蜱抗凝血肽与RGDS肽的重组基因构建及真核表达

发布时间：2018-01-19 13:29

本文关键词： 蜱肽抗凝血巴斯德毕赤酵母菌重组　出处：《重庆医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 蜱抗凝血肽(tick anticoagulant peptide,TAP)是从软蜱唾液内分离鉴定出的抗凝分子,为60个氨基酸组成的单链酸性多肽,是一种慢速而结合紧密的具有高度选择性的Ⅹa因子(FⅩa )抑制剂,与Kunitz型抑制剂有同源性。在进行提高TAP对FⅩa抑制活性方面的研究时发现,将TAP的第一位酪氨酸残基替换为色氨酸,把第十位天冬氨酸替换为精氨酸,产生的TAP双重突变体,可使其抗Ⅹa的活性增加37倍。血小板黏附和聚集的一个重要成分是其表面的糖蛋白Ⅱb/Ⅲa(GpIIb/IIIa)受体。研究发现GpⅡb/Ⅲa与其配体的结合主要是通过构象改变而识别其配体中的精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp, RGD)序列来完成的,通过基因工程或人工合成含有以上序列的多肽能抑制纤维蛋白原等配体与GpⅡb/Ⅲa受体的结合,从而具有抗凝作用。目前含RGD序列的多肽是临床研究的热点。本研究采用环嫁接的方法,将RGDS(精氨酸-甘氨酸-天冬氨酸-丝氨酸)序列在一定的构象限制下导入TAP活性位点以外具有稳定的β折叠指状结构的顶端。将此构建的嵌合体基因在酵母表达系统中进行表达,以期获得一种既有抗Ⅹa的作用又有抑制血小板聚集功能(兼具纤溶酶激活功能)的双功能、高效抗栓物质。首先参照天然TAP的氨基酸顺序,设计将TAP的Y1的酪氨酸基因替换为色氨酸基因以及D10的天冬氨酸基因替换为精氨酸基因,产生双重突变体TAP的基因序列。在编码TAP指状结构的K30的赖氨酸和G31的甘氨酸之间插入编码RGDS氨基酸的基因序列。在5’及3’端分别加入EcoRI、NotI限制性酶切位点,全长共64个氨基酸残基编码序列共216个碱基。全基因分4个片段人工合成,并设计三个相应接头。磷酸化后用T4-DNA连接酶16℃连接过夜。PCR扩增、胶回收后与PMD18-T质粒连接,再转入大肠杆菌DH5a。质粒抽提后用聚合酶链反应(Polymerase chain reaction, PCR)技术特异性扩增TAP基因片段,胶回收、纯化PCR反应产物后用限制性内切酶EcoRI和NotI双酶切并与同样经EcoRI和NotI双酶切的pPIC9K真核表达载体连接,将连接产物再次转化大肠杆菌DH5α。筛选阳性克隆质粒,经双酶切,电泳及DNA测序鉴定,证实插入序列为目的基因序列。将筛选后的表达载体质粒pPIC9K-TAP经限制性内切酶SalⅠ酶切线性化后用电转化的方法转入感受态GS115酵母菌,得到的转化子经MD平板与MM平板、G418抗性筛选,PCR鉴定后,得到重组酵母工程菌GS115/pPIC9K-TAP,用甲醇诱导分泌表达目标蛋白。经SDS-PAGE分析后,将表达产物进行初步的分离纯化后用正常人的血浆进行白陶土部分凝血酶原时间测定(APTT)和血浆凝血酶原时测定间(PT)试验检测产物抑制凝血酶活化的作用,采用血小板聚集试验(platelet aggregation test, PAgT)检测表达产物是否有结合血小板的活性。研究结果:经过酶切,PCR,序列分析证实成功构建了含TAP-RGDS基因的重组分泌型表达质粒pPIC9K/TAP,重组质粒电转化GS115酵母菌,并经MD平板与MM平板、G418抗性筛选,及PCR鉴定后,得到重组酵母工程菌GS115/pPIC9K-TAP,用甲醇诱导分泌表达目标蛋白。经SDS-PAGE分析,在标准分子量8kD左右有一条特异蛋白带,说明培养上清中确有外源蛋白表达。表达产物进行抗凝血初步试验,证明表达产物有抗凝血因子活性和抗血小板的作用。研究结论:①采用基因合成的方式成功构建rTAP-RGDS的真核表达载体。②通过诱导该载体表达了重组基因产物。③经研究表明该表达产物的粗提物具有抗抗凝血因子活性和抗血小板的作用。
[Abstract]:Tick anticoagulant peptide (tick anticoagulant peptide, TAP) is separated out from the anticoagulant molecular identification of soft ticks in the saliva, composed of 60 amino acid polypeptide chain, is a slow and closely combined with the highly selective a factor Xa (F x a) inhibitor, homology with Kunitz inhibitors. Found in the study to improve the TAP of F x a inhibitory activities when the first TAP tyrosine residues replaced by tryptophan, tenth aspartic acid is replaced with arginine, TAP double mutants were generated, the anti Xa activity of a increased by 37 times.
