地鳖虫纤溶活性蛋白抗血栓、抗肿瘤及其基因克隆与表达的研究
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摘要
地鳖虫(Eupolyphaga sinensis walker)，为《中华人民共和国药典》(2000版)记载的正品药材，是传统的活血化瘀类动物药，其功效主要为逐瘀、破积、通络、理伤。现代药理试验已充分证明地鳖虫具有溶解静脉血栓、抑制血小板聚集和抗凝血等药用功效。但是传统中医用药均以全虫入药，对其药效成分不明确，且对其作用机理研究不充分，本文从地鳖虫体内提取出纤溶活性蛋白(Eupolyphaga sinesis Walker Hbrinolytic Protein，EFP)成分进行实验，首先研究它在抗血栓形成、抗凝血和增强纤溶方面的作用。抗肿瘤血管生成的研究是当前抗肿瘤研究的热门课题，随着中药抗肿瘤研究的不断深入，发现某些中药，特别是一些具有活血化瘀作用的中药均具有抗肿瘤功效，其抗肿瘤的机制在于抑制肿瘤血管的生成。基于此，本文还研究了地鳖虫纤溶活性蛋白在抑制抑制血管生成和抗肿瘤方面的作用。为深入研究地鳖虫纤溶活性蛋白，本文还克隆了地鳖虫纤溶活性蛋白基因cDNA序列，并用大肠杆菌和毕赤酵母表达系统进行了表达。
本文主要研究结果如下：
(1)用地鳖虫纤溶活性蛋白(ig.)作用于角叉菜胶所致血栓的小鼠模型和正常小鼠，结果表明地鳖虫纤溶活性蛋白能够抑制角叉菜胶所致的小鼠尾部血栓的形成，能明显延长正常小鼠凝血酶时间，增强其组织型纤溶酶原激活剂(t-PA)的活性、抑制其组织型纤溶酶原激活剂抑制剂(PAI)的活性。显示了地鳖虫纤溶活性蛋白具有良好的体内抗血栓作用。
(2)以MTT法检测地鳖虫纤溶活性蛋白对体外培养的人微血管内皮细胞(MVEC)增殖的影响，结果表明EFP能有效抑制MVEC的增殖，实验浓度(0.02-200μg/ml)范围内最高抑制率达45.93％，且呈剂量依赖关系。
(3)利用Annexin V-FITC/PI双荧光染色法和单细胞凝胶电泳(SCGE)法检测了EFP对MVEC凋亡的影响，结果表明EFP能显著促进MVEC细胞的凋亡。
(4)通过流式细胞术检测了EFP对MVEC细胞周期的影响，结果EFP可将MVEC细胞阻滞在S期和_G2/M期，使细胞不能进行分裂，抑制其增殖。
(5)用鸡胚尿囊膜(CAM)模型检测EFP对血管形成的抑制作用，结果发现EFP能显著抑制鸡胚尿囊膜的血管生成，且具有一定的量应关系。
(6)体内抑瘤实验结果表明，EFP能显著抑制S180和H22荷瘤小鼠实体瘤的生长，最高抑制率分别达48.54％和42.16％。
(7)检测H22荷瘤小鼠肝脏丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性，结果表明，EFP还能够增强小鼠体内抗氧化能力；检测H22荷瘤小鼠血清抗H22抗体水平和测量小鼠脾脏系数，结果表明EFP还能增强小鼠机体的体液免疫能力。
(8)分析多种动物纤溶酶氨基酸序列，根据其同源性，获得保守序列，利用保守序列设计简并引物，进行PCR扩增。由于多种动物纤溶酶成熟肽N端具有一段非常保守的序列，在设计兼并引物时选取了该段保守序列，所以PCR扩增得到是具有完整5’末端的cDNA序列。然后利用3’末端快速扩增法(RACE)获得了该序列的3’末端，经拼接得到了完整的EFP基因cDNA序列。
(9)利用大肠杆菌表达系统对EFP基因进行表达，首先构建了原核表达载体pET28a-EFP，转化表达菌株Rossetta，用IPTG进行诱导表达，结果经SDS-PAGE和Western blot鉴定，表明目的蛋白获得了表达，最高表达量约占细菌总蛋白的10％。但目的蛋白以包涵体的形式表达，无生物学活性。
(10)将EFP cDNA基因与分泌型酵母表达载体pPICZαA进行重组，转化毕赤酵母表达菌株GS115，得到的转化子经甲醇诱导后，诱导表达上清经SDS-PAGE和Western blot分析证明目的蛋白获得了表达，经纤维蛋白平板法检测，发现目的蛋白具有纤溶活性。
上述研究结果从不同方面表明地鳖虫纤溶活性蛋白具有抗血栓、抗肿瘤和抗肿瘤血管生成的作用；另外，成功克隆了EFP cDNA基因，并用毕赤酵母表达系统表达出了具有纤溶活性的蛋白。
Eupolyphaga sinensis Walker, a Chinese traditional medicine, which is used to activate bloodflow and remove blood stasis as recorded in Chinese Pharmacopoeia (2000 edition). It also exhibitsthe effects of melting vein thrombosis, restraining blood platelet aggregation and anticoagulationthrough the modem pharmacological researches. But the effective component and the underlyingmechanisms are not very clear. Here we purified a protein with fibrinolytic activity fromEupolyphaga sinesis Walker (EFP) and detected the antithrombotic, anticoagulatic andfibrinolytic effects respectively. We also analysed the activities of EFP on anti-angiogenesis andanticancer. Moreover, we cloned EFP gene and expressed it both in E. coli and in Yeast PichiaPastoris. The results are summarized as follows.
