【摘要】：背景和目的胰蛋白酶抑制劑(trypsin inhibitor,TI)為絲氨酸蛋白酶抑制劑家族的成員之一,普遍存在于自然界的動(dòng)植物及微生物中,能參與調(diào)解體內(nèi)的多重生命活動(dòng)過(guò)程。其中關(guān)于胰蛋白酶抑制劑的抗癌作用,是近年來(lái)的研究熱點(diǎn)。吸血蛭類在醫(yī)療領(lǐng)域的應(yīng)用歷史悠久,我國(guó)藥用蛭最早見(jiàn)于《神農(nóng)本草經(jīng)》。隨著現(xiàn)代藥理學(xué)研究的進(jìn)展,水蛭的抗腫瘤作用得到了大量實(shí)驗(yàn)研究證實(shí),水蛭提取物以及水蛭中提取的小分子蛋白質(zhì)水蛭素顯示出直接抑制腫瘤生長(zhǎng)和增殖,誘導(dǎo)腫瘤細(xì)胞凋亡,抑制腫瘤血管生成等多方面的抗腫瘤作用。迄今為止,對(duì)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,在抗腫瘤方面的作用少有報(bào)道。本研究將探討菲牛蛭中胰蛋白酶抑制劑的分離純化及其功能。方法本文運(yùn)用陰離子交換層析(DEAE Sephadex A-50)、反相高壓液相色譜(RP-HPLC)等分離純化手段首次從菲牛蛭的頭部勻漿液中純化得到一個(gè)具有胰蛋白酶抑制活性的蛋白,隨后通過(guò)基質(zhì)輔助激光解析電離飛行時(shí)間質(zhì)譜(MALDI-TOF-MS)確定其分子量。通過(guò)Edman降解法測(cè)定的該蛋白N端部分氨基酸序列,利用測(cè)定獲得的氨基酸序列設(shè)計(jì)特異性引物,從構(gòu)建的菲牛蛭頭部c DNA文庫(kù)中擴(kuò)增得到編碼該蛋白的c DNA序列。將其編碼序列通過(guò)http://web.expasy.org/compute_pi/網(wǎng)站預(yù)測(cè)獲得其理論分子量,再運(yùn)用NCBI中BLAST功能以及Lasergene軟件中Meg Align進(jìn)行序列比對(duì)分析其結(jié)構(gòu)特點(diǎn)。通過(guò)蛋白酶活性檢測(cè),確定Bdellin-HM對(duì)胰蛋白酶,彈性蛋白酶,胰凝乳蛋白酶,激肽釋放酶,凝血酶,血纖維蛋白溶酶,FXIIa,FXIa,FXa活性的影響,并運(yùn)用Dixon plot酶動(dòng)力學(xué)曲線計(jì)算得出其抑制常數(shù)。結(jié)果1.通過(guò)分離純化方法成功獲得胰蛋白酶抑制劑Bdellin-HM的純品。2.通過(guò)菲牛蛭頭部c DNA文庫(kù)的構(gòu)建與篩選得到編碼Bdellin-HM的c DNA序列,該序列編碼的成熟肽由149個(gè)氨基酸組成,含6個(gè)半胱氨酸。網(wǎng)站預(yù)測(cè)其理論分子量為17,438.04Da,比天然蛋白的分子量大6Da,表明天然蛋白形成了三對(duì)分子內(nèi)二硫鍵。3.結(jié)構(gòu)分析表明,Bdellin-HM屬于Kazal型蛋白酶抑制劑,并同Bdellin B-3以及Bdellin-KL具有高度相似性。4.酶活性研究顯示,Bdellin-HM具有顯著并專一的胰蛋白酶抑制作用,抑制常數(shù)Ki達(dá)到(8.12±0.18)*10-9 M,而對(duì)彈性蛋白酶、胰凝乳蛋白酶、激肽釋放酶、凝血因子(F)XIIa、FXIa、FXa、凝血酶與血纖維蛋白溶酶無(wú)明顯抑制作用。結(jié)論本文首次從菲牛蛭中純化得到胰蛋白酶抑制劑Bdellin-HM,其對(duì)胰蛋白酶具有顯著且特異的抑制作用。該工作為進(jìn)一步研究Bdellin-HM的抗腫瘤作用、作用機(jī)制以及菲牛蛭在腫瘤中的研究提供了基礎(chǔ)。
[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 鹵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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R914

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