摘要
目的
血栓性疾病,如心肌梗塞、脑梗塞和深静脉血栓等,严重危害着人类的健康,溶栓治疗是血栓性疾病安全而有效的治疗手段。目前临床上常用的主要有链激酶、尿激酶和瑞替普酶(rt-PA)等,但存在半衰期短,需反复给药,费用昂贵等缺点。因此,人们又把研究的热点转向其他来源的纤溶酶原激活剂上来,如纳豆激酶(NK)、吸血蝙蝠唾液纤溶酶原激活剂(bat-PA)和蛇毒纤溶酶原激活剂等。蛇毒是多种生物活性蛋白和多肽的混合物,其中许多的组分可作用于人的凝血和纤溶系统,特别是在蝮科和蝰科蛇毒中尤为突出。本研究旨在通过传统的分离纯化方法和现代的基因工程方法从江浙产短尾蝮蛇毒中获得短尾蝮蛇毒纤溶酶原激活剂(Plasminogen activator of Gloydius brevicaudus venom, GBV-PA),并对其理化性质及抗栓作用进行初步研究,为寻找新的抗血栓药物提供实验依据。
方法
本研究应用亲和层析和反相高效液相色谱(RP-HPLC)两步法分离纯化蛇毒纤溶酶原激活剂,纤维蛋白平板法鉴定活性。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定蛋白质的纯度及分子量大小,聚丙烯酰胺凝胶盘状电泳测定其等电点,发色底物法鉴定GBV-PA激活人纤溶酶原为纤溶酶的活性,GBV-PA与抑制剂的相互作用和纤维蛋白结合实验测定GBV-PA的部分理化性质。应用FeC13诱导的大鼠颈总动脉血栓、兔脑粉浸液诱发的大鼠下腔静脉血栓和犬实验性急性脑梗塞等不同的血栓模型对蛇毒纤溶酶原激活剂的抗栓作用进行研究。采用经典的Born法,测定GBV-PA对ADP和凝血酶诱导的体外血小板聚集的影响。采用ADP诱导的急性肺栓塞和大鼠颈动静脉回路血栓模型观察GBV-PA对体内血小板聚集的影响。利用基因工程技术,从江浙短尾蝮蛇毒中获得GBV-PA的cDNA序列,将该基因定向克隆到原核表达载体pET42a(+)中,在大肠杆菌中表达rGBV-PA蛋白。
结果
从江浙短尾蝮蛇毒中分离纯化到分子量为32kDa左右,等电点为5.2的纤溶酶原激活剂,命名为GBV-PA。发色底物法(S- 2390)法证明GBV-PA能特异的激活人纤溶酶原为纤溶酶而降解纤维蛋白,其比活性为2.87 t-PA IU·mg-1。GBV-PA为一种丝氨酸蛋白酶,与纤维蛋白无特异性的亲和力。
应用三种不同的血栓模型研究蛇毒纤溶酶原激活剂的抗栓作用,结果表明:分离纯化到的蛇毒纤溶酶原激活剂对于动静脉血栓均有良好的溶解作用,在一定范围内呈明显的量效和时效关系,且对血栓的形成有抑制作用。采用经典的Born法,测定GBV-PA对ADP和凝血酶诱导的体外血小板聚集的影响,结果显示:GBV-PA对ADP和凝血酶诱导的血小板聚集均有抑制作用,呈明显的量效关系。ADP诱导的急性肺栓塞和大鼠颈动静脉回路血栓模型观察GBV-PA对体内血小板聚集的影响,实验结果表明GBV-PA可抑制体内血小板聚集。
成功地从江浙短尾蝮蛇毒中获得GBV-PA的核苷酸序列,GBV-PA的cDNA大小为777bp,推导出的氨基酸数目为259个。GBV-PA核苷酸序列与TSV-PA和Haly-PA的同源性分别为91%,和96%。将该基因定向克隆到原核表达载体pET42a(+)中,并在大肠杆菌中得到表达,SDS-PAGE凝胶电泳显示rGBV-PA主要以包涵体形式存在,分子量约61.7kDa。rGBV-PA经His亲和层析柱纯化后,获得的rGBV-PA融合蛋白具有蝮蛇毒的抗原性。
结论
⑴成功地从江浙短尾蝮蛇毒中分离纯化到分子量为32kDa左右,等电点为5.2的纤溶酶原激活剂,命名为GBV-PA。
⑵GBV-PA为一种丝氨酸蛋白酶,对纤维蛋白无亲和性。
⑶GBV-PA对实验性动、静脉血栓均有治疗作用,GBV-PA可明显抑制血小板聚集。
⑷在大肠杆菌中成功表达得到rGBV-PA蛋白,为rGBV-PA进一步的药效及药物动力学研究提供了充足的原料。
Objective
Thromboembolic disease,such as Myocardial infarction,stroke and deep vein thrombosis,does great harm to people’s health. The most common thrombolytic agents have been streptokinase,urokinase and reteplase. But most of them have short half-life.In the quest for new thrombolytic agents, scientists turned to extracting natural thrombolytic agents from animals and insects and from snake venoms. Snake venoms were complex mixtures containing many different biologically active proteins and peptides. A number of these proteins act on components of the haemostatic system in humans.This study was designed to isolate and purify novel plasminogen activator from Gloydius brevicaudus venom(GBV-PA)and analyse its characterization and biological activities.
