【作者】 许建强；

【导师】 杨青； 安利佳；

【作者基本信息】 大连理工大学 ， 生物化工， 2008， 博士

【摘要】 蛇毒类凝血酶属于纤维蛋白原裂解酶，是一种丝氨酸蛋白酶。它们降解纤维蛋白原但不激活凝血因子FactorⅩⅢ，形成的纤维蛋白块能被后继的血液纤溶系统清除。由于它们在血浆中能够降低纤维蛋白原，可以作为抗凝剂使用。从珍稀树栖毒蛇——大连蛇岛蝮蛇（Gloydius shedaoensis）毒液中分离制备的蛇毒类凝血酶制品已经在血栓病临床治疗应用了很多年。可是鉴于国家法律法规禁止捕蛇，天然酶生产面临严峻的挑战，因而基因工程方法是解决这一困境的最佳选择之一。本论文内容主要就获得可溶性、活性重组大连蛇岛蝮蛇类凝血酶的制备，尤其是其纤维蛋白原裂解活性开展研究工作。（一）在大肠杆菌体系中可溶、活性大连蛇岛蝮蛇类凝血酶的克隆与表达使用Wilkinson-Harrison蛋白质可溶性概率模型，对带有不同商业化融合标签的重组大连蛇岛蝮蛇类凝血酶的可溶性趋势进行预测，结果显示在N端融合有NusA、GST和TrxA的重组大连蛇岛蝮蛇类凝血酶具有更高的可溶性。因而选择NusA、GST和TrxA三种标签用于促进大连蛇岛蝮蛇类凝血酶在大肠杆菌中的可溶性表达。相应地，构建了pQYNusA-glo-a、pQYGST-Glo-a和pQYTrxA-Glo-a三种表达质粒，转化获得了相应的大肠杆菌BL21-Gold（DE3）重组菌株。SDS-PAGE分析和Western blotting检测确定pQYNusA-Glo-a的可溶性表达状况最佳。（二）重组蛇毒类凝血酶的分离纯化1、首次使用人工核酸酶R5对重组大连蛇岛蝮蛇类凝血酶样品预处理：在400 nm紫外光激发条件下，1 mM R5可以高效去除重组大连蛇岛蝮蛇类凝血酶样品中的核酸杂质，而对重组大连蛇岛蝮蛇类凝血酶的活力没有造成损伤。由于合成成本低，人工核酸酶R5在蛋白质／酶的生产中具有很好的应用前景。2、采用四步柱层析方法获得高纯度重组大连蛇岛蝮蛇类凝血酶：柱层析依次包括Phenyl Sepharose FF疏水层析、Q Sepharose HP阴离子交换层析、Mono Q高分辨阴离子交换层析和Superdex 200分子排阻层析，最终获得重组蛇岛蝮蛇类凝血酶在SDS-PAGE胶上呈现单一条带，产率为0．1 mg／L。其表观分子量90 kDa，比活力为506 U／mg。（三）重组大连蛇岛蝮蛇类凝血酶的生化性质表征：1、重组大连蛇岛蝮蛇类凝血酶酶学性质：具有纤维蛋白原凝结活性、纤维蛋白原裂解活性以及酰胺水解活性。以酰胺水解活性底物BApNA为模式底物，该酶催化活性最适温度为40℃，最适pH为8．0。重组酶对纤维蛋白原裂解呈现剂量依赖和时间依赖，其裂解方式为：优先裂解Aα-链，然后裂解Bβ-链，但不裂解γ-链。2、抑制剂对酰胺水解酶活力的影响：研究了多种丝氨酸蛋白酶抑制剂、螯合剂、还原剂及凝血酶抑制剂对重组大连蛇岛蝮蛇类凝血酶的影响。丝氨酸蛋白酶抑制剂PMSF（1 mM）和TPCK（1 mM）分别抑制了重组酶100％和77％的酰胺水解活力，表明它属于丝氨酸蛋白酶家族。抑制剂苯脒、3-氨基苯脒、4-氨基苯脒和咪唑只抑制了<15％酶活力。EDTA不影响酰胺水解活力，表明大连蛇岛蝮蛇类凝血酶不是金属依赖型蛋白水解酶。还原剂二硫苏糖醇和β-巯基乙醇对酶活力没有影响，表明重组酶具有高度稳定性。凝血酶天然抑制剂如肝素等，不影响重组酶的酰胺水解活力，表明重组大连蛇岛蝮蛇类凝血酶与凝血酶在结构上存在差异。3、抑制剂对纤维蛋白原裂解酶活力的影响：通过SDS-PAGE的方法，研究了丝氨酸蛋白酶抑制剂、螯合剂、还原剂及凝血酶抑制剂对重组酶纤维蛋白原裂解活性的影响。丝氨酸蛋白酶抑制剂中只有PMSF具有抑制作用，表明Ser182既是酰胺水解活性又是纤维蛋白原裂解活性的关键氨基酸催化残基。同源模建预测表明PMSF与Ser182之间形成共价键阻碍了底物分子的趋近。可是TPCK只影响了酰胺水解活力，却对纤维蛋白原裂解活力没有影响。EDTA阻碍了重组酶的纤维蛋白原裂解活力，提示金属离子尤其是过渡金属离子在纤维蛋白原裂解过程发挥了作用。EDTA的抑制作用可能与EDTA螯合去除痕量金属离子从而引起底物纤维蛋白原的构象改变有关。还原剂二硫苏糖醇和β-巯基乙醇不抑制酰胺水解活力，表明重组大连蛇岛蝮蛇类凝血酶的高稳定性。肝素和水蛭素不影响纤维蛋白原裂解活力，提示重组大连蛇岛蝮蛇类凝血酶可与肝素或水蛭素联合应用于肝素相关血小板减少症和心肺血栓疾病的临床治疗。4、金属离子对酰胺水解活性和纤维蛋白原裂解活性的影响：1)1 mM过渡金属离子对重组大连蛇岛蝮蛇类凝血酶酰胺水解活力产生抑制的强弱顺序为：Fe²⁺>Cu²⁺≈Zn²⁺>Hg²⁺>>Ni²⁺，EDTA的引入可以恢复>80％被过渡金属离子抑制的重组大连蛇岛蝮蛇类凝血酶酰胺水解活力：其他金属离子Co²⁺和Mn²⁺分别抑制了重组酶酰胺水解活力的38％和25％；2)过渡金属离子抑制重组酶的纤维蛋白原裂解活力的强弱顺序为：Cu²⁺≈Ni²⁺>Zn²⁺>Co²⁺；其他二价金属离子如Ca²⁺和Mg²⁺对两种活力都没有影响。根据Irving-Williams晶体场理论，结合同源模建获得的大连蛇岛蝮蛇类凝血酶模型，抑制效应的产生可能和Zn²⁺与酶催化中心Ser182侧链上O原子、His43咪唑环上N原子以及两分子H₂O中O原子之间形成四对配位键有关。综上，本研究通过在目标蛋白N端融合NusA标签的表达策略，获得了重组蛇岛蝮蛇类凝血酶可溶性、活性表达；人工核酸酶R5可以高效地去除重组大连蛇岛蝮蛇类凝血酶粗样品中的核酸杂质，并对重组酶活力没有损伤，表明人工核酸酶R5在生化工程领域中具有很好的应用潜力；利用四步柱层析纯化获得了高纯度的可溶性重组大连蛇岛蝮蛇类凝血酶，表现出高的酰胺水解活性和纤维蛋白原裂解活性；系统研究了丝氨酸蛋白酶抑制剂、螯合剂、还原剂、凝血酶抑制剂及二价金属离子对重组大连蛇岛蝮蛇类凝血酶活力的影响，结合同源模建手段揭示抑制效应的作用机制。本研究可以为蛇毒类凝血酶的重组制备和临床应用提供依据。更多还原

【Abstract】 Snake venom thrombin-like enzymes belong to the fibrinogenolytic enzyme family and are serine proteases. They do not activate factor VIII and possess the fibrinogenolytic ability to hydrolyze fibrinogen into non-crossing linked fibrin, which are then eliminated by succeeding blood fibrinolytic system. They are applied as anti-coagulates due to their "defibrinogenation" in the blood. Snake venom thrombin-like enzyme preparations, gloshedobin, from the venom of the rare arboreal pit-viper, Gloydius shedaoensis, has been applied in clinic therapy for many years. However, upon the recent domestic decree of forbidding snake capture, production of these enzymes from snake venoms faces big challenge. Therefore, genetic engineering is one of the best alternatives to solve this problem. In this work, the preparation of recombinant gloshedobin in soluble and active form, and its