摘要
动脉发生栓塞是血栓类疾病(如心肌梗塞)的一个重要原因。溶栓治疗被认为是治疗血栓最为有效的方法,临床上所用的溶栓药物大多为纤维蛋白溶解酶原(简称纤溶酶原)激活剂类药物,它们可以将纤溶酶原转化为纤溶酶,纤溶酶可以溶解栓塞位置的纤维蛋白,从而恢复血液的畅通。目前常用的溶栓药物有链激酶、组织型纤溶酶原激活剂(t-PA)、尿激酶型纤溶酶原激活剂(u-PA)、重组组织型纤溶酶原激活剂(rt-PA)、对-甲氧苯甲酰纤溶酶原链激酶激活剂复合物(APSAC) 和重组链激酶(r-SK)等,它们的溶栓疗效肯定,但是还存在一些缺陷(如初始再灌注延迟或失败、出血、再梗塞等),阻碍了溶栓疗效的进一步提高。因此,随着对溶栓药物结构和功能的研究,寻找和研制溶栓效率高、特异性强、具有纤维蛋白选择性、半衰期长的溶栓治疗药物对于更有效治疗血栓性疾病具有十分重要的意义。
尿激酶原(pro-UK)是单链的u-PA,属于丝氨酸蛋白酶,能够激活纤溶酶原,使之成为纤溶酶,从而溶解血栓。体内和体外的实验均证明当用量控制在一定范围时,pro-UK 可以使纤维蛋白特异性溶解,但用量加大时,pro-UK 就转化为尿激酶(UK),失去了对纤维蛋白的特异性。对pro-UK 的研究表明,它由411 个氨基酸残基组成;经激肽释放酶或纤溶酶激活可在Lys158-Ile159 断开形成UK;凝血酶可催化pro-UK 的Arg156-Phe157 之间肽键的水解,产生凝血酶断裂型的UK 双链,该双链仅具有微弱的激活纤溶酶原的活性;为了防止系统性纤溶,pro-UK 受到纤溶酶原激活剂抑制剂(PAI)的调控;PAI-1 可通过与pro-UK 共价结合形成复合物而使之失活,是pro-UK主要的抑制剂;一项研究显示pro-UK 表面可变环中的许多带正电荷氨基酸残基(R178RHRGGS184)可与PAI-1 分子中的带负电荷氨基酸残基形成“盐桥”,该盐桥在pro-UK 与PAI-1 结合过程中起决定性作用;用苯甲酰甲醛处理pro-UK,改变四个Arg 残基可使衍生物在血浆中最稳定,且具有更高的溶栓活性,推测该四个Arg残基中有3 个位于pro-UK 表面的可变环(R178RHRGGS184)中。
基于以上结果,本研究设计带有突变位点的引物,以天然pro-UK cDNA为模板,采用PCR 介导的定点突变技术对pro-UK 基因进行了突变,构建了三种人pro-UK 的突变体,分别将蛋白序列中的凝血酶作用位点Arg156 突变成His156 ,构建成pro-UKM1;将PAI-1 的作用位点Arg178、Arg179、Arg181 突变为Lys178、Lys179、His181构建成pro-UKM3;同时将凝血酶和PAI-1 的作用位点突变构建成pro-UKM13。
由于蛋白翻译后修饰(糖基化、形成二硫键、亚基组装等)对产物的性质至关重
Arterial thrombosis is the underlying cause of a wide variety of cardiovascular diseases including myocardial infarction. Most of the currently used thrombolytic agents are plasminogen activators, which are very efficient in restoring the blood flew. They can convert plasminogen into plasmin and thus degrade fibrin. Despite the widespread use of a established thrombolytic agents such as streptokinase, tissue-type plasminogen activators (t-PA), urokinase-type plasminogen activators (u-PA), recombinant tissue-type plasminogen activator(rt-PA),anisoylated plasminogen streptokinase activator complex (APSAC),all these agents suffer from a number of inadequacies including resistance to reperfusion, occurrence of coronary reocclusion and bleeding complications.The quest continues for plasminogen activators with higher potency, specific thrombolytic activity, fibrin selectivity and longer half-life time. Based on the detail understanding of the relationship between structure and function of thrombolytic agents, the new generation of thrombolytic agents showed considerable potential with regard to improving to the efficacy of thrombolytic therapy.
