一种骨桥蛋白小分子肽的重组表达与生物学活性研究
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摘要
目的:血管损伤部位新生内膜的形成是动脉粥样硬化(AS)和血管成形术后再狭窄(PTCA)病理过程中的重要事件之一,当血管内膜受损时,位于血管中膜的血管平滑肌细胞(VSMC)在生长因子和细胞因子的刺激下由收缩表型转化为合成表型,并获得向内膜下迁移、增殖及合成和分泌大量细胞外基质(ECM)的能力。VSMC迁移主要是由ECM蛋白和细胞表面整合素受体(主要是αvβ3)相互作用所介导的。已经证明,在各种ECM蛋白中,骨桥蛋白(OPN)与细胞迁移和黏附的关系最为密切。OPN通过它所含有的RGD序列与整合素αvβ3、αvβ5相互作用,通过SVVYGLR序列与整合素α9β1、α4β1结合。已经证实,含RGD和SVVYGLR序列的短肽能够竞争性的抑制OPN与多种整合素的结合,从而抑制VSMC以及炎性细胞的黏附和迁移。以小分子肽作为阻断剂研究蛋白质的功能是揭示细胞活动调节机制的重要手段。本实验利用基因重组技术,将含有编码RGD序列的cDNA片段与携带2×His-Trx-S·tag编码序列的原核表达载体pET-32c(+)重组,构建pET-32c-RGD重组子,将其转化大肠杆菌BL21(DE3) ,表达产物为His-Trx-S·tag-RGD融合蛋白(简称His-RGD)。经分离纯化后获得His-RGD纯品,该短肽可作为阻断剂用于研究OPN在体内的生物学作用。
方法:
1重组表达质粒的构建与鉴定
将编码RGD13肽的DNA(以下简称RGD)片段经NcoⅠ/BamH I酶切位点克隆入pET-32c(+)载体,构建His-RGD融合蛋白表达质粒。通过双酶切和序列分析对重组质粒进行鉴定。
2 His-RGD融合蛋白在大肠杆菌中的表达
2.1 His-RGD融合蛋白的诱导表达
将pET-32c-RGD转化大肠杆菌BL21(DE3),在不同条件下,用IPTG诱导后可检测His-RGD融合蛋白的表达活性,并对IPTG浓度、诱导时间和诱导温度进行筛选优化,确定His-RGD融合蛋白诱导表达的最佳条件。表达产物经SDS-PAGE检查融合蛋白在细胞中的存在形式。
2.2 His-RGD融合蛋白的分离纯化
取含有表达产物的细菌裂解液上清直接经Ni-NTA His Bind Resin金属离子螯合层析进行纯化。
3 His-RGD融合蛋白生物学活性检测
3.1黏附抑制实验
取3-4代体外培养的VSMC分别与0、150、300、450 mg/L的His-RGD融合蛋白预孵育1 h,然后将细胞接种到用BSA (20 mg/L)或OPN (20 mg/L)包被的96孔板中,检测单位时间( 2h )内黏附至孔板上的细胞数,以此表示细胞黏附活性。以融合蛋白洗脱液和2×His-Trx-S·tag蛋白作为平行对照,以确定His-RGD抑制细胞黏附的特异性。
3.2伤口愈合实验
将VSMC接种于带有玻片的孔板内,待细胞生长至100%汇合后,分别加入0、150、300、450 mg/L的His-RGD预孵育1 h。取出玻片,用无菌吸头在玻片上划痕。随即将玻片放回孔板内,每孔加入20 mg/L OPN或20 mg/L BSA 100μl,继续孵育24 h后,于低倍镜下观察细胞伤口愈合程度,以此表示细胞的迁移活性。以融合蛋白洗脱液和2×His-Trx-S·tag蛋白作为平行对照,以确定His-RGD抑制细胞迁移的特异性。
结果:
1重组表达质粒pET-32c-RGD的构建与鉴定
重组表达质粒经NcoI和BamHI双酶切,产物经核酸电泳鉴定时出现长度为50 bp的插入片段,与预期结果相一致。测序结果显示插入片段的核酸序列正确无误。重组质粒命名为pET-32c-RGD。
2 His-RGD融合蛋白的诱导表达
转化pET-32c-RGD的宿主菌经IPTG诱导后可表达约18.71 kD的蛋白质,与His-RGD融合蛋白分子量相一致,该融合蛋白主要以可溶性蛋白形式存在于胞浆。取培养物(OD600=0.6)按1:50接种于含0.01 mmol/L IPTG的LB培养液中,25℃诱导培养7 h时,His-RGD融合蛋白的表达量最大,此条件作为His-RGD融合蛋白诱导表达的最佳条件。
3 His-RGD融合蛋白的纯化
经Ni-NTA His Bind Resin金属离子螯合层析进行纯化后,每100 ml培养物可得到约25 mg电泳纯的可溶性His-RGD融合蛋白,纯度为95%。
4 His-RGD融合蛋白对细胞黏附的影响
黏附抑制实验结果显示,在所观察的浓度范围内,His-RGD融合蛋白能剂量依赖性的抑制VSMC在OPN包被板上的黏附,在融合蛋白为450 mg/L时,抑制效果最明显。
5 His-RGD融合蛋白对细胞迁移的影响
伤口愈合实验结果表明,His-RGD能剂量依赖性的抑制VSMC迁移,在融合蛋白为450 mg/L时,抑制效果最明显。
结论:
1成功构建了含有OPN RGD序列13肽基因单拷贝的pET-32c-RGD原核表达质粒。
2经过对IPTG浓度、诱导温度、诱导时间等条件进行优化,His-RGD融合蛋白在大肠杆菌中得到有效表达。
3每100 ml培养物经亲和层析纯化可得到约25 mg电泳纯的可溶性融合蛋白,纯度为95%。
4 His-RGD融合蛋白能够剂量依赖性的抑制OPN诱导的VSMC的黏附与迁移。