The antithrombotic experiments in vivo indicated that EFP could inhibit the thrombus forming onmice tail induced by carrageenin, delay thrombin time in mice obviously, enhance tissueplasminogen activator (t-PA) activity and inhibit plasminogen activator inhibitor activitysignificantly. The results suggest that EFP could suppress thrombosis.
The proliferation ability of human microvascular endothelial cell (MVEC)/n vitro was examinedby MTr assay. The result showed that EFP could inhibit MVEC proliferation efficiently, in adose-dependent manner. The inhibitory rate reached 45.93％under the experimentalconcentration (0.02-200μg/ml). EFP could induce the apoptosis of MVEC as indicated by AnnexinV-FITC/PI fluorescence staining and single cell gel electrophoresis (SCGE) assay. The flowcytometry analysis showed that EFP could block the cell cycle at S and G_2/M phases, inhibit celldivision and proliferation. Embryo chorioallantoic membrane (CAM) test showed that EFP couldinhibit angiogenesis effectively in a dose-dependent manner.
The anti-tumor experiments in vivo indicated that EFP could inhibit the tumor growth in S180and H22-bearing mice significantly, and the inhibition rate could reach 48.54％and 42.16％respectively. At the same time, we also examined MDA and SOD level in the mice's liver, and theresults showed that EFP could enhance the antioxidation ability in H22-bearing mice. By measuringthe level of anti-H22 antibody in serum and the spleen index in H22-bearing mice, the resultsshowed that EFP could enhance the immunity in those mice.
By analyzing the homogeneity and the conserved sequence of fibrinolytic enzyme amino acidsequences of several animals, the primers wcrc designed. A fragment of ElF cDNA sequence wasamplified by PCR. Because the 5' primer is the sequence of N terminal, the fragment contains awhole 5' terminal. Wc obtained the 3' terminal of the cDNA sequence by using 3'RACE. Then thewhole cDNA sequence of ElF was acquired.
With the gcnc of EFP cloned, we constructed a recombinant vector pET28a-EFP, and transformedit into E. coli Rossctta. Transformants were induced by IPTG. The target protein was examined bySDS-PAGE and Western blot analysis. Nearly 10％of the total bacterial protein was recombinantprotein expressed in inclusion body form but without activity.
