究将探讨菲牛蛭中胰蛋白酶抑制剂的分离纯化及其功能。方法本文运用阴离子交换层析(DEAE Sephadex A-50)、反相高压液相色谱(RP-HPLC)等分离纯化手段首次从菲牛蛭的头部匀浆液中纯化得到一个具有胰蛋白酶抑制活性的蛋白,随后通过基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)确定其分子量。通过Edman降解法测定的该蛋白N端部分氨基酸序列,利用测定获得的氨基酸序列设计特异性引物,从构建的菲牛蛭头部c DNA文库中扩增得到编码该蛋白的c DNA序列。将其编码序列通过http://web.expasy.org/compute_pi/网站预测获得其理论分子量,再运用NCBI中BLAST功能以及Lasergene软件中Meg Align进行序列比对分析其结构特点。通过蛋白酶活性检测,确定Bdellin-HM对胰蛋白酶,弹性蛋白酶,胰凝乳蛋白酶,激肽释放酶,凝血酶,血纤维蛋白溶酶,FXIIa,FXIa,FXa活性的影响,并运用Dixon plot酶动力学曲线计算得出其抑制常数。结果1.通过分离纯化方法成功获得胰蛋白酶抑制剂Bdellin-HM的纯品。2.通过菲牛蛭头部c DNA文库的构建与筛选得到编码Bdellin-HM的c DNA序列,该序列编码的成熟肽由149个氨基酸组成,含6个半胱氨酸。网站预测其理论分子量为17,438.04Da,比天然蛋白的分子量大6Da,表明天然蛋白形成了三对分子内二硫键。3.结构分析表明,Bdellin-HM属于Kazal型蛋白酶抑制剂,并同Bdellin B-3以及Bdellin-KL具有高度相似性。4.酶活性研究显示,Bdellin-HM具有显著并专一的胰蛋白酶抑制作用,抑制常数Ki达到(8.12±0.18)*10-9 M,而对弹性蛋白酶、胰凝乳蛋白酶、激肽释放酶、凝血因子(F)XIIa、FXIa、FXa、凝血酶与血纤维蛋白溶酶无明显抑制作用。结论本文首次从菲牛蛭中纯化得到胰蛋白酶抑制剂Bdellin-HM,其对胰蛋白酶具有显著且特异的抑制作用。该工作为进一步研究Bdellin-HM的抗肿瘤作用、作用机制以及菲牛蛭在肿瘤中的研究提供了基础。
[Abstract]:Background and objective trypsin inhibitor (trypsin inhibitor,TI) is a member of the serine protease inhibitor family. It is widely found in animals, plants and microorganisms in nature, and can participate in the regulation of multiple life processes in vivo. Among them, the anticancer effect of trypsin inhibitors is a hot topic in recent years. Blood-sucking leeches have a long history of application in the medical field. The medicinal leeches were first found in Shennong Herbal Classic in China. With the development of modern pharmacology, the antitumor effect of Hirudo has been confirmed by a large number of experimental studies. Hirudin, a small molecular protein extracted from leech extract, has been shown to directly inhibit tumor growth and proliferation. It can induce tumor cell apoptosis and inhibit tumor angiogenesis. Up to now, the studies on Leech and its extracts are mainly focused on anticoagulant activity, antihyperlipidemic effect, antithrombotic and thrombolytic effect, etc. In this study, the purification and function of trypsin inhibitors from Leech were studied. Methods A protein with trypsin inhibitory activity was first purified from the head homogenate of Leech by anion exchange chromatography (DEAE Sephadex A-50) and reversed-phase high performance liquid chromatography (RP-HPLC). Then the molecular weight was determined by matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS). The N-terminal amino acid sequence of the protein was determined by Edman degradation method. A specific primer was designed using the amino acid sequence to amplify the c DNA sequence of the protein from the constructed c DNA library of the head of Leech. Its theoretical molecular weight is obtained by predicting its coding sequence through http://web.expasy.org/compute_pi/ website, and its structural characteristics are analyzed by using BLAST function in NCBI and Meg Align in Lasergene software. The effects of Bdellin-HM on the activities of trypsin, elastase, chymotrypsin, kallikrein, thrombin, blood fibrinolytic enzyme and FXIIa,FXIa,FXa were determined. The inhibition constant was calculated by using the kinetic curve of Dixon plot enzyme. Result 1. The purified trypsin inhibitor Bdellin-HM was obtained by isolation and purification. 2. The cDNA sequence of c DNA encoding Bdellin-HM was obtained by construction and screening of c DNA library in the head of Leech. The mature peptide encoded by the sequence consists of 149 amino acids and contains 6 cysteine. Its theoretical molecular weight was 17438.04 Daa, which was 6Da. higher than that of natural protein, indicating that the natural protein formed three pairs of intramolecular disulfide bonds. Structural analysis showed that Bdellin-HM belongs to Kazal type protease inhibitor and has high similarity with Bdellin B-3 and Bdellin-KL. 4. The enzyme activity showed that Bdellin-HM had significant and specific trypsin inhibitory effect, and the inhibitory constant Ki was (8.12 卤0.18) * 10-9 M. however, it could inhibit elastase, chymotrypsin and kallikrein. (F) XIIa,FXIa,FXa, thrombin and fibrinolytic enzyme had no obvious inhibitory effect. Conclusion Bdellin-HM, a trypsin inhibitor, was purified from Leech for the first time and has a remarkable and specific inhibitory effect on trypsin. This work provides a basis for further study on the antitumor effect and mechanism of Bdellin-HM and the study of Leech in tumor.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914