Objective: The formation of neointima induced by endothelium injury is an important cause of percutaneous transluminal coronary angioplasty(PTCA). When the vessels are injured, vascular smooth muscle cells (VSMC) in media which are stimulated by growth factors and cytokines reverse from the contractile (differentiated) state to the synthetic (dedifferentiated) state, and migrate into endothelium and release cytokines and extracellular matrix (ECM) proteins. Migration of VSMC is regulated by the interaction of OPN with integrinαvβ3 on surface of cells. OPN plays a key role in VSMC adhesion and migration by interactions of RGD motif in OPN withαvβ3 andαvβ5. In addition, the SVVYGLR sequence of OPN regulates many cell behaviors by interactions withα9β1 andα4β1. Previous studies indicated that peptides containing a RGD sequence and peptides with a SVVYGLR sequence blocked the binding of OPN to the integrins and inhibited VSMC adhesion, migration, and macrophage infiltration. Small peptides that display inhibitory roles as decoy oligopeptides may be used to study the function of proteins. This study constructed prokaryotic expression plasmids containing RGD and SVVYGLR cDNA sequences. E. coli transformed with the construct could be induced to express His-RGD fusion protein.
Methods:
1 Construction of recombinant plasmids
His-RGD prokaryotic expression plasmid was constructed by cloning synthesized OPN 13 peptide-coding sequence into prokaryotic expression vector pET-32c(+). The recombinant plasmid was identified by DNA sequence determination.
2 Expression of His-RGD fusion protein in E.coli
2.1 Expression of His-RGD fusion protein
E.coli BL21(DE3) transformed by the construct was induced by IPTG under different conditions. Supernatant and precipitation of pET-32c-RGD transformant lysates were analyzed by SDS-PAGE.
2.2 Purification of His-RGD fusion protein
The fusion proteins were purified from the supernatants of the culture lysates with Ni-NTA His Bind Resin metal chelation chromatography. 25 mg of purified soluble His-RGD fusion protein was obtained from 100 ml bacteria culture.
3 Detection of biological activities of His-RGD fusion protein
3.1 Adhesion assay
VSMCs suspended in 1% NCS medium were preincubated with His-RGD fusion proteins at 0, 150, 300 and 450 mg/L for 1 h, and then the cells were seeded in 96-well plate coated by BSA (20 mg/L) or OPN (20 mg/L). The number of the adhesion cells per well was detected, which represented the activity of the cell adhesion. The fusion proteins elution buffer and 2×His-Trx-S·tag protein represented the parallel controls.