Prourokinase(pro-UK) is a single-chain urokinase-type plasminogen activators belonging to serine protease, able to promote the dissolution of fibrin clot through the activation of plasminogen to plasmin which has a broad application in thrombolysis therapy. In-vitro and in-vivo, pro-UK induces fibrin specific clot lysis, when dosage is limited to a range at which activation of pro-UK to urokinase(UK) is restricted to the fibrin surface. At higher doses, systemic conversion to UK takes place,some of the benefits of pro-UK are lose. Characterization of pro-UK showed that it was made up of 411 residues with a plasmin or kallikrein susceptible site at Lys158-Ile159 .Another functionally important cleavage site at Arg156-Phe157 is hydrolyzed by thrombin and generates a two-chain form with little catalytic activity against plasminogen.To prevent systemic activation of
plasminogen, the regulation of pro-UK activity involves three plasminogen activator inhibitors(PAIs).PAI-1 is the major PAI of plasma, forming inactive covalent complexes with the activator. A reseach showed that the pro-UK with four modified Arg residues by phenylglyoxal under mild conditions was found to be the most stable derivative in human blood plasma; it causes a more efficient lysis of plasma clots than the native activator. Three of four modified Arg residues are supposed to be within the 178RRHRGGS184 cluster, which was localized in the superficial loop of the pro-UK globule and was shown to interact with the complementary series of negatively charged residues in the molecule of the main plasma inhibitor PAI-1. The neutralization of positively charged Arg residues in this cluster decreases the affinity of pro-UK for PAI-1, which results in an enhancement of the stability in plasma and the fibrinolytic efficiency of the activator. Based on the above researches, three pro-UK mutant (pro-UKm) genes were constructed in this experiment with the PCR point-mutant method. The thrombin cleavage site Arg156 in pro-UK was mutated into His156, and named as pro-UKM1; PAI binding site Arg178, Arg179,Arg181 was mutated into Lys178, Lys179, His181, named as pro-UKM3; The mutant containing His156, Lys178, Lys179, His181 as pro-UKM13. The primers with mutant sites were designed, then the mutant cDNA was constructed through PCR. Because post-translation modification such as glycosylation ,disulfide bond formation, specific proteolysis, and formation of higher-order structure are important for protein activity, eukaryotic cell—-chinese hamster ovary cell—-has been used as a host cell to produce heterologous proteins—pro-UKm. Three mutants were analyzed with fibrin plate assay , mutant antigenic specificity assay ,SDS-PAGE fibrin plate assay, western blot, and Lysis in vitro of human blood clots after inhibition with thrombin and/or PAI. The fibrin plate assay , antigenic specificity assay displayed that the three mutant can dissolute fibrin and combine with pro-UK primary antigen.The SDS-PAGE fibrin plate assay and western blot results showed that the three mutants and the native pro-UK have the same single electrophoresis band indicating most of the pro-UK was single chain. In vitro plasma clot lysis assays indicated that the pro-UKM1 have the ability to resistant against thrombin digestion; pro-UK3 could resist against PAI inhibition; while pro-UK13 improved resistances against both thrombin and PAI. It looks very promising that the pro-UK13 can be a new medicine of dissolving thrombus. To produce transgenic animals which can express pro-UKm in animal mammary gland, two kinds of vector were constrcted. In vectorⅠ, pro-UKM1, pro-UKM3, pro-UKM13 were respectively inserted to the PBCⅠvector by the XhoⅠcloning site. The PBCⅠvector uses the goat ?-casein promoter to drive high-level expression of
pro-UKm.The goat ?-casein promoter is a tissue-specific promoter that target expression of pro-UKm almost exclusively to the lactating mammary gland. The vector Ⅰwere identified by PCR, and can be used to produce transgenic animals by microinjection or sperm-media technology. Rous Sarcoma Virus enhancer/promoter, modified HIV-1 5’and 3’Long Terminal Repeat(LTR), HIV-1 psi(ψ) packageing sequence, HIV Rev response element were amplified by PCR, then partial goat ?-casein promoter, partial goat ?-casein genomic sequence, the PCR products, BGH poly(A), pro-UKM13 cDNA, and pGRM-7ZF were used to construct the vector Ⅱ-another mammary gland expression lentivirus vector, which can be transfected into package 293FT cell to get recombinant lentivirus used to infect zygote to produce transgenic animal. To efficiently construct cloned human type 5 ’recombinant adenovirus bearing pro-UK mutant by employing homologous recombination in Escherichia coli, pro-UKM13 cDNA was first subcloned into vector pShuttle -CMV. After identification, the resultant plasmid was linearized by Pme I and subsequently cotransformed in to E. coli BJ 5183 cells by electroporation with adenoviral backbone plasmid pAdEasy-1. To directly confirm recombination, pro-UKM13 cDNA was amplified by PCR with oligonucleotide in pro-UKM13 cDNA . After the cloned recombinant adenovirus plasmid DNA was obtained, it was digested with P ac I for releasing inverted terminal repeat(ITR) and transfected into package 293 cell to get recombinant virus. As a result, PCR products indicated that pro-UKM13 cDNA was successfully integrated into the adenoviral genome. Homogenous recombinant virus could be easily obtained by transfecting 293 cell with this infectious DNA. The resultant recombinant adenovirus could reach 4.6×106 pfu/ml in 293 cells.This experiments suggest that construction of cloned recombinant adenovirus genome by homologous recombination in Escherichia coli is a very efficient method to construct homogeneous recombinant adenovirus. This recombinant adenovirus can be injected into mammary gland of mice or goat directly, allowed for the efficient secretion of pro-UKM13 in the milk. ,
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