Method
A novel plasminogen activator from Gloydius brevicaudus venom was identified and purified to homogeneity by Affinity chromatography and RP-HPLC. The biological activity of GBV-PA was assayed by The fibrin plate method. molecular weight and isoelectric point were assayed by SDS-PAGE and disc polyacrylamide gel eletrophoresis. The biological activities were assayed by the chromogenic substrate method and Fibrin binding experiments. The study on thrombolytic effect of GBV-PA by using three models which were thrombus of common carotid artery induced by FeC13 in rat,and thrombus of inferior caval vein induced by powder of rabbit brain and cerebral thrombus in dog. The platelet aggregation induced by ADP and thrombin were assayed by the Born’s method. The effect on t platelet aggregation was observed by the model of pulmonary thrombus in rats and thrombus of man-made artery-vein circulation in rat. Isolated the complementary DNA encoding PA precursor from the Gloydius brevicaudus By RT-PCR. The cDNA of GBV-PA were subcloned into PET-42a expression vector and then transfected into Escherichia coli. The inclusion of the transfected cells turned out biological activity of GBV-PA as assayed by The fibrin plate method.
Results
A novel plasminogen activator from Gloydius brevicaudus venom has been identified and purified to homogeneity by Affinity chromatography and RP-HPLC. SDS-PAGE shows that GBV-PA was homogeneous as a single band in non-reducing conditions and reducing conditions.It is a single chain protein with an apparent molecular weight of 32600 and an isoelectric point of pH 5.2. GBV-PA specifically activates plasminogen through an enzymatic reaction that leads to plasmin generation and displays an indirect fibrinolysis activity through using the chromogenic substrate method. The activity of GBV-PA was 2.87 t-PA IU·mg-1 according the chromogenic substrate S- 2390. The activity of GBV-PA was not affected by a metal chelator (EDTA).In the contrast, PMSF, the irreversible serine protease inhibitor,can completely inhibite the fibrinolysis activity of GBV-PA. These observations show that GBV-PA is not a metallo-protease and indicated that it is a serine protease. Fibrin binding experiments demonstrate that GBV-PA has not fibrin binding capacity.
The study on thrombolytic effect of GBV-PA by using three models .The results indicated that GBV-PA have the thrombolytic effect which are time-response and dose-response relationships.And it also can inhibit the formation of thrombus. According to the Born’s method,the effects of GBV-PA on platelet aggregation induced by ADP and thrombin were assayed. The results showed that GBV-PA dose-dependently inhibited ADP- induced and thrombin-induced platelet aggregation.We also certified that GBV-PA can inhibit the accumulation of platelet by the model of pulmonary thrombus in rats and thrombus of man-made artery-vein circulation in rat.
By RT-PCR, we successfully isolated the complementary DNA encoding PA precursor from the Gloydius brevicaudus. Sequence analysis revealed the obtained DNA consisted of 777bp .The nucleotide sequence which presents96% and 91% homology to that of Agkistrodon halys and Trimeresurus Stejnegeri respectively. The cDNA inserts of GBV-PA were subcloned into PET-42a expression vector and then transfected into Escherichia coli. SDS - PAGE showed that the fusion protein was expressed as inclusion body formation in E. coli BL21(DE3). The rGBV-PA fusion protein was obtained by His-bind Purification system,which has the antigenicity of Agkistrodon halys pallas venom.
Conclusions
In summary, A novel plasminogen activator from Gloydius brevicaudus venom has been identified and purified to homogeneity by Affinity chromatography and HPLC.It is a single chain serine proteases with an apparent molecular weight of 32600 and an isoelectric point of pH 5.2,but it has not fibrin binding capacity .It can specifically activates plasminogen to plasmin generation and displays an indirect fibrinolysis activity. It has the thrombolylic effect of both venous and arterial thrombosis . It can inhibite platelet aggregation and the thrombus formulation. a novel plasminogen activator of Gloydius brevicaudus had been successfully over expressed in Escherichia col.The obtained protein can be used as an instrument under investigation of pharmacokinetics and pharmacodynamics of GBV-PA.
引文
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