3.2 Wound healing assay
VSMCs grown to 100% confluent monolayers were preincubated with His-RGD fusion proteins at 0, 150, 300 and 450 mg/L for 1 h,and then scratched to form a wound. After addition of OPN (20 mg/L) or BSA (20 mg/L) for 24 h, wound healing was observed by microscope, which represented the activity of the cell migration. The fusion proteins elution buffer and 2×His-Trx-S·tag protein represented the parallel controls to identify the specificity of migration inhibition.
Results:
1 Construction and identification of recombinant plasmids pET-32c-RGD.
The recombinant plasmids were identified by NcoⅠ/BamH I digestion. DNA sequence determination and restriction enzyme analysis showed that the inserted fragment of pET-32c-RGD was correct.
2 Expression and purification of His-RGD fusion protein
His-RGD fusion proteins were effectively expressed in E.coli transformed by pET-32c-RGD after induction with 10μ mol/L IPTG for 7 h at 25℃.
The fusion proteins were purified by Ni-NTA His Bind Resin metal chelation chromatography. The final yield of His-RGD fusion proteins was 25 mg/100 ml culture, and the purity of His-RGD fusion protein was over 95%.
3 Effect of His-RGD fusion protein on VSMC adhesion and migration
The adhesion assay indicated that His-RGD fusion protein dose-dependently decreased the adhesion and migration of VSMC, the inhibitiory effect reached the peak after preincubation by 450 mg/ml OPN-13-peptide.
Conclusions:
1 Prokaryotic expression plasmid pET-32c-RGD is successfully constructed.
2 His-RGD fusion protein is effectively expressed in E.coli after IPTG induction.
3 His-RGD fusion protein is purified with Ni-NTA His Bind Resin metal chelation chromatography. The yield of purified His-RGD fusion proteins is 25 mg/100 ml culture, and the purity of fusion protein is over 95%.
4 His-RGD fusion protein can dose-dependently decrease adhesion and migration of VSMC.
方法:
1重组表达质粒的构建与鉴定
将编码RGD13肽的DNA(以下简称RGD)片段经NcoⅠ/BamH I酶切位点克隆入pET-32c(+)载体,构建His-RGD融合蛋白表达质粒。通过双酶切和序列分析对重组质粒进行鉴定。
2 His-RGD融合蛋白在大肠杆菌中的表达
2.1 His-RGD融合蛋白的诱导表达
将pET-32c-RGD转化大肠杆菌BL21(DE3),在不同条件下,用IPTG诱导后可检测His-RGD融合蛋白的表达活性,并对IPTG浓度、诱导时间和诱导温度进行筛选优化,确定His-RGD融合蛋白诱导表达的最佳条件。表达产物经SDS-PAGE检查融合蛋白在细胞中的存在形式。
2.2 His-RGD融合蛋白的分离纯化
取含有表达产物的细菌裂解液上清直接经Ni-NTA His Bind Resin金属离子螯合层析进行纯化。
3 His-RGD融合蛋白生物学活性检测
3.1黏附抑制实验
取3-4代体外培养的VSMC分别与0、150、300、450 mg/L的His-RGD融合蛋白预孵育1 h,然后将细胞接种到用BSA (20 mg/L)或OPN (20 mg/L)包被的96孔板中,检测单位时间( 2h )内黏附至孔板上的细胞数,以此表示细胞黏附活性。以融合蛋白洗脱液和2×His-Trx-S·tag蛋白作为平行对照,以确定His-RGD抑制细胞黏附的特异性。
3.2伤口愈合实验
将VSMC接种于带有玻片的孔板内,待细胞生长至100%汇合后,分别加入0、150、300、450 mg/L的His-RGD预孵育1 h。取出玻片,用无菌吸头在玻片上划痕。随即将玻片放回孔板内,每孔加入20 mg/L OPN或20 mg/L BSA 100μl,继续孵育24 h后,于低倍镜下观察细胞伤口愈合程度,以此表示细胞的迁移活性。以融合蛋白洗脱液和2×His-Trx-S·tag蛋白作为平行对照,以确定His-RGD抑制细胞迁移的特异性。
结果:
1重组表达质粒pET-32c-RGD的构建与鉴定
重组表达质粒经NcoI和BamHI双酶切,产物经核酸电泳鉴定时出现长度为50 bp的插入片段,与预期结果相一致。测序结果显示插入片段的核酸序列正确无误。重组质粒命名为pET-32c-RGD。
2 His-RGD融合蛋白的诱导表达
转化pET-32c-RGD的宿主菌经IPTG诱导后可表达约18.71 kD的蛋白质,与His-RGD融合蛋白分子量相一致,该融合蛋白主要以可溶性蛋白形式存在于胞浆。取培养物(OD600=0.6)按1:50接种于含0.01 mmol/L IPTG的LB培养液中,25℃诱导培养7 h时,His-RGD融合蛋白的表达量最大,此条件作为His-RGD融合蛋白诱导表达的最佳条件。
