【作者】 郭强；

【导师】 韩曜平；

【作者基本信息】 苏州大学 ， 动物学， 2012， 硕士

【摘要】 丝氨酸蛋白酶抑制剂(Serine Protease Inhibitor)作为一类具有抑制蛋白酶活性作用的物质,在动物、植物、微生物中广泛存在，能与体内参与各种调控作用的蛋白酶形成一定的动态平衡,调节生物体内许多重要的生命过程。根据其一级结构、三维模型和作用机制，可将其分为至少18个家族。蛋白酶抑制剂作为调节者，在生物体内一系列的生理作用中起着重要作用。在脊椎动物体内，与血液凝固，纤维蛋白溶解作用，炎症，补体激活有关；在非脊椎动物的免疫中，能调节蛋白酶活性包括血淋巴凝集，酚氧化酶活性以及细胞活素类和抗菌肽合成。由于外源性蛋白酶是重要的病原因子，许多蛋白酶抑制剂能直接抑制病原生物的外源性蛋白酶的水解作用，起到病害防御作用。研究表明，鱼卵中存在着种类丰富的蛋白酶抑制剂成分，在鱼卵中除执行生理调节功能外，在病原生物防御中也起到了重要作用。然而，截止目前只对少数鱼卵中的蛋白酶抑制剂进行了研究，有关鱼卵自身的防御机制依然存疑。因此，本研究采用鲫鱼卵作为主要研究对象，利用生物化学，分子生物学手段和研究方法，从鲫鱼卵中成功获得了一种丝氨酸蛋白酶抑制剂，并对其进行进一步性质研究，为进一步探讨其生理、防御功能，及其在食品、医药等领域的利用提供基础数据。本实验主要内容及结果如下：1、收集四种常见鱼卵（沙塘鳢，鲫鱼，草鱼和乌鳢），以发色底物对其进行丝氨酸蛋白酶抑制活性测定，包括胰蛋白酶、弹性蛋白酶、枯草杆菌蛋白酶和胰凝乳蛋白酶。结果表明，除胰凝乳蛋白酶外，四种鱼卵提取物对其余3种丝氨酸蛋白酶都具有较强抑制活性。2、通过ConA Sepharose4B亲和层析、Sephadex G-75凝胶过滤、反相高压液相等生化技术手段，从鲫鱼卵中获得一个表观分子量约为43kDa的单链蛋白。我们将其命名为CaSPI。CaSPI对胰蛋白酶具有专一性的抑制活性。Edman降解法测序结果显示CaSPI的N-末端氨基酸序列为EAHPSHSGED。糖蛋白检测结果表明CaSPI属于糖蛋白。接合活性测定中，CaSPI在SDS-PAGE电泳时能与胰蛋白酶形成稳定的复合物，该结果提示它可能属于Serpins蛋白超家族中的一员。3、利用RT-PCR和RACE技术，获得了一条全长为1397bp，其中包括1233bp完整的开放阅读框的核酸序列，编码410个氨基酸。对由核酸序列推测的氨基酸序列分析显示，在52-408处具有SERPIN结构域。经过BLAST比对，该蛋白与来源于鲤鱼围脑膜液的CP9等Serpins超家族的丝氨酸蛋白酶抑制剂具有高度同源性，并且在P1位置处为精氨酸（Arg），推测该蛋白属于Sepins超家族一员。4、对所获得的蛋白酶抑制剂CaSPI进行结合比和及抑制常数K_i测定，结果为CaSPI与胰蛋白酶结合比为1.15：1时能完全抑制蛋白酶水解活性，K_i为0.46μM，表明CaSPI为竞争性抑制剂。并对其进行稳定性（时间、温度、pH、金属离子及变性剂）检测，表明CaSPI具有相当的稳定性。抑菌活性检测结果显示CaSPI能分别抑制革兰氏阴性菌（Escherichia coli）和革兰氏阳性菌（Bacillus subtilis）的生长，具有一定的抑菌作用。更多还原

【Abstract】 As a kind of inhibition substances, Serine protease inhibitors are distributed widelyin animals, plants and microorganisms. Based on the protease inhibitor primary andthree-dimensional structures, together with inhibition mechanisms, they are classifiedinto at least18families. They act as modulators, playing key roles in a variety ofphysiological functions in vivo, such as blood coagulation, fibrinolysis, apoptosis,development, inflammation and complement activation in vertebrate and Bloodlymphatic agglutinate, polyphenoloxidase activity, cytokines and antimicrobial peptidessynthesis in invertebrate. Due to exogenous protease is an important pathogenic factor,a major function of proteinase inhibitors is to avoid the hydrolysis of protease bypathogenic organisms.Studies show that there exist a variety of protease inhibitors in fish eggs. Inaddition to perform physical adjustment function, also play an important role in defenseof pathogens. However, only a few kinds of eggs have been studied so far, and themechanism of the defense itself is still doubtful. Therefore, carp eggs are used as themain object in this study, using biochemistry and molecular biology methods,successfully obtained a serine protease inhibitor from carp eggs and further studiedthe characterization of it. To further explore the physiological defense function, andprovides experimental basis on food, medicine and other applications in the future. Themain contents and the results of this experiment are as follows:1. Collected four common fish eggs (Odontobutis obscurus, Carassius auratus,Ctenopharyngodon idellus, Channa argus), and measured serine protease(trypsin,elastase, subtilisin,chymotrypsin) inhibitory activity by chromogenic substrate. Theresults showed that in addition to chymotrypsin, the other three kinds of serine proteaseswere strongly inhibitored.2. A inhibitor with an apparent molecular weight of43kDa was purified from theegg of carp by a combination of ConA Sepharose4B chromatography, gel filtrationchromatography and reverse phase high performance liquid chromatography (RP-HPLC) steps. It was named CaSPI and showed a trypsin inhibitor activity. The partialN-terminal amino acid sequence of43kDa protein was EAHPSHSGED. Glycoproteinstaining results showed that the CaSPI a glycoprotein. In the determination of jointactivity, CaSPI formed a stable complex with trypsin in SDS-PAGE electrophoresis.This result suggests that it may belong to a superfamily of Serpins.3. A1397bp length of cDNA was got by RT-PCR and RACE. It contains a1233bpopen reading frame corresponding to a deduced protein of410amino acid. Analysisto the deduced amino acid sequences from the nucleic acid sequence showed that thereexists SERPIN domain in position52-408. The BLAST search at NCBI showed that theprotein encoded by the cDNA has a high degree of homology with the Serpinssuperfamily of serine protease inhibitor such as CP9from the carp meningeal fluid. Andthe P1position was occupied by Arg. So we speculated that the protein belongs to aSerpins superfamily.4. The CaSPI/proteinase molar ratio and the inhibitory constant Kiwas determinedand calculated. When the molar ratio up to1.15:1, it could completely inhibit theprotease hydrolytic activity. And Kiwas0.46μM. This indicated that CaSPI is acompetitive inhibitor. The stability of CaSPI (time, temperature, pH, metal ions anddenaturing agents) was detected. The results showed that CaSPI is considerable stability.The antibacterial activity test results showed CaSPI could inhibited gram-negativebacteria Escherichia coli and gram-positive bacteria Bacillus subtilis.更多还原