本文關(guān)鍵詞： 蜱 肽 抗凝血 巴斯德畢赤酵母菌 重組　出處：《重慶醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 蜱抗凝血肽(tick anticoagulant peptide,TAP)是從軟蜱唾液內(nèi)分離鑒定出的抗凝分子,為60個(gè)氨基酸組成的單鏈酸性多肽,是一種慢速而結(jié)合緊密的具有高度選擇性的Ⅹa因子(FⅩa )抑制劑,與Kunitz型抑制劑有同源性。在進(jìn)行提高TAP對FⅩa抑制活性方面的研究時(shí)發(fā)現(xiàn),將TAP的第一位酪氨酸殘基替換為色氨酸,把第十位天冬氨酸替換為精氨酸,產(chǎn)生的TAP雙重突變體,可使其抗Ⅹa的活性增加37倍。 血小板黏附和聚集的一個(gè)重要成分是其表面的糖蛋白Ⅱb/Ⅲa(GpIIb/IIIa)受體。研究發(fā)現(xiàn)GpⅡb/Ⅲa與其配體的結(jié)合主要是通過構(gòu)象改變而識別其配體中的精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp, RGD)序列來完成的,通過基因工程或人工合成含有以上序列的多肽能抑制纖維蛋白原等配體與GpⅡb/Ⅲa受體的結(jié)合,從而具有抗凝作用。目前含RGD序列的多肽是臨床研究的熱點(diǎn)。 本研究采用環(huán)嫁接的方法,將RGDS(精氨酸-甘氨酸-天冬氨酸-絲氨酸)序列在一定的構(gòu)象限制下導(dǎo)入TAP活性位點(diǎn)以外具有穩(wěn)定的β折疊指狀結(jié)構(gòu)的頂端。將此構(gòu)建的嵌合體基因在酵母表達(dá)系統(tǒng)中進(jìn)行表達(dá),以期獲得一種既有抗Ⅹa的作用又有抑制血小板聚集功能(兼具纖溶酶激活功能)的雙功能、高效抗栓物質(zhì)。 首先參照天然TAP的氨基酸順序,設(shè)計(jì)將TAP的Y1的酪氨酸基因替換為色氨酸基因以及D10的天冬氨酸基因替換為精氨酸基因,產(chǎn)生雙重突變體TAP的基因序列。在編碼TAP指狀結(jié)構(gòu)的K30的賴氨酸和G31的甘氨酸之間插入編碼RGDS氨基酸的基因序列。在5’及3’端分別加入EcoRI、NotI限制性酶切位點(diǎn),全長共64個(gè)氨基酸殘基編碼序列共216個(gè)堿基。全基因分4個(gè)片段人工合成,并設(shè)計(jì)三個(gè)相應(yīng)接頭。磷酸化后用T4-DNA連接酶16℃連接過夜。PCR擴(kuò)增、膠回收后與PMD18-T質(zhì)粒連接,再轉(zhuǎn)入大腸桿菌DH5a。質(zhì)粒抽提后用聚合酶鏈反應(yīng)(Polymerase chain reaction, PCR)技術(shù)特異性擴(kuò)增TAP基因片段,膠回收、純化PCR反應(yīng)產(chǎn)物后用限制性內(nèi)切酶EcoRI和NotI雙酶切并與同樣經(jīng)EcoRI和NotI雙酶切的pPIC9K真核表達(dá)載體連接,將連接產(chǎn)物再次轉(zhuǎn)化大腸桿菌DH5α。篩選陽性克隆質(zhì)粒,經(jīng)雙酶切,電泳及DNA測序鑒定,證實(shí)插入序列為目的基因序列。將篩選后的表達(dá)載體質(zhì)粒pPIC9K-TAP經(jīng)限制性內(nèi)切酶SalⅠ酶切線性化后用電轉(zhuǎn)化的方法轉(zhuǎn)入感受態(tài)GS115酵母菌,得到的轉(zhuǎn)化子經(jīng)MD平板與MM平板、G418抗性篩選,PCR鑒定后,得到重組酵母工程菌GS115/pPIC9K-TAP,用甲醇誘導(dǎo)分泌表達(dá)目標(biāo)蛋白。經(jīng)SDS-PAGE分析后,將表達(dá)產(chǎn)物進(jìn)行初步的分離純化后用正常人的血漿進(jìn)行白陶土部分凝血酶原時(shí)間測定(APTT)和血漿凝血酶原時(shí)測定間(PT)試驗(yàn)檢測產(chǎn)物抑制凝血酶活化的作用,采用血小板聚集試驗(yàn)(platelet aggregation test, PAgT)檢測表達(dá)產(chǎn)物是否有結(jié)合血小板的活性。 研究結(jié)果:經(jīng)過酶切,PCR,序列分析證實(shí)成功構(gòu)建了含TAP-RGDS基因的重組分泌型表達(dá)質(zhì)粒pPIC9K/TAP,重組質(zhì)粒電轉(zhuǎn)化GS115酵母菌,并經(jīng)MD平板與MM平板、G418抗性篩選,及PCR鑒定后,得到重組酵母工程菌GS115/pPIC9K-TAP,用甲醇誘導(dǎo)分泌表達(dá)目標(biāo)蛋白。經(jīng)SDS-PAGE分析,在標(biāo)準(zhǔn)分子量8kD左右有一條特異蛋白帶,說明培養(yǎng)上清中確有外源蛋白表達(dá)。表達(dá)產(chǎn)物進(jìn)行抗凝血初步試驗(yàn),證明表達(dá)產(chǎn)物有抗凝血因子活性和抗血小板的作用。 研究結(jié)論:①采用基因合成的方式成功構(gòu)建rTAP-RGDS的真核表達(dá)載體。②通過誘導(dǎo)該載體表達(dá)了重組基因產(chǎn)物。③經(jīng)研究表明該表達(dá)產(chǎn)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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R346

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相關(guān)期刊論文 前1條

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本文編號：1444385