biochemical properties in particular fibrinogenolytic activity was investigated.Firstly, cloning and expression of soluble and active gloshedobin in E. coli was investigated. The solubility tendency of recombinant gloshedobin with various commercial fusion proteins was predicted using Wilkinson-Harrison protein solubility predication model, which showed that recombinant gloshedobin fused with NusA, TrxA or GST tags at its N terminus had higher solubility. NusA, TrxA and GST were thus chosen for facilitating the expression of soluble gloshedobin in Escherichia coli. Accordingly, three expression vectors, pQYNusA-Glo-a, pQYGST-Glo-a and pQYTrxA-Glo-a were constructed and then transformed into Escherichia coli BL21-Gold （DE3）. pQYNusA-glo-a was determined to be the best by both SDS-PAGE analysis and Western blotting assay.Secondly, separation and purification of the recombinant gloshedobin was performed. （1） Removal of nucleic acids contaminants from crude recombinant gloshedobin samples by artificial nuclease R5 was investigated: Under 400nm UV-light, 1 mM of R5 could efficiently eliminate nucleic acids contaminants without impairing the activity of recombinant gloshedobin. Since the cost of synthesis is low, artificial nuclease R5 shows application potentials in the production of protein/enzymes; （2） Four-step column chromatographic purification of the recombinant gloshedobin with high purity: The column chromatographies included hydrophobic interaction chromatography on Phenyl Sepharose FF, ion exchange chromatography on Q Sepharose HP and Mono Q, and size exclusion chromatography on Superdex 200 were used. And the purified recombinant gloshedobin with the activity yield of 0.1mg/L were obtained, which appeared as one single band on SDS-PAGE. Its apparent molecular weight was determined to be 90 kDa and its specific amidolytic activity was 506 U/mg.Thirdly, biochemical characterization of recombinant gloshedobin was investigated: （1） Enzymatic properties: The recombinant gloshedobin exhibited fibrinogen clotting, fibrinogenolytic and amidolytic activities. By using N-a-benzoyl-DL-arginine p-nitroanilide （BApNA） as model substrate, its optimum temperature and pH for amidolytic activity were 40℃and pH 8.0, respectively. The recombinant gloshedobin preferentially cleaved bovine fibrinogen’s Aα-chain, then Bβ-chain, but did not cleaveγ-chain at all, which was dose-dependent and time-dependent. （2） Effects of serine proteases inhibitors, reducing agents, chelator and thrombin inhibitors, on the amidolytic activity of recombinant gloshedobin were determined. Serine protease inhibitors PMSF （1 mM） and TPCK （1 mM） inhibited the amidolytic activity of recombinant gloshedobin by 100 % and 77 %, respectively, indicating recombinant gloshedobin belongs to serine proteases family. Inhibitors including benzamidine, 3-aminobenzamidine, 4-aminobenzamidine and imidazole inhibited <15 % amidolytic activity of recombinant gloshedobin. EDTA did not affect recombinant gloshedobin, suggesting gloshedobin is not metalloproteinase. Reducing agents dithiothreitol andβ-mercaptoethanol did not affect recombinant gloshedobin, suggesting recombinant gloshedobin is highly stable. Thrombin natural inhibitors such as heparin also did not affect this enzyme’s amidolytic activity, suggesting recombinant gloshedobin differs from thrombin in structure; （3） Effects of serine proteases inhibitors, reducing agents, chelator and