重组人纤溶酶原Kringle1-5的制备及其

为了研究重组人纤溶酶原 Kringle1-5(K1-5)的抗血管生成活性及其对内皮细胞增殖的影响, 通过PCR扩增人纤溶酶原K1-5 cDNA,定向克隆于原核表达载体pET30a(+)中,构建重组表达载体pET-K1-5, 转化E.coli BL21(DE3), IPTG诱导表达,SDS-PAGE 和Western 杂交检测K1-5的表达。鸡胚尿囊膜 (CAM) 实验和MTT实验分别检测重组人纤溶酶原Kringle1-5对鸡胚新生血管生成和内皮细胞的抑制作用。结果表明,IPTG诱导原核表达载体pET-K1-5在E.coli BL21(DE3)中的表达量约占菌体总蛋白量的32%, K1-5主要以包涵体形式存在,包涵体经过洗涤、溶解、Ni-spin 亲合柱层析纯化以及蛋白质复性等步骤后,获得了纯度约为96%的重组K1-5蛋白。CAM实验表明,原核表达的重组人K1-5能有效地按剂量依赖的方式抑制鸡胚新生血管的形成。MTT实验结果显示,重组人K1-5特异地抑制内皮细胞的增殖, 而对非内皮细胞无抑制作用。     

重组小鼠canstatin N端片段对鸡胚新生血管生成的抑制作用(英)

为了研究重组小鼠canstatin N端片段的体内抗血管生成活性, 通过PCR扩增小鼠canstatin N端片段cDNA,定向克隆于原核表达载体pET30a(+)中,构建小鼠canstatin N端片段重组表达载体pET-mCanN, 转化E.coli BL21(DE3), IPTG诱导表达,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹检测小鼠canstatin N端片段的表达. 结果表明,IPTG诱导原核表达载体pET-mCanN在大肠杆菌E.coli BL21(DE3)中高效表达, 小鼠canstatin N端片段表达量约占菌体总蛋白量的18%, 小鼠canstatin N端片段主要以包涵体形式存在,包涵体经过洗涤、裂解、Ni-spin column亲合柱层析以及蛋白质复性等步骤纯化后,获得了纯度约为92%的重组小鼠canstatin N端片段. 鸡胚绒毛尿囊膜(chicken embryo choriollantoic membrane,CAM)实验表明,原核表达的小鼠canstatin N端片段能有效地按剂量依赖的方式抑制鸡胚新生血管的形成.     

Expression of recombinant kringle 1-5 domains of human plasminogen by a prokaryote expression system

Kringle1-5 (K1-5), a proteolytic fragment containing five kringle domains of human plasminogen generated by plasmin-mediated proteolysis, has been already identified by Cao et al. with relation to anti-angiogenesis and proliferation of endothelial cells. To investigate anti-angiogenesis activity of recombinant human K1-5 (rhK1-5) expressed in Escherichia coli BL21, the cDNA of human K1-5 obtained from cloning vector pUC57-K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E. coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody and an anti-hexahistidine tag antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed, purified, and refolded to a purity of 96% as estimated by capillary electrophoresis and the final purification yield of K1-5 in E. coli system was approximately 5.8 mg/L. Purified K1-5 protein was tested on chicken embryo chorioallantoic membranes (CAMs), and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, the rhK1-5 potently inhibited endothelial cell proliferation but not non-endothelial cells. For the first time, these findings demonstrate that the rhK1-5 produced by a prokaryote expression system effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells. This fact derived from the present study further suggests the rhK1-5 can be used for anti-angiogenesis therapy of cancer.     

Functional expression of recombinant canstatin in stably transformed Drosophila melanogaster S2 cells

We describe the expression and in vitro activity of recombinant canstatin from stably transformed Drosophila melanogaster S2 cells. Southern blot analysis indicated that transformed S2 cells contained multiple copies of the canstatin gene in the genome. Recombinant canstatin with a molecular weight of 29kDa was secreted into the culture medium. Recombinant canstatin was purified to homogeneity using a simple one-step Ni(2+) affinity fractionation. Purified recombinant canstatin inhibited human umbilical vein endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED(50)) for recombinant canstatin expressed in stably transformed Drosophila S2 cells was approximately 0.37mug/ml. A maximum production level of 76mg/l of recombinant canstatin was obtained in a T-flask culture of Drosophila S2 cells 6 days after induction with 0.5mM CuSO(4).     

Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth

We isolated and identified an endogenous 24-kDa human basement membrane-derived inhibitor of angiogenesis and tumor growth, termed canstatin. Canstatin, a fragment of the alpha2 chain of type IV collagen, was produced as a recombinant molecule in Escherichia coli and 293 embryonic kidneys cells. Canstatin significantly inhibited human endothelial cell migration and murine endothelial cell tube formation. Additionally, canstatin potently inhibited 10% fetal bovine serum-stimulated endothelial cell proliferation and induced apoptosis, with no inhibition of proliferation or apoptosis observed on non-endothelial cells. Inhibition of endothelial proliferation was not concomitant with a change in extracellular signal-regulated kinase activation. We demonstrate that apoptosis induced by canstatin was associated with a down-regulation of the anti-apoptotic protein, FLIP. Canstatin also suppressed in vivo growth of large and small size tumors in two human xenograft mouse models with histology revealing decreased CD31-positive vasculature. Collectively, these results suggest that canstatin is a powerful therapeutic molecule for suppressing angiogenesis.     

小鼠canstatin C端片段基因的克隆及其在大肠杆菌中的表达

为了构建小鼠canstatinC端片段的原核表达载体并在大肠杆菌中表达。以小鼠肝脏组织总RNA为模板,通过RT-PCR扩增小鼠canstatinC端片段(mCan-C)基因,克隆到pMD18-T载体中并进行序列分析。将mCan-C基因定向克隆于原核表达载体pET30a(+)中,构建表达载体pET／mCan-C,转化大肠杆菌BL21(DE3),IPTG诱导表达。结果表明,小鼠canstatinC端片段的cDNA长度为399bp,含有1个终止密码,编码132个氨基酸,与已知的人canstatinC端片段氨基酸的同源性为61％。IPTG诱导mCan-C在大肠杆菌E．coliBL21中表达,表达量约占菌体总蛋白量的28％,重组蛋白主要以包涵体形式存在。首次克隆了小鼠canstatinC端片段的cDNA,IPTG诱导mCan-C在大肠杆菌E．coliBL21中高效表达。小鼠canstatinC端片段的cDNA序列已收入GenBank,接受号为:AY502947。     

人血管能抑素基因的克隆、表达及其表达产物的纯化和生物活性测定

以人胎盘脐带组织为材料,提取组织总RNA,用netRTPCR方法合成人血管能抑素cDNA基因,将该cDNA克隆进pSP72载体获得重组质粒pSP72C, DNA序列分析结果与预期序列一致。用BamHⅠ和NdeⅠ双酶切,切下pSP72C上的血管能抑素cDNA,插入pET3c载体的相应位点获得重组表达质粒pETC, 转化E. coli BL21(DE3), SDSPAGE分析显示：在IPTG诱导下,血管能抑素基因获得了高效表达,表达量约占菌体总蛋白的 27.9 %,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白复性以及Sephadex G75凝胶过滤层析等步骤后,获得了纯度达91.4 %的人血管能抑素。CAM实验证明10 μg纯化蛋白就能显著抑制鸡胚新生血管生成。     

小鼠canstatin及其N端片段在大肠杆菌BL21 中的表达

以小鼠肝脏组织总RNA为模板,通过RT-PCR扩增小鼠canstatin及其N端片段基因,克隆到pMD18-T载体中并进行序列分析。将小鼠canstatin及其N端片段基因定向克隆于原核表达载体pET30a(+)中,分别构建表达质粒pET/Can和pET/Can-N, 转化大肠杆菌BL21(DE3), IPTG诱导表达。结果表明: 小鼠canstatin的cDNA长度为684bp,编码227个氨基酸,与已知的人canstatin cDNA同源性为89%,氨基酸的同源性为96%。小鼠canstatin N端片段(1-95aa)与人的同源性为100%。 IPTG诱导原核表达载体pET/Can和pET/Can-N在大肠杆菌 BL21(DE3)中的表达量约占菌体总蛋白量的35% 和 18%, 重组蛋白主要以包涵体形式存在。文中报道的小鼠canstatin 及其N端片段核苷酸序列已收入GenBank, 接受号分别为: AY375463和AY502946。Abstract:The mouse canstatin and its N-domain cDNA were amplified from total RNA of mouse liver by RT- PCR and cloned into vector pMD18-T for sequencing. Prokaryotic expression vectors pET/Can and pET/Can-N were constructed and expressed in E.coli BL21(DE3) with induction of IPTG.. Mouse canstatin cDNA is 684bp in length encoding 227 amino acids. The sequences of both cDNA and amino acids share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. N-domain of mouse canstatin is the same amino acid sequence as that of human canstatin. In the present study, prokaryotic expression vector pET/Can and pET/Can-N were expressed in E.coli BL21 with amount of 35% and 18% of the total bacterial proteins after being induced by IPTG for 4h. The expressed products existed mainly as inclusion bodies. This work has laid down the basis for further study of its angiogenic activity and potential application for tumor dormancy therapy.     

