【作者】 王俊杰；

【导师】 孙树汉；

【作者基本信息】 第二军医大学 ， 遗传学， 2009， 硕士

【摘要】 心脑血管疾病如脑中风、冠心病以及外周血管的血栓栓塞等已成为了严重影响人类健康的主要疾病。作为自然界的野生资源之一,毒蛇毒液中含有许多生物活性蛋白质和多肽,其中就包括对血液系统有重要影响的组分。利用蛇毒开发新的治疗血栓的药物,成为国内外研究的热点之一。本研究分离了广西尖吻蝮蛇毒腺组织,利用T7Select(?)Phage Display System首次构建了尖吻蝮蛇蛇毒毒腺噬菌体展示文库,以纤维蛋白/纤维蛋白原为底物,经过三轮淘选,获得了一个新的尖吻蝮蛇蛇毒类凝血酶基因;利用生物信息学技术,分析了类凝血酶基因结构特点,构建了尖吻蝮蛇蛇毒类凝血酶基因的原核表达载体pET-32a-TLE,诱导表达,亲和纯化和分子筛纯化后,获得了具有生物学活性的重组蛇毒类凝血酶蛋白。本研究的主要实验结果如下:1.最先构建了广西尖吻蝮蛇毒腺噬菌体展示文库,文库滴度为2.5×10~6 pfu/ml。2.建立了使用纤维蛋白/纤维蛋白原淘选抗凝组分的方法:在固相介质上包被(ELISA板)100μl的10μg/ml纤维蛋白原后,加入扩增的噬菌体文库的裂解产物(100μl),洗去为非特异性结合的噬菌体,将特异性结合的噬菌体洗脱下来进行下一轮淘选,经过三轮“吸附一洗脱一扩增”,富集了特异性结合的噬菌体。3.获得了一个新的尖吻蝮蛇蛇毒类凝血酶基因,基因长783 bp,能够编码260个氨基酸,GenBank Accession Number为AY861138.1,序列同源性分析,发现与GenBank登录号为AF159060类凝血酶的同源性为97%,运用互联网的http://www.expasy.org/cgi-bin/protscale.pl分析了此类凝血酶基团的亲疏水性,具有明显的亲水性,同源建模分析,发现其有三个结构域,信号肽,类胰岛素丝氨酸蛋白酶,内在的结构。4.构建了原核表达载体pET-32a-TLE,在0.7 mmol/L的IPTG诱导下,25℃下低温诱导表达类凝血酶蛋白,在该条件下大大降低了类凝血酶蛋白的包涵体表达,为后面简便快捷的纯化蛋白奠定了基础。重组尖吻蝮蛇蛇毒类凝血酶的生物活性分析:血浆体外凝固实验表明,37℃下,4.4μg的重组类凝血酶使200μl血浆在65s钟内凝固;纤维蛋白原水解活性表明8μg重组类凝血酶蛋白在60分钟内可以降解纤维蛋白原的Aα链。更多还原

【Abstract】 Cardiovascular and cerebrovascular diseases such as stroke,coronary heart disease and peripheral vascular thrombosis embolism have seriously impacted on quality of people life,cause a heavy burden for families.As one precious nature resources,Snake venoms contain lots of biologically active proteins and peptides,including on the components of interacting vascular system.Using snake venom to the develop new thrombosis-treatment drugs has emerged as the important researches.In this study,we separated the tissue of Agkistrodon acutus venom gland,and constructed a phage display library of Agkistrodon acutus venom gland by use of Novagen’s T7 Select(?) Phage Display System.After three rounds of panning,we obtained a new Agkistrodon acutus snake venom thrombin-like enzyme gene.Whereafter,we analyzed the gene structure of thrombin-like enzyme using bioinformatics.In order to express the thrombin-like enzyme,we constructed a prokaryon vector of pET-32a-TLE, then expressing induced by IPTG.After affinity purification and molecular sieve purification,we obtained the recombinant snake venom thrombin-like enzyme protein which keeped a goog biological activity.The main results of our study are as follws:1.Firstly constructed phage display library of Guangxi Agkistrodon acutus venom,after identifying,the titer of library reaching 2.5×10~6 pfu/ml.2.Set up approach to pan anticoagulant components using the ways of using fibrin/fibrinogen:Coat 100μl 10μg/m fibrin/fibrinogen on the solid-phase medium such as ELISA plate,add amplified phage library into coated plate,wash away the non-specific phage which is binded to the plate,after three rounds of biopanning "Adsorption -Elution-Amplificationion",enrich the specific phage which is binded to the plate.3.Obtaining a new thrombin-like enzyme gene from Agkistrodon acutus snake venom. Length is 783bp,it encodes 260 amino acids.Acquaired the GenBank Accession Number AY861138.1 after submission.It has 97%homology with the thrombin-like enzyme of Gen Bank accession nuber AF159060 through the Blast.we analysised the Hydrophilicity and hydrophobicity by the web of http://www.expasy.org/cgi-bin/protscale.pl,and found it obvious Hydrophilicity.Homology modeling analysis indicted finding that there are three structural domains,signal peptide,Tryp_Spc(Trypsin-like serine protease),and intrinsic disorder.4.Constructed the prokaryotic expression vector pET-32a-TLE,optimized expression conditions at 0.7 mmol/L of IPTG and 25℃.Protein inclusions expression was cut down under this condition,which laid the foundation for the simple and efficient purification of the thrombin-like enzyme.5.The biological activity of recombination Agkistrodon acutus snake venom thrombin-like was analyzed:plasma coagulation experiments in vitro showed that 4.4μg of recombinant thrombin-like enzyme could make 200μl plasma coagulation in 65 seconds at 37℃;Fibrinogen hydrolysis activity assay showed that 8μg thrombin-like enzyme could degrade the Aαchain of fibrinogen in 60 minutes throughly.更多还原