3 His-RGD融合蛋白的纯化
经Ni-NTA His Bind Resin金属离子螯合层析进行纯化后,每100 ml培养物可得到约25 mg电泳纯的可溶性His-RGD融合蛋白,纯度为95%。
4 His-RGD融合蛋白对细胞黏附的影响
黏附抑制实验结果显示,在所观察的浓度范围内,His-RGD融合蛋白能剂量依赖性的抑制VSMC在OPN包被板上的黏附,在融合蛋白为450 mg/L时,抑制效果最明显。
5 His-RGD融合蛋白对细胞迁移的影响
伤口愈合实验结果表明,His-RGD能剂量依赖性的抑制VSMC迁移,在融合蛋白为450 mg/L时,抑制效果最明显。
结论:
1成功构建了含有OPN RGD序列13肽基因单拷贝的pET-32c-RGD原核表达质粒。
2经过对IPTG浓度、诱导温度、诱导时间等条件进行优化,His-RGD融合蛋白在大肠杆菌中得到有效表达。
3每100 ml培养物经亲和层析纯化可得到约25 mg电泳纯的可溶性融合蛋白,纯度为95%。
4 His-RGD融合蛋白能够剂量依赖性的抑制OPN诱导的VSMC的黏附与迁移。
Objective: The formation of neointima induced by endothelium injury is an important cause of percutaneous transluminal coronary angioplasty(PTCA). When the vessels are injured, vascular smooth muscle cells (VSMC) in media which are stimulated by growth factors and cytokines reverse from the contractile (differentiated) state to the synthetic (dedifferentiated) state, and migrate into endothelium and release cytokines and extracellular matrix (ECM) proteins. Migration of VSMC is regulated by the interaction of OPN with integrinαvβ3 on surface of cells. OPN plays a key role in VSMC adhesion and migration by interactions of RGD motif in OPN withαvβ3 andαvβ5. In addition, the SVVYGLR sequence of OPN regulates many cell behaviors by interactions withα9β1 andα4β1. Previous studies indicated that peptides containing a RGD sequence and peptides with a SVVYGLR sequence blocked the binding of OPN to the integrins and inhibited VSMC adhesion, migration, and macrophage infiltration. Small peptides that display inhibitory roles as decoy oligopeptides may be used to study the function of proteins. This study constructed prokaryotic expression plasmids containing RGD and SVVYGLR cDNA sequences. E. coli transformed with the construct could be induced to express His-RGD fusion protein.
Methods:
1 Construction of recombinant plasmids
His-RGD prokaryotic expression plasmid was constructed by cloning synthesized OPN 13 peptide-coding sequence into prokaryotic expression vector pET-32c(+). The recombinant plasmid was identified by DNA sequence determination.
2 Expression of His-RGD fusion protein in E.coli
2.1 Expression of His-RGD fusion protein
E.coli BL21(DE3) transformed by the construct was induced by IPTG under different conditions. Supernatant and precipitation of pET-32c-RGD transformant lysates were analyzed by SDS-PAGE.
2.2 Purification of His-RGD fusion protein
The fusion proteins were purified from the supernatants of the culture lysates with Ni-NTA His Bind Resin metal chelation chromatography. 25 mg of purified soluble His-RGD fusion protein was obtained from 100 ml bacteria culture.
3 Detection of biological activities of His-RGD fusion protein
3.1 Adhesion assay
VSMCs suspended in 1% NCS medium were preincubated with His-RGD fusion proteins at 0, 150, 300 and 450 mg/L for 1 h, and then the cells were seeded in 96-well plate coated by BSA (20 mg/L) or OPN (20 mg/L). The number of the adhesion cells per well was detected, which represented the activity of the cell adhesion. The fusion proteins elution buffer and 2×His-Trx-S·tag protein represented the parallel controls.