Expression, purification, and characterization of recombinant fibulin-5 in a prokaryote expression system

Fibulin-5 is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. To investigate anti-angiogenesis activities and context-specific activities on responsive cells of recombinant fibulin-5 (rfibulin-5) expressed in Escherichia coli, the cDNA of fibulin-5 cloned from a human placenta cDNA library was inserted into the pET32a (+) vector to allow fibulin-5 expression as a Trx fusion protein. The fusion protein Trx-fibulin-5, expressed as insoluble inclusion bodies, was solubilized and its resulting expression level reached to 15% of the total cell protein. The Trxfibulin-5 was purified effectively by N²⁺-chelating chromatography and then identified by Western blotting analysis with an anti-His tag antibody. The purified Trx-fibulin-5 was refolded by dialysis against redox reagents, and the rfibulin-5 released from the fusion protein by enterokinase cleavage was purified using a RESOURCE RPC column. The final purified rfibulin-5 effectively inhibited angiogenesis in chicken embryos in a dose-dependent manner through a chorioallantoic membrane (CAM) assay. Additionally, rfibulin-5 potently suppressed in vitro proliferation of human umbilical vein endothelial cells, but stimulated that of human dermal fibroblasts. The expression and in vitro refolding of rfibulin-5 resulted in production of an active molecule with a yield of 2.1 mg/L.     

人血管能抑素基因N端片段的表达、纯化及其生物活性测定

以中国人胎盘脐带组织为材料 ,提取组织总RNA ,用RT PCR方法合成人血管能抑素cDNA ,将该cD NA克隆进 pSP72载体获得重组质粒 pSP72C。以pSP72C为模板 ,PCR方法合成编码血管能抑素N端 189aa的基因片段 ,将其克隆进pET 3c载体获得重组表达质粒 pET CN ,转化E .coliBL2 1(DE3) ,SDS PAGE分析显示 ,在IPTG诱导下 ,人血管能抑素N端基因片段获得了有效表达 ,表达量约占菌体总蛋白质的 35 .3% ,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白质复性以及SephadexG 10 0凝胶过滤层析等步骤纯化后 ,获得了纯度约92 .6 %的人血管能抑素N端片段 ,CAM实验证明具有显著抑制鸡胚新生血管生成活性。     

Expression, purification, and bioactivity of human tumstatin from Escherichia coli

Tumstatin is a M(r) 28,000 C-terminal NC1 fragment of type alpha3 (IV) collagen that inhibits pathological angiogenesis and suppresses proliferation of endothelial cells and growth of tumors. We report here high cytoplasmic expression of recombinant human tumstatin in Escherichia coli and its purification, in vitro refolding, and inhibitory activity analysis. Human tumstatin was expressed in the bacterial cytoplasm as an insoluble N-terminal polyhistidine tagged protein, which accounted for more than 30% of total bacterial protein in BL21 (DE3) cells. After extraction and solubilization in guanidine-HCl, recombinant protein was purified to homogeneity using a simple one-step Ni(2+)-chelate affinity chromatography and then refolded by dialysis against acidic pH buffers with gradually decreasing concentrations of denaturant. The renatured recombinant tumstatin could specifically inhibit endothelial cell proliferation in a dose-dependent manner, and suppress bFGF-induced angiogenesis in chick embryo chorioallantoic membrane and tumor growth in mouse B16 melanoma xenograft models.     

Inhibition of endothelial cell proliferation by the recombinant kringle domain of tissue-type plasminogen activator

Tissue-type plasminogen activator (tPA) is a multidomain serine protease that converts the zymogen plasminogen to plasmin. tPA contains two kringle domains which display considerable sequence identity with those of angiostatin, an angiogenesis inhibitor. TK1-2, a recombinant kringle domain composed of t-PA kringles 1 and 2 (Ala(90)-Thr(263)), was produced by both bacterial and yeast expression systems. In vitro, TK1-2 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, and epidermal growth factor. It did not inhibit proliferation of non-endothelial cells. TK1-2 also inhibited in vivo angiogenesis in the chick embryo chorioallantoic membrane model. These results suggest that the recombinant kringle domain of t-PA is a selective inhibitor of endothelial cell growth and identifies this molecule as a novel anti-angiogenic agent.     