本文编号：2417920
[Abstract]:Background and objective trypsin inhibitor (trypsin inhibitor,TI) is a member of the serine protease inhibitor family. It is widely found in animals, plants and microorganisms in nature, and can participate in the regulation of multiple life processes in vivo. Among them, the anticancer effect of trypsin inhibitors is a hot topic in recent years. Blood-sucking leeches have a long history of application in the medical field. The medicinal leeches were first found in Shennong Herbal Classic in China. With the development of modern pharmacology, the antitumor effect of Hirudo has been confirmed by a large number of experimental studies. Hirudin, a small molecular protein extracted from leech extract, has been shown to directly inhibit tumor growth and proliferation. It can induce tumor cell apoptosis and inhibit tumor angiogenesis. Up to now, the studies on Leech and its extracts are mainly focused on anticoagulant activity, antihyperlipidemic effect, antithrombotic and thrombolytic effect, etc. In this study, the purification and function of trypsin inhibitors from Leech were studied. Methods A protein with trypsin inhibitory activity was first purified from the head homogenate of Leech by anion exchange chromatography (DEAE Sephadex A-50) and reversed-phase high performance liquid chromatography (RP-HPLC). Then the molecular weight was determined by matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS). The N-terminal amino acid sequence of the protein was determined by Edman degradation method. A specific primer was designed using the amino acid sequence to amplify the c DNA sequence of the protein from the constructed c DNA library of the head of Leech. Its theoretical molecular weight is obtained by predicting its coding sequence through http://web.expasy.org/compute_pi/ website, and its structural characteristics are analyzed by using BLAST function in NCBI and Meg Align in Lasergene software. The effects of Bdellin-HM on the activities of trypsin, elastase, chymotrypsin, kallikrein, thrombin, blood fibrinolytic enzyme and FXIIa,FXIa,FXa were determined. The inhibition constant was calculated by using the kinetic curve of Dixon plot enzyme. Result 1. The purified trypsin inhibitor Bdellin-HM was obtained by isolation and purification. 2. The cDNA sequence of c DNA encoding Bdellin-HM was obtained by construction and screening of c DNA library in the head of Leech. The mature peptide encoded by the sequence consists of 149 amino acids and contains 6 cysteine. Its theoretical molecular weight was 17438.04 Daa, which was 6Da. higher than that of natural protein, indicating that the natural protein formed three pairs of intramolecular disulfide bonds. Structural analysis showed that Bdellin-HM belongs to Kazal type protease inhibitor and has high similarity with Bdellin B-3 and Bdellin-KL. 4. The enzyme activity showed that Bdellin-HM had significant and specific trypsin inhibitory effect, and the inhibitory constant Ki was (8.12 卤0.18) * 10-9 M. however, it could inhibit elastase, chymotrypsin and kallikrein. (F) XIIa,FXIa,FXa, thrombin and fibrinolytic enzyme had no obvious inhibitory effect. Conclusion Bdellin-HM, a trypsin inhibitor, was purified from Leech for the first time and has a remarkable and specific inhibitory effect on trypsin. This work provides a basis for further study on the antitumor effect and mechanism of Bdellin-HM and the study of Leech in tumor.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914
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