Then we rccombined EFP gcnc with Yeast Pichia Pastoris vector pPICZαA, and transformed itinto Yeast Pichia Pastoris, the transformants were screened and cultured by methanol. Expressionproducts wcrc analyzed by SDS-PAGE and Western blot, and the results showed that the targetprotein has bccn expressed and with fibrinolytic activity by a fibrin plate assay.
All the results showed that EFP exhibits effects of anti-thrombus, anti-tumor andanti-angiogcncsis. The EFP gcnc has bccn cloned successfully, and EFP has bccn expressed byYeast Pichia Pastoris expression system.
本文主要研究结果如下：
(1)用地鳖虫纤溶活性蛋白(ig.)作用于角叉菜胶所致血栓的小鼠模型和正常小鼠，结果表明地鳖虫纤溶活性蛋白能够抑制角叉菜胶所致的小鼠尾部血栓的形成，能明显延长正常小鼠凝血酶时间，增强其组织型纤溶酶原激活剂(t-PA)的活性、抑制其组织型纤溶酶原激活剂抑制剂(PAI)的活性。显示了地鳖虫纤溶活性蛋白具有良好的体内抗血栓作用。
(2)以MTT法检测地鳖虫纤溶活性蛋白对体外培养的人微血管内皮细胞(MVEC)增殖的影响，结果表明EFP能有效抑制MVEC的增殖，实验浓度(0.02-200μg/ml)范围内最高抑制率达45.93％，且呈剂量依赖关系。
(3)利用Annexin V-FITC/PI双荧光染色法和单细胞凝胶电泳(SCGE)法检测了EFP对MVEC凋亡的影响，结果表明EFP能显著促进MVEC细胞的凋亡。
(4)通过流式细胞术检测了EFP对MVEC细胞周期的影响，结果EFP可将MVEC细胞阻滞在S期和_G2/M期，使细胞不能进行分裂，抑制其增殖。
(5)用鸡胚尿囊膜(CAM)模型检测EFP对血管形成的抑制作用，结果发现EFP能显著抑制鸡胚尿囊膜的血管生成，且具有一定的量应关系。
(6)体内抑瘤实验结果表明，EFP能显著抑制S180和H22荷瘤小鼠实体瘤的生长，最高抑制率分别达48.54％和42.16％。
(7)检测H22荷瘤小鼠肝脏丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性，结果表明，EFP还能够增强小鼠体内抗氧化能力；检测H22荷瘤小鼠血清抗H22抗体水平和测量小鼠脾脏系数，结果表明EFP还能增强小鼠机体的体液免疫能力。
(8)分析多种动物纤溶酶氨基酸序列，根据其同源性，获得保守序列，利用保守序列设计简并引物，进行PCR扩增。由于多种动物纤溶酶成熟肽N端具有一段非常保守的序列，在设计兼并引物时选取了该段保守序列，所以PCR扩增得到是具有完整5’末端的cDNA序列。然后利用3’末端快速扩增法(RACE)获得了该序列的3’末端，经拼接得到了完整的EFP基因cDNA序列。
(9)利用大肠杆菌表达系统对EFP基因进行表达，首先构建了原核表达载体pET28a-EFP，转化表达菌株Rossetta，用IPTG进行诱导表达，结果经SDS-PAGE和Western blot鉴定，表明目的蛋白获得了表达，最高表达量约占细菌总蛋白的10％。但目的蛋白以包涵体的形式表达，无生物学活性。
(10)将EFP cDNA基因与分泌型酵母表达载体pPICZαA进行重组，转化毕赤酵母表达菌株GS115，得到的转化子经甲醇诱导后，诱导表达上清经SDS-PAGE和Western blot分析证明目的蛋白获得了表达，经纤维蛋白平板法检测，发现目的蛋白具有纤溶活性。
上述研究结果从不同方面表明地鳖虫纤溶活性蛋白具有抗血栓、抗肿瘤和抗肿瘤血管生成的作用；另外，成功克隆了EFP cDNA基因，并用毕赤酵母表达系统表达出了具有纤溶活性的蛋白。
Eupolyphaga sinensis Walker, a Chinese traditional medicine, which is used to activate bloodflow and remove blood stasis as recorded in Chinese Pharmacopoeia (2000 edition). It also exhibitsthe effects of melting vein thrombosis, restraining blood platelet aggregation and anticoagulationthrough the modem pharmacological researches. But the effective component and the underlyingmechanisms are not very clear. Here we purified a protein with fibrinolytic activity fromEupolyphaga sinesis Walker (EFP) and detected the antithrombotic, anticoagulatic andfibrinolytic effects respectively. We also analysed the activities of EFP on anti-angiogenesis andanticancer. Moreover, we cloned EFP gene and expressed it both in E. coli and in Yeast PichiaPastoris. The results are summarized as follows.