thrombin inhibitors on the fibrinogenolytic activity of recombinant gloshedobin were determined by using SDS-PAGE analysis: none of the serine protease inhibitors except PMSF had any effect on the fibrinogenolytic activity of recombinant gloshedobin, suggesting Ser182 is a key amino acid catalytic residue for both amidolytic and fibrinogenolytic activities. The convalent bond formed between PMSF and Ser182 was predicted to hamper the approach of substrates according to homology modeling. However, TPCK did not affect the fibrinogenolytic activity but affect amidolytic activity. EDTA hampered the activity, suggesting that metal ion（s）, in particular transition metal ions, played a role in the fibrinogenolytic process. The inhibitory effect of EDTA might relate to the removal of divalent metal ions by EDTA causing the configuration change of substrate fibrinogen. Reducing agents dithiothreitol andβ-mercaptoethanol did not affect recombinant gloshedobin, suggesting recombinant gloshedobin is highly stable. The fact that heparin as well as hirudin did not show any effect on the fibrinogenolytic activity of recombinant gloshedobin allowed the combined application of recombinant gloshedobin with heparin or hirudin in the clinical treatment of heparin-associated thrombocytopenia and cardiopulmonary diseases; （4） Effects of metal ions on recombinant gloshedobin were determined by amidolytic activity and SDS-PAGE analysis: Gloshedobin’s amidolytic activity toward BApNA was greatly inhibited by 1 mM of transition metal ions in the order: Fe²⁺>Cu²⁺≈Zn²⁺>Hg²⁺>>Ni²⁺. The addition of EDTA can reverse this inhibitory effect to >80 % of the original amidolytic activity which could be inhibited by transition metal ions. Other ions such as Co²⁺ and Mn²⁺ inhibited the amidolytic activity of recombinant gloshedobin by 38 % and 25 %, respectively. Besides, its fibrinogenolytic activity was inhibited by divalent transition metal ions in the order: Cu²⁺≈Ni²⁺>Zn²⁺>Co²⁺. Other ions such as Ca²⁺ and Mg²⁺ have almost no effect on either activity. According to the Irving-Williams crystal field theory and gloshedobin 3-D structure model generated by homology modeling, the inhibitory effects by metal ions like Zn²⁺ might be due to four intermolecular coordination bonds formed between Zn²⁺ and oxygen atom in the side chain of Ser182, Zn²⁺ and nitrogen atom in the imidazole ring of His43, Zn²⁺ and oxygen atoms in two water molecules.In conclusion, the production of recombinant gloshedobin in a soluble and active form was achieved by the strategy of fusing NusA tag at its N-terminal. Artificial nuclease R5 proved to be highly efficient in the removal of nucleic acids contaminants from crude recombinant gloshedobin samples and non-impairment on the recombinant gloshedobin’s activity, showing its application potentials in the field of biochemical engineering. The recombinant gloshedobin with high purity was obtained by four-step column chromatographic purification, which exhibited high amidolytic and fibrinogenolytic activities. The effects of serine protease inhibitors, reducing agents, chelator, thrombin inhibitors and divalent metal ions on recombinant gloshedobin were systematically investigated and the inhibitory mechanisms were also proposed using homology modeling techniques. The data obtained in this study might provide essential assistances in the preparation and clinic application of recombinant thrombin-like enzyme.更多还原