好食脉孢菌纤溶酶的高效纯化、功能性及基因的克隆表达研究

Fibrinolytic enzymes have the ability to dissolve blood clots. Along with the age or improper diet, some human body will tend to increase blood clots. We found that Neurospora sitophila can produce fibrinolytic enzymes by fermentation. A method for the enzyme purification has been established, a small amount of high purity enzyme has been obtained, partial amino acid sequence of the enzyme has been determined. It has been found that the enzyme is a novel fibrinolytic enzyme based on NCBI comparison. This project will apply expansion bed, ion exchange, hydrophobic interaction chromatographies to build efficient purification method with larger loading capacity, higher speed and better yields. Based on obtaining more purified enzyme, characteristics of the enzyme will be determined, animal models with blood clots tendency will be established, and the functionality of the fibrinolytic enzyme to decrease tendency of blood clots, in the whole animal levels, will be explored with biological methods. Based on partial amino acid sequence of the enzyme, gene cloning, expression and the recombinant enzyme purification will be explored. Based on the conserved domains, degenerate primers will be generated; and will be used to amplify the partial gene. Total RNA will be extracted and transcribed with reverse transcriptase. To obtain the full-length cDNA sequence of the fibrinolytic enzyme, 5′and 3′RACE (Rapid Amplification of cDNA Ends) will be carried out. To express the cloned fibrinolytic gene, the coding sequence will be amplified by PCR using specific primers. The recombinant plasmid, containing the fibrinolytic enzyme gene will be transformed into host competent cells. Transformants will be identified by enzymatic digestion and PCR analysis and further confirmed by DNA sequencing. For the purification of recombinant enzyme, the transformed host cells will be grown and the recombinant enzyme will be expressed, purified and further analyzed. This project will provide a good foundation for the application of this enzyme in functional food, and provide reference method for other fibrinolytic enzyme efficient separation, functional evaluation, gene cloning and expression.

纤溶酶具有溶解血栓的能力。前期发现脉孢菌产纤溶酶，已获得少量高纯度酶，经部分氨基酸序列测定和比对，发现该酶是新纤溶酶，具有很高的应用价值。本项目拟采用扩张床、离子交换、疏水相互作用色谱方法，创立载量大、速度快、活力回收率高的纯化酶的方法。在获得较多高纯度酶的前提下，阐明酶的基本性质；依据生物体纤溶系统功能低下与高血脂之间的关联性，建立具有血栓倾向的动物模型，在整体动物水平阐明脉孢菌纤溶酶逆转血栓倾向的功能性。根据脉孢菌纤溶酶的部分氨基酸序列，设计特异性引物，提取脉孢菌总RNA逆转录ｃDNA，用RACE技术扩增酶基因，构建重组载体转化宿主细胞，对重组子鉴定并进行DNA测序后，培养转化子诱导表达重组酶，纯化后分析活性。本项目为脉孢菌纤溶酶在功能食品中的应用奠定理论基础，为其它微生物来源纤溶酶的高效分离纯化和逆转血栓倾向的功能性分析评估提供借鉴方法。

血栓倾向症威胁人类健康。利用分离自发酵食品的Neurospora sitophila发酵获溶栓功能纤溶酶，针对酶的纯化与性质、酶的逆转血栓倾向功能进行研究，构建基因工程重组菌。获如下重要结果。.1.建立了N.sitophila纤溶酶纯化方法。发酵液分级盐析后，用疏水色谱分离到酶I和酶II，酶I和II再顺次经离子交换色谱和凝胶色谱纯化，最终两酶均达电泳纯。酶I比活力288.3 U/mg，纯化31.5倍，活力回收1.5%；酶II比活力688 U/mg，纯化75.2倍，活力回收1.1%。.2.优化了酶的纯化方法。用盐析和三级疏水色谱依次对酶II分离。纯化的酶II比活力2011.64 U/mg，纯化61.65倍，活力回收16.95%。.3.研究了酶的基本性质。酶I和II分子量分别为49 kDa和34 kDa，等电点分别为7.9±0.2和9.3±0.2，最适作用温度分别为50℃和41℃，最适作用pH分别为7.4和7.6；酶I和II均是丝氨酸蛋白酶，均能水解纤维蛋白Aα、Bβ链和γ链；其均是糖蛋白，含糖量分别为1.9437%和2.196% (w/v)。确定了酶II三个肽段氨基酸序列，经比对发现其为新纤溶酶。.4.以高血脂大鼠为模型，研究了酶II对血栓倾向的逆转功能。抗血栓倾向和降血栓倾向试验均表明，酶能降低大鼠血清中甘油三脂、总胆固醇和低密度脂蛋白含量，升高高密度脂蛋白含量；延长凝血酶时间、部分凝血活酶时间和凝血酶原时间；降低纤溶酶原激活物抑制剂含量，升高纤溶酶原激活剂和纤溶酶原含量。这表明酶具有防止机体形成血栓倾向作用。酶对试验大鼠内脏具有一定保护作用。.5.进行了N.sitophila纤溶酶基因的克隆与表达。经总RNA 抽提和mRNA纯化，用RT PCR合成cDNA。据N.sitophila部分氨基酸序列和丝氨酸蛋白酶保守序列设计简并引物，获500 bp酶基因片段。用RACE方法获酶全基因序列，成功克隆其到pET32a 载体并在E. coli 中表达。但表达的酶未显示活性。.6.进行了C.militaris纤溶酶基因克隆并在毕赤酵母中表达。通过PCR获酶基因，构建重组载体，内切酶双酶切重组载体及酵母表达载体，构建重组质粒，将其转入毕赤酵母感受态细胞，阳性重组菌诱导表达的酶活力达155.47 U/mL。.本项目研究成果为开发降低血栓倾向的功能食品奠定了理论基础。