The antithrombotic experiments in vivo indicated that EFP could inhibit the thrombus forming onmice tail induced by carrageenin, delay thrombin time in mice obviously, enhance tissueplasminogen activator (t-PA) activity and inhibit plasminogen activator inhibitor activitysignificantly. The results suggest that EFP could suppress thrombosis.
The proliferation ability of human microvascular endothelial cell (MVEC)/n vitro was examinedby MTr assay. The result showed that EFP could inhibit MVEC proliferation efficiently, in adose-dependent manner. The inhibitory rate reached 45.93％under the experimentalconcentration (0.02-200μg/ml). EFP could induce the apoptosis of MVEC as indicated by AnnexinV-FITC/PI fluorescence staining and single cell gel electrophoresis (SCGE) assay. The flowcytometry analysis showed that EFP could block the cell cycle at S and G_2/M phases, inhibit celldivision and proliferation. Embryo chorioallantoic membrane (CAM) test showed that EFP couldinhibit angiogenesis effectively in a dose-dependent manner.
The anti-tumor experiments in vivo indicated that EFP could inhibit the tumor growth in S180and H22-bearing mice significantly, and the inhibition rate could reach 48.54％and 42.16％respectively. At the same time, we also examined MDA and SOD level in the mice's liver, and theresults showed that EFP could enhance the antioxidation ability in H22-bearing mice. By measuringthe level of anti-H22 antibody in serum and the spleen index in H22-bearing mice, the resultsshowed that EFP could enhance the immunity in those mice.
By analyzing the homogeneity and the conserved sequence of fibrinolytic enzyme amino acidsequences of several animals, the primers wcrc designed. A fragment of ElF cDNA sequence wasamplified by PCR. Because the 5' primer is the sequence of N terminal, the fragment contains awhole 5' terminal. Wc obtained the 3' terminal of the cDNA sequence by using 3'RACE. Then thewhole cDNA sequence of ElF was acquired.
With the gcnc of EFP cloned, we constructed a recombinant vector pET28a-EFP, and transformedit into E. coli Rossctta. Transformants were induced by IPTG. The target protein was examined bySDS-PAGE and Western blot analysis. Nearly 10％of the total bacterial protein was recombinantprotein expressed in inclusion body form but without activity.
Then we rccombined EFP gcnc with Yeast Pichia Pastoris vector pPICZαA, and transformed itinto Yeast Pichia Pastoris, the transformants were screened and cultured by methanol. Expressionproducts wcrc analyzed by SDS-PAGE and Western blot, and the results showed that the targetprotein has bccn expressed and with fibrinolytic activity by a fibrin plate assay.
All the results showed that EFP exhibits effects of anti-thrombus, anti-tumor andanti-angiogcncsis. The EFP gcnc has bccn cloned successfully, and EFP has bccn expressed byYeast Pichia Pastoris expression system.
引文
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