人肿瘤抑素(Tumstatin)在E.coli中的克隆、表达及活性分析

从人胚肾2 93细胞中扩增肿瘤抑素(tumstatin)基因,进行原核表达,纯化和生物活性检测.利用原核表达载体pMAL c2在大肠杆菌BL2 1中表达肿瘤抑素,经AmyloseResin亲和层析柱和QSepharoseFastFlow柱纯化,通过体外内皮细胞增殖、内皮细胞凋亡和鸡尿囊绒膜新生血管生成试验检测其抑制活性.MBP tumstatin在BL2 1中表达率约2 0 % ,肿瘤抑素纯度可达95 % .肿瘤抑素可明显抑制内皮细胞增殖(IC50 约为15 μg ml)、诱导内皮细胞凋亡和抑制鸡尿囊绒膜新生血管生成.研究结果表明,肿瘤抑素对内皮细胞具有明显的抑制作用,提示其在肿瘤治疗中有潜在的应用前景.     

Production and on-column re-folding of human vascular endothelial growth factor 165 in Escherichia coli

Vascular endothelial growth factors (VEGFs) are a family of proteins that promote angiogenesis and participate in a variety of physiological and pathological processes. In this work, the gene encoding the human VEGF isoform 165 (hVEGF₁₆₅) was cloned into the expression vector pET32a (+) to construct a fusion expression plasmid that induced the thioredoxin (Trx) gene and transformed into Escherichia coli. The recombinant fusion protein TrxhVEGF₁₆₅ was expressed optimally as inclusion bodies in the case of being cultivated for 4 h at 30°C and 1 mM IPTG concentration. The Trx-hVEGF₁₆₅ was refolded and purified effectively from urea-solubilized inclusion bodies by the immobilized metal affinity chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant hVEGF₁₆₅ (rhVEGF₁₆₅) was biologically active as assessed by the human umbilicalvein endothelial cells (HUVECs) proliferation and the chicken chorioallantoic membrane (CAM) assay. The expression and in vitro refolding of rhVEGF₁₆₅ resulted in production of an active molecule in a yield of 4.04 mg/L flask cultivation.     

基质金属蛋白酶-2 C端片段PEX的原核表达及其对血管发生的抑制作用

为了克隆表达鸡的基质金属蛋白酶-2(MMP-2)的C端片段PEX,并探讨其对血管发生的抑制作用,利用RT-PCR从鸡胚成纤维细胞克隆MMP-2 C端片段PEX,构建原核表达载体pCal-n-PEX;转化大肠杆菌BL21(DE3)-pLys,异丙基β-D硫代半乳糖苷（IPTG）诱导产生PEX融合蛋白,包涵体蛋白用盐酸胍法变性、复性;生长曲线观察PEX融合蛋白对人脐静脉血管内皮细胞增殖的影响;鸡胚绒毛尿囊膜血管发生实验研究其对血管发生的抑制作用.结果表明融合蛋白CBP/PEX具有抑制人脐静脉血管内皮细胞的生长和鸡胚绒毛尿囊膜血管发生的作用.提示PEX是有待进一步开发的潜在抑制血管发生的药物.     

Expression and purification of human endostatin from Hansenula polymorpha A16

Endostatin (EDN), an endogenous angiogenesis inhibitor of 20 kDa, was originally isolated from the supernatant of culture murine hemangioendothelioma cell line. Interest in EDN arises from its therapeutic potential as anti-tumor and anti-angiogenesis agents. However, it is difficult to obtain sufficient quantities of native EDN from its natural resources. We report here the construction of a pMIRH-type vector pLRG29 by introducing the 25s rDNA of Hansenua wingei and the Km(R) gene into the YIp vector pSML12 and the EDN expression vector pLRG-EDN by cloning the human EDN cDNA into pLRG29. Human EDN was expressed in Hansenula polymorpha (H.p.) A16 (pLRG-EDN) as a secreted soluble protein. The yield of the secreted EDN was 65 mg/L in shake flask. The secreted EDN was purified to a purity of 98 % by the use of SP Sepharose FF ion-exchange chromatography and Sepharose-heparin Hi Trap affinity chromatography. The MTT and chicken embryo chorioallantoic membrane assay demonstrated that the human EDN produced from H. polymorpha inhibited in vitro the proliferation of human umbilical vein endothelial cells and in vivo the neovascularization induced by bFGF.     

