蚓激酶基因的克隆及异源表达研究

发布时间：2018-03-07 08:46

  本文选题：赤子爱胜蚓　切入点：蚓激酶　出处：《安徽工程大学》2017年硕士论文　论文类型：学位论文

【摘要】：蚓激酶(Lumbrukinase,LK)是从蚯蚓粗提物中分离得到的一类具有纤溶活性的蛋白质。其不但能激活纤维蛋白溶酶原转变为纤溶酶,刺激血管内皮细胞释放组织纤溶酶原激活物(t-PA),间接促进纤维蛋白的溶解,且能够直接水解纤维蛋白。此外,蚓激酶拥有良好的溶栓效果,可以改善微循环,增加血管弹性,降低血液粘度,抑制血栓再次形成,因而广泛应用于临床,且越来越多地用在心脑血管、内分泌,呼吸系统等疾病的预防和治疗中。论文主要研究了野生型赤子爱胜蚓(Eisenia foetida)蚓激酶EFLK在大肠杆菌和巴斯德毕赤酵母中的表达,对重组毕赤酵母的发酵条件进行了优化,并对蚓激酶进行分离纯化及酶学特性的初步探究。主要工作如下:(1)从野生蚯蚓中提取基因组,利用分子生物学手段进行物种鉴定及系统发育树的构建,确定该野生蚯蚓属于赤子爱胜蚓。(2)提取赤子爱胜蚯蚓总RNA,利用反转录试剂盒获得单链cDNA作为为模板,克隆得到蚓激酶基因Eflk(GenBank登录号:KY452025)。利用生物信息学软件分析蚓激酶基因Eflk全长为738 bp,编码245个氨基酸,其中含有一个由7个氨基酸组成的信号肽。蚓激酶EFLK与粉正蚓的蚓激酶F-Ⅲ-2(GenBank登录号:AB045719)相似性最高达96.21%。经DS2.5软件建模及分子对接,确定蚓激酶EFLK 的活性中心为:Arg15-Phe19-Pro20-Glu66-Asn113。(3)构建重组质粒pET-28a(+)-Eflk,并将其导入大肠杆菌BL21(DE3)中,成功实现蚓激酶EFLK在大肠杆菌的表达。在培养基中添加山梨醇、蔗糖、甘氨酸和乙醇可有效提高酶活,最高达到15.6 U/mL。(4)构建重组质粒pPIC9K-Eflk,经Sac I线性化后导入巴斯德毕赤酵母GS115中,成功实现蚓激酶EFLK在酵母中的表达。在重组菌GS115-pPIC9K-EflK发酵上清液中检测到了纤溶活性,其中发酵第72 h达到最高酶活为224.8 U/mL。(5)利用单因素法和水平正交法优化毕赤酵母发酵条件,经单因素实验优化确定4个关键因素:初始pH、诱导温度、接种量和甲醇添加量。就此进行4因素3水平正交实验,得到最佳发酵条件:初始pH5.5、诱导温度30℃、接种量OD_(600)=2.0、甲醇添加量1%,优化后发酵72 h酶活最高达到259.4 U/mL。(6)利用Ni~+柱纯化得到蚓激酶EFLK蛋白,比酶活为4545.84 U/mg。酶学性质研究表明,蚓激酶EFLK最适pH为8.0,酶液在pH 7.4条件下37 ℃保温12 h时蚓激酶活力快速损失20%,第48 h蚓激酶残余活力为67.33%左右。蚓激酶EFLK在低温(低于40 ℃)条件下较为稳定。酶液在最适pH 8.0条件下4 ℃保存48 h蚓激酶活力损失41.63%,仍保持较高的活力。
[Abstract]:LumbrukinaseLK is a kind of fibrinolytic protein isolated from earthworm extract, which not only activates plasminogen transformation into plasminogen, Stimulating vascular endothelial cells to release tissue plasminogen activator t-PAN indirectly promotes fibrinolysis and directly hydrolyzes fibrin. In addition, lumbrokinase has a good thrombolytic effect, which can improve microcirculation and increase vascular elasticity. It is widely used in clinic, and is more and more used in cardiovascular, cerebrovascular, endocrine, etc. In the prevention and treatment of respiratory diseases, the expression of wild type Eisenia foetida EFLK in Escherichia coli and Pichia pastoris was studied, and the fermentation conditions of recombinant Pichia pastoris were optimized. The main work is as follows: extracting genome from wild earthworm, using molecular biological methods to identify species and construct phylogenetic tree. It was determined that the wild earthworm belongs to Aisheng vermis. 2) the total RNAs of Aisheng earthworm were extracted, and the single-stranded cDNA was obtained by reverse transcription kit as template. Eflk(GenBank accession number: KY452025 was cloned. The total length of Eflk was 738 BP, encoding 245 amino acids, using bioinformatics software. It contains a signal peptide consisting of seven amino acids. The similarity between EFLK and F- 鈪，

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