One of the important components of platelet adhesion and aggregation of the surface glycoprotein b/ receptor A (GpIIb/IIIa). The study found that the combination of Gp II b/ III A and its ligand is mainly by changing the conformation of the ligand in the identification of amino acid glycine aspartic acid (Arg-Gly-Asp, RGD) to complete the sequence the above sequence polypeptide containing, through genetic engineering or synthetic inhibition of fibrinogen binding to ligand and Gp II b/ III a receptor, which has anticoagulant effect. The polypeptide containing RGD sequence is a hot topic in clinical research.
This research adopts the method of ring grafting, RGDS (arginine glycine aspartic acid serine) outside the sequence into the TAP in conformationally restricted active sites with stable beta folding top like structure. The construction of the chimeric gene in the yeast expression system for expression, in order to a is the role of a and anti Xa inhibition of platelet aggregation (both plasminogen activation function) dual function, efficient antithrombotic substances.
First of all according to the amino acid sequence of natural TAP, the design will replace the TAP gene Y1 for tyrosine tryptophan aspartic acid gene and D10 gene replacement for arginine gene, gene sequence of double mutant TAP. Between the gene sequences of glycine like structure K30 lysine and G31 insert encoding RGDS amino acids in the encoding TAP. EcoRI were added in the 5 'and 3' end, NotI restriction sites, a total length of 64 amino acid residues encoding sequence of 216 base pairs. The whole gene was divided into 4 fragments synthesized, and design three corresponding connectors. After phosphorylation by T4-DNA ligase 16 C connection for.PCR amplification, gel recovery after connected with PMD18-T plasmid, then transformed into Escherichia coli DH5a. plasmid after extraction by polymerase chain reaction (Polymerase chain reaction, PCR) amplified TAP gene fragment, gel recovery, purification of PCR reaction products with restrictions Endonuclease EcoRI and NotI double enzyme digestion and pPIC9K via EcoRI and NotI eukaryotic expression vector with double enzyme digestion, connect the product once again transformed into Escherichia coli DH5 alpha. Positive clones plasmid by double enzyme digestion, electrophoresis and DNA sequencing confirmed the insertion sequence gene sequence. After screening the expression vector plasmid pPIC9K-TAP by restriction endonuclease Sal linearized using electroporation method into competent GS115 yeast, the transformants by MD plate and MM plate, G418 resistance screening, PCR identification, get the recombinant yeast GS115/ pPIC9K-TAP induced secretion expression of target protein by SDS-PAGE with methanol. After analysis, the expression product was purified by preliminary separation of normal human plasma was measured kaolin partial thromboplastin time (APTT) and prothrombin time (PT) determination between test product inhibition of coagulation The effect of blood enzyme activation was tested by platelet aggregation test (platelet aggregation test, PAgT) to detect whether the expressed product had the activity of binding platelets.
Results: after enzyme digestion, PCR and sequence analysis confirmed successful construction of the TAP-RGDS gene containing recombinant secretory expression plasmid pPIC9K/TAP, recombinant plasmid was electroporated into GS115 yeast, and the MD plate and MM plate, G418 resistance screening and PCR identification, obtain recombinant yeast engineering bacteria GS115/pPIC9K-TAP, induced secretion expression of target protein by methanol. By SDS-PAGE analysis, there is a specific protein with a standard molecular weight of about 8kD, that is the expression of heterologous protein in culture supernatant. The expression product blood clotting experiment showed that the expression product has anticoagulant and antiplatelet activity factor role.
Conclusion: (1) the eukaryotic expression vector of rTAP-RGDS was successfully constructed by gene synthesis. Secondly, the recombinant gene product was expressed by inducting the vector.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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