肝素黄杆菌肝素酶基因在大肠杆菌中表达的研究

发布时间：2018-02-25 23:18

  本文关键词： 肝素酶 克隆 表达 优化 纯化 酶学性质　出处：《浙江工商大学》2012年硕士论文　论文类型：学位论文

【摘要】：肝素酶(heparanase, Hpa)是近年来研究较多的一类-D糖苷内切酶(endo-D-glucuronidase),它既存在于正常组织,如胎盘和淋巴器官,对胚胎的形成,神经系统的发育,新生血管的形成,炎症反应等都发挥着正常的生理功能；同时也表达于多种恶性肿瘤细胞的表面,能够促进细胞的侵袭和转移,诱导肿瘤血管生成,降低肿瘤患者的生存率。肝素用于治疗和预防血栓已经有很长的历史,但是研究表明肝素有一定的副作用,例如影响血小板的稳定,从而导致手术后大出血。而分子量在4000-8000的低分子量肝素(LMWH)则能够有效地治疗血栓,但是没有明显的副作用。目前我国LMWH主要依靠进口或者来自合资企业生产,因此LMWH的生产研究具有重要的工业应用前景。而肝素酶的重要用途之一便是用于生产LMWH。 针对以上问题,本课题组构建了高效表达可溶性肝素酶的重组大肠杆菌系统,获得了纯度高,活力高,性质稳定的肝素酶,极大降低了肝素酶的生产成本,使得肝素酶用于LMWH的生产成为可能。本课题组已完成了以大肠杆菌表达系统克隆表达黄杆菌肝素酶基因的工作,并以该基因作为起始材料,利用酶活测定方法筛选出高活力菌株；然后对其发酵培养基进行优化；最后对重组蛋白进行表达、纯化并研究其酶学性质。主要研究结果如下所述： 提取黄杆菌肝素酶的全基因组,设计合适的引物(引入HindⅢ和EcoR Ⅰ两个酶切位点),利用PCR技术扩增出肝素酶的ORF;同时用上述两种酶双酶切pET-28a载体和肝素酶基因,在T4连接酶的作用下连接上述目的基因与载体片段。将所得到的克隆载体转化大肠杆菌BL21菌株,在含Kan的固体LB培养基上筛选阳性可克隆子。经PCR、酶切鉴定成功后测序确定重组表达载体构建成功。 以适当浓度的IPTG诱导表达肝素酶,用SDS-PAGE检测不同的诱导时间下肝素酶的表达量,从而确定最佳的诱导时间。结合酶活测定方法,得到表达肝素酶最高活性的菌株。 采用两水平部分因子实验设计研究了发酵培养基各组分对肝素酶活力的影响,得出主要影响因子为蛋白胨、酵母粉和葡萄糖。三者在95%的概率水平上对菌体产酶都有正效应。然后采用BBD确定各主要影响因子的最佳作用浓度,得到优化的发酵培养基组成(g/L)：蛋白胨11.95,酵母粉7.4,葡萄糖2.74,Na2HPO4·12H2O10, MgSO4·7H2O0.5, NH4Cl3, KH2P042。在优化发酵培养基中供试菌株的酶活力是未优化发酵培养基中的1.95倍。 发酵液经镍柱亲和层析纯化得到电泳纯肝素酶,SDS-PAGE电泳结果显示,经纯化得到一条单一的目的蛋白条带,经分析得该酶的分子量为42.9kDa。对纯化后肝素酶的酶学性质进行研究,发现该酶的最适反应温度为30℃,最适反应pH为7.0。该酶热稳定性较差,当温度高于60℃时,30min可完全丧失酶活。当pH范围在7-10之间时,1h内酶活基本不受影响,偏离此范围则对酶活影响较大。对比8种不同金属离子对优化后酶的活性影响,发现Ca2+和Cu2+对该酶有比较明显的激活作用,Mn2+和Pb2+对该酶有明显的抑制作用。
[Abstract]:Heparanase (heparanase, Hpa) is a kind of -D endoglycosidase much studied in recent years (endo-D-glucuronidase), which exists in normal tissues such as placenta and embryo formation of lymphoid organs, nervous system development, angiogenesis, inflammation and other play a normal physiological function and surface; expressed in various malignant tumor cells, can promote cell invasion and metastasis, tumor induced angiogenesis, reduce the survival rate of cancer patients. Heparin for the treatment and prevention of thrombosis has a long history, but the research shows that heparin has certain side effects, such as platelet stability, resulting in massive hemorrhage after surgery while the molecular weight of low molecular weight heparin 4000-8000 (LMWH) can effectively treat thrombosis, but no obvious side effects. At present LMWH mainly rely on imports or from joint ventures Therefore, the production research of LMWH has an important industrial application prospect. One of the important uses of heparanase is to produce LMWH..
To solve the above problems, the research group to construct a recombinant Escherichia coli system for efficient expression of soluble heparin enzyme, has high purity, high activity, heparanase stable, greatly reduces the production cost of heparanase, the heparin enzyme for LMWH production possible. The research group has completed the expression of Flavobacterium heparinase the gene expressed in Escherichia coli and the gene cloning system, as the starting material, determination of the activity of high activity strains were screened by enzyme; then the fermentation medium was optimized; finally the recombinant protein expression, purification and characterization. The main results are as follows:
鎻愬彇榛勬潌鑿岃倽绱犻叾鐨勫叏鍩哄洜缁，

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