3.2 Wound healing assay
VSMCs grown to 100% confluent monolayers were preincubated with His-RGD fusion proteins at 0, 150, 300 and 450 mg/L for 1 h,and then scratched to form a wound. After addition of OPN (20 mg/L) or BSA (20 mg/L) for 24 h, wound healing was observed by microscope, which represented the activity of the cell migration. The fusion proteins elution buffer and 2×His-Trx-S·tag protein represented the parallel controls to identify the specificity of migration inhibition.
Results:
1 Construction and identification of recombinant plasmids pET-32c-RGD.
The recombinant plasmids were identified by NcoⅠ/BamH I digestion. DNA sequence determination and restriction enzyme analysis showed that the inserted fragment of pET-32c-RGD was correct.
2 Expression and purification of His-RGD fusion protein
His-RGD fusion proteins were effectively expressed in E.coli transformed by pET-32c-RGD after induction with 10μ mol/L IPTG for 7 h at 25℃.
The fusion proteins were purified by Ni-NTA His Bind Resin metal chelation chromatography. The final yield of His-RGD fusion proteins was 25 mg/100 ml culture, and the purity of His-RGD fusion protein was over 95%.
3 Effect of His-RGD fusion protein on VSMC adhesion and migration
The adhesion assay indicated that His-RGD fusion protein dose-dependently decreased the adhesion and migration of VSMC, the inhibitiory effect reached the peak after preincubation by 450 mg/ml OPN-13-peptide.
Conclusions:
1 Prokaryotic expression plasmid pET-32c-RGD is successfully constructed.
2 His-RGD fusion protein is effectively expressed in E.coli after IPTG induction.
3 His-RGD fusion protein is purified with Ni-NTA His Bind Resin metal chelation chromatography. The yield of purified His-RGD fusion proteins is 25 mg/100 ml culture, and the purity of fusion protein is over 95%.
4 His-RGD fusion protein can dose-dependently decrease adhesion and migration of VSMC.
引文
1 Renault MA,Jalvy S,Belloc I,et a1. AP-1 is involved in UTP-induced esteopontin expression in arterial smooth muscle cells. Circ Res, 2003, 93:674~681
2 Li G, Oparil S, Kelpke SS,et al. Fibroblast growth factor receptor-1 signaling induces esteopontin expression and vascular smooth muscle cel1-dependent adventitial fibroblast migration in vitro. Circulation,2002,106:854~859
3 Myers DL, Harmon KJ, Lindner V, et al. Alterations of arterial physiology in osteopontin-null mice. Arterioscler Thromb Vasc Biol, 2003, 23:1021~8
4 Yokosaki Y, Nariaki M, Tomohiro S, et al. The integrin α9β1 binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of osteopontin. J Biol Chem, 1999, 274: 36328~36334
5 Byzova TV, Plow EF. Networking in the hemostatic system: integrin α Ⅱ bβ3 binds prothrombin and influences its activation. J Biol Chem, 1997, 272: 27183~27188
6 Stawowy P, Kallisch H, Veinot JP, et al. Endoproteolytic activation of alpha(v) integrin by proprotein convertase PC5 is required for vascular smooth muscle cell adhesion to vitronectin and integrin-dependent signaling. Circulation, 2004, 109:770~6
7 Qian Y,Zhong X S,Flynn D C,et a l. ILK mediates actin filament rearrangement and cell migration and invasionthrough PI3K / Akt / Rac1 signaling. Oncogene,2005,24: 3154~3165
8 柴锡庆, 郑斌, 韩梅, 杨淑丽, 温进坤. 骨桥蛋白的制备及功能研究. 中国老年学杂志, 2004, 8: 725~727
9 刘智敏, 韩梅, 温进坤. 细胞外基质促血管平滑肌细胞迁移及 β3 整合素和黏着斑激酶表达. 中国病理生理杂志, 2003, 19: 163~166
10 Bauters C, Isner JM. The biology of restenosis. Prog Cardiovasc Dis, 1997, 40: 107～116
11 Saito Y. Mechanism of phenotype formation of smooth muscle cells. Annals of the New Academy of Sciences, 1995, 748: 7～10
12 Shanahan CM, Weissberg PL, Metealfe JC. Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells. Circ Res, 1993, 73: 193～204