重组人角质细胞生长因子-2基因克隆、表达、纯化与活性分析

 从人胚肺二倍体细胞KMB17中抽提总RNA ,经RT PCR扩增获得编码人角质细胞生长因子 2 (keratinocytegrowthfactor 2 ,KGF 2 )的cDNA .克隆于硫氧环蛋白表达载体pThioHisA ,序列分析表明与文献报道一致 .经IPTG诱导 ,在大肠杆菌BL2 1中实现高效表达 ,表达量可达菌体总蛋白 10 %～15 % .菌体超声破碎 ,上清经CM SepharoseFF阳离子交换 ,Heparin Sepharose亲和层析 ,Superdex 75凝胶过滤层析纯化得到重组人KGF 2 ,纯度高于 95 % .生物活性分析表明 ,它能够促进成纤维细胞NIH 3T3的增殖 ,诱导鸡胚背根神经结神经轴突的生长 ,促进鸡胚尿囊膜血管生成 .研究结果表明 ,获得了 95 %纯度的具有生物学活性的重组人KGF 2 ,为进一步的基础与应用研究提供了基础     

Vascular basement membrane-derived multifunctional peptide, a novel inhibitor of angiogenesis and tumor growth

Vascular basement membrane-derived multifunctional peptide(VBMDMP)gene(fusion geneof the human immunoglobulin G3 upper hinge region and two tumstatin-derived fragments)obtained bychemical synthesis was cloned into vector pUC19,and introduced into the expression vector pGEX-4T-1 toconstruct a prokaryotic expression vector pGEX-4T-1-VBMDMP.Recombinant VBMDMP produced inEscherichia coli has been shown to have significant activity of antitumor growth and antimetastasis inLewis lung carcinoma transplanted into mouse C57B1/6.In the present study,we have studied the ability ofrVBMDMP to inhibit endothelial cell tube formation and proliferation,to induce apoptosis in vitro,and tosuppress tumor growth in vivo.The experimental results showed that rVBMDMP potently inhibited prolif-eration of human endothelial(HUVEC-12)cells and human colon cancer(SW480)cells in vitro,with noinhibition of proliferation in Chinese hamster ovary(CHO-K1)cells.rVBMDMP also significantly inhibitedhuman endothelial cell tube formation and suppressed tumor growth of SW480 cells in a mouse xenograftmodel.These results suggest that rVBMDMP is a powerful therapeutic agent for suppressing angiogenesisand tumor growth.     

Syndecan-2 is expressed in the microvasculature of gliomas and regulates angiogenic processes in microvascular endothelial cells

Angiogenesis is the formation of new blood vessels from the existing vasculature and is necessary for tumor growth. Syndecan-2 (S2) is highly expressed in the microvasculature of mouse gliomas. When S2 expression was down-regulated in mouse brain microvascular endothelial cells (MvEC), this inhibited cell motility and reduced the formation of capillary tube-like structures in vitro. Pro-angiogenic growth factors and enzymes up-regulated during glioma tumorigenesis stimulated shedding of the S2 ectodomain from endothelial cells in vitro. The effect of shed S2 on angiogenic processes was investigated by incorporating recombinant S2 ectodomain (S2ED) into in vitro angiogenesis assays. S2ED promoted membrane protrusion, migration, capillary tube formation, and cell-cell interactions. We therefore propose that S2 is necessary for angiogenesis of MvEC, proangiogenic factors expressed during glioma progression regulate S2 shedding, and shed S2 ectodomain may increase endothelial cell angiogenic processes.     

Inhibition of angiogenesis by a mouse sprouty protein

Sprouty negatively modulates branching morphogenesis in the Drosophila tracheal system. To address the role of mammalian Sprouty homologues in angiogenesis, another form of branching morphogenesis, a recombinant adenovirus engineered to express murine Sprouty-4 selectively in endothelial cells, was injected into the sinus venosus of embryonic day 9.0 cultured mouse embryos. Sprouty-4 expression inhibited branching and sprouting of small vessels, resulting in abnormal embryonic development. In vitro, Sprouty-4 inhibited fibroblast growth factor and vascular endothelial cell growth factor-mediated cell proliferation and migration and prevented basic fibroblast growth factor and vascular endothelial cell growth factor-induced MAPK phosphorylation in endothelial cells, indicating inhibition of tyrosine kinase-mediated signaling pathways. The ability of constitutively activated mutant Ras(L61) to rescue Sprouty-4 inhibition of MAPK phosphorylation suggests that Sprouty inhibits receptor tyrosine kinase signaling upstream of Ras. Thus, Sprouty may regulate angiogenesis in normal and disease processes by modulating signaling by endothelial tyrosine kinases.