13 Reinholt FP, Hultenby K, Oldberg A, et al. Osteopontina possible anchor of osteoclasts to bone. Proc Natl Acad Sci USA, 1990, 87: 4473～4475
14 Miyauchi A, Alvarez J, Greenfield E, et al. Recognition of osteopontin and related peptides by αVβ3 integrin stimulates immediate cell signals in osteoclasts. J Biol Chem, 1991, 266: 20369~20374
15 Oldberg A, Franzen A, Heinegard D. Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence. Proc Natl Sci, 1986, 83: 8819~8823
16 Ellen MG. Osteopontin bridges between bone and immune system. J Clin Invest, 2003, 112: 147~149
17 Kayla JB, George ED. Identification of dual α4β1 integrin binding sites within a 38 amino acid domain in the N-terminal thrombin fragment of human osteopontin. J Biol Chem, 2001, 276: 13483~13489
18 Nobuchika Y, Fumihiko S, Shigeyuki K, et al. Essential role of the cryptic epitope SLAYGLR with osteopontin in a murine model of rheumatoid arthritis. J Clin Invest, 2003, 112: 181~188
19 Michelle P, Colleen I, Michael R, et al. Smooth muscle cell matrix metalloproteinase production is stimulated via αvβ3 integrin. Arterioscler Thromb Vasc Biol, 2000, 20: 1467~1472
1 Perotti M, Mancini N, Diotti RA, et al. Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein. J Virol, 2008, 82:1047~52
2 Naz RK, Chauhan SC. Human sperm-specific peptide vaccine that causes long-term reversible contraception. Biol Reprod, 2002, 67:674~80
3 Naz R, Aleem A. Effect of immunization with six sperm peptide vaccines on fertility of female mice. Soc Reprod Fertil Suppl, 2007, 63:455~64
4 Radulovic S, Cai RZ, Serfozo P,et al. Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonist with N-terminal D-Trp or D-Tpi. Int J Pept Protein Res, 1991, 38: 593~600
5 Szepeshazi K, Schally AV, Cai RZ,et al. Inhibitory effect of bombesin/gastrin-releasing peptide antagonist RC-3095 and high dose of somatostatin analogue RC-160 on nitrosamine-induced pancreatic cancers in hamsters. Cancer Res, 1991, 51: 5980~5986
6 Dietrich B, Hildebrad P, Jeker LB, et al. Effect of BIM26226, a potent and specific bombesin receptor antagonist, on anylase release and binding of bombesin-like peptide to AR4-2J cells. Regul Pept, 1994, 53: 165~173
7 Llinàs-Brunet M, Bailey M, Déziel R, et al. Studies on the C-terminal of hexapeptide inhibitors of the hepatitis C virus serine protease. Bioorg Med Chem Lett, 1998, 8:2719~24
8 Rancourt J, Cameron DR, Gorys V, et al. Peptide-based inhibitors of the hepatitis C virus NS3 protease: structure-activity relationship at the C-terminal position. J Med Chem, 2004, 47:2511~22
9 Li BC, Zhang SQ, Dan WB, et al. Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori. Biotechnol Lett, 2007, 29:1031~6
10 Li ML, Liao RW, Qiu JW, et al. Antimicrobial activity of synthetic all-D mastoparan M. Int J Antimicrob Agents,2000, 13:203~8
11 Steiner H, Hultmark D, Engstr?m A, et al. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature,1981,292:246~8
12 Charnley M, Moir AJ, Douglas CW, et al. Anti-microbial action of melanocortin peptides and identification of a novel X-Pro-d/l-Val sequence in Gram-positive and Gram-negative bacteria. Peptides,2008,Feb 15 [Epub ahead of print]
13 Nedjar-Arroume N, Dubois-Delval V, Adje EY, et al. Bovine hemoglobin: An attractive source of antibacterial peptides. Peptides, 2008, Jan 31 [Epub ahead of print]
14 Cruz-Gonzalez I, Sanchez-Ledesma M, Baron SJ, et al. Efficacy and safety of argatroban with or without glycoprotein IIb/IIIa inhibitor in patients with heparin induced thrombocytopenia undergoing percutaneous coronary intervention for acute coronary syndrome. J Thromb Thrombolysis, 2007, 15
15 Kaluski E, Uriel N, Hendler A, et al. Interventional cardiology in Israel at 2005 - state of practice. Acute Card Care, 2007, 9:104~10
16 Berkowitz SD. Current knowledge of the platelet glycoprotein Ⅱb/Ⅲa reporter antagonists for the treatment of coronary artery disease. Haemostasis, 2000, 30: 27~43
17 Hackalamannil S. G-protein coupled receptor antagonists-1: protease activated receptor-1 (PAR-1) antagonists as novel cardiovascular therapeutic agents. Curr Top Med Chem, 2003, 3: 1115~1123
18 O'Shea JC, Tcheng JE. Eptifibatide: a potent inhibitor of the platelet receptor integrin glycoprotein IIb/IIIa. Expert Opin Pharmacother, 2002, 3:1199~210
19 Stiernberg J, Norfleet AM, Redin WR, et al. Acceleration of full-thickness wound healing in normal rats by the synthetic thrombin peptide, TP508. Wound Repair Regen, 2000, 8:204~15
20 Pfister RR, Sommers CI.. L-arginine-threonine-arginine (RTR) tetramer peptide inhibits ulceration in the alkali-injured rabbit cornea. Cornea, 2006, 25:1187~92
21 Carron CP, Meyer DM, Engleman VW, et al. Peptidomimetic antagonists of alphavbeta3 inhibit bone resorption by inhibiting osteoclast bone resorptive activity, not osteoclast adhesion to bone. J Endocrinol, 2000, 165:587~98
22 Ebinuma H, Nakamoto N, Li Y, et al. Identification and in-vitro expansion of functional antigen-specific CD25+FoxP3+ regulatory T-cells in hepatitis C virus infection. J Virol, 2008 Mar 12 [Epub ahead of print]
23 Haslin C, Lévêque M, Ozil A, A recombinant humanmonoclonal anti-R7V antibody as a potential therapy for HIV infected patients in failure of HAART. Hum Antibodies,2007,16:73~85
24 《生物多样性公约(1992)》第 2 条,1992
25 Zou Y, Chen Y, Jiang Y, et al. Targeting matrix metalloproteinases and endothelial cells with a fusion peptide against tumor. Cancer Res, 2007, 67:7295~300
26 Groleau PE, Morin P, Gauthier SF, et al. Effect of physicochemical conditions on peptide-peptide interactions in a tryptic hydrolysate of beta-lactoglobulin and identification of aggregating peptides. J Agric Food Chem, 2003, 51:4370~5
27 Leblanc J, Fliss I, Matar C. Induction of a humoral immune response following an Escherichia coli O157:H7 infection with an immunomodulatory peptidic fraction derived from Lactobacillus helveticus-fermented milk. Clin Diagn Lab Immunol,2004, 11:1171~81
28 Symmank H, Franke P, Saenger W, et al. Modification of biologically active peptides: production of a novel lipohexapeptide after engineering of Bacillus subtilis surfactin synthetase. Protein Eng, 2002, 15:913~21
29 Cortese R, Monaci P, Luzzago A, et al. Selection of biologically active peptides by phage display of random peptide libraries. Curr Opin Biotechnol, 1996, 7:616~21
30 Baek H, Suk KH, Kim YH, et al. An improved helper phage system for efficient isolation of specific antibody moleculesin phage display. Nucleic Acids Res, 2002, 30: 8
31 李家大,王克夷. 从噬菌体多肽文库筛选α2葡萄糖苷酶的抑制剂. 生物化学与生物物理学报, 2001, 33 :5132~518
32 Zhan JB , Xia Z , Wang KY. Using phage display to identify peptides binding to human natural antibodies. Chinese Peptide Symposium Program, 2002, 62
33 Livnah O, Stura EA, Johnson DL, et al. Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8 A. Science, 1996, 273:464~71
34 郝文波,徐伟文,陈白虹等。噬菌体展示ICAM-1模拟肽的制备及活性鉴定。细胞与分子免疫学杂志(Hao WB, Xu WW, Chen BH, et al. Preparation and identification of phage displayed ICAM-1-mimic peptide. Chin J cell Mol Immunol),2003,19
2 Li G, Oparil S, Kelpke SS,et al. Fibroblast growth factor receptor-1 signaling induces esteopontin expression and vascular smooth muscle cel1-dependent adventitial fibroblast migration in vitro. Circulation,2002,106:854~859
3 Myers DL, Harmon KJ, Lindner V, et al. Alterations of arterial physiology in osteopontin-null mice. Arterioscler Thromb Vasc Biol, 2003, 23:1021~8
4 Yokosaki Y, Nariaki M, Tomohiro S, et al. The integrin α9β1 binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of osteopontin. J Biol Chem, 1999, 274: 36328~36334
5 Byzova TV, Plow EF. Networking in the hemostatic system: integrin α Ⅱ bβ3 binds prothrombin and influences its activation. J Biol Chem, 1997, 272: 27183~27188
6 Stawowy P, Kallisch H, Veinot JP, et al. Endoproteolytic activation of alpha(v) integrin by proprotein convertase PC5 is required for vascular smooth muscle cell adhesion to vitronectin and integrin-dependent signaling. Circulation, 2004, 109:770~6
7 Qian Y,Zhong X S,Flynn D C,et a l. ILK mediates actin filament rearrangement and cell migration and invasionthrough PI3K / Akt / Rac1 signaling. Oncogene,2005,24: 3154~3165
8 柴锡庆, 郑斌, 韩梅, 杨淑丽, 温进坤. 骨桥蛋白的制备及功能研究. 中国老年学杂志, 2004, 8: 725~727
9 刘智敏, 韩梅, 温进坤. 细胞外基质促血管平滑肌细胞迁移及 β3 整合素和黏着斑激酶表达. 中国病理生理杂志, 2003, 19: 163~166
10 Bauters C, Isner JM. The biology of restenosis. Prog Cardiovasc Dis, 1997, 40: 107～116
11 Saito Y. Mechanism of phenotype formation of smooth muscle cells. Annals of the New Academy of Sciences, 1995, 748: 7～10
12 Shanahan CM, Weissberg PL, Metealfe JC. Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells. Circ Res, 1993, 73: 193～204
13 Reinholt FP, Hultenby K, Oldberg A, et al. Osteopontina possible anchor of osteoclasts to bone. Proc Natl Acad Sci USA, 1990, 87: 4473～4475
14 Miyauchi A, Alvarez J, Greenfield E, et al. Recognition of osteopontin and related peptides by αVβ3 integrin stimulates immediate cell signals in osteoclasts. J Biol Chem, 1991, 266: 20369~20374
15 Oldberg A, Franzen A, Heinegard D. Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence. Proc Natl Sci, 1986, 83: 8819~8823
16 Ellen MG. Osteopontin bridges between bone and immune system. J Clin Invest, 2003, 112: 147~149
17 Kayla JB, George ED. Identification of dual α4β1 integrin binding sites within a 38 amino acid domain in the N-terminal thrombin fragment of human osteopontin. J Biol Chem, 2001, 276: 13483~13489
18 Nobuchika Y, Fumihiko S, Shigeyuki K, et al. Essential role of the cryptic epitope SLAYGLR with osteopontin in a murine model of rheumatoid arthritis. J Clin Invest, 2003, 112: 181~188
19 Michelle P, Colleen I, Michael R, et al. Smooth muscle cell matrix metalloproteinase production is stimulated via αvβ3 integrin. Arterioscler Thromb Vasc Biol, 2000, 20: 1467~1472
1 Perotti M, Mancini N, Diotti RA, et al. Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein. J Virol, 2008, 82:1047~52
2 Naz RK, Chauhan SC. Human sperm-specific peptide vaccine that causes long-term reversible contraception. Biol Reprod, 2002, 67:674~80
3 Naz R, Aleem A. Effect of immunization with six sperm peptide vaccines on fertility of female mice. Soc Reprod Fertil Suppl, 2007, 63:455~64
4 Radulovic S, Cai RZ, Serfozo P,et al. Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonist with N-terminal D-Trp or D-Tpi. Int J Pept Protein Res, 1991, 38: 593~600
5 Szepeshazi K, Schally AV, Cai RZ,et al. Inhibitory effect of bombesin/gastrin-releasing peptide antagonist RC-3095 and high dose of somatostatin analogue RC-160 on nitrosamine-induced pancreatic cancers in hamsters. Cancer Res, 1991, 51: 5980~5986
6 Dietrich B, Hildebrad P, Jeker LB, et al. Effect of BIM26226, a potent and specific bombesin receptor antagonist, on anylase release and binding of bombesin-like peptide to AR4-2J cells. Regul Pept, 1994, 53: 165~173
7 Llinàs-Brunet M, Bailey M, Déziel R, et al. Studies on the C-terminal of hexapeptide inhibitors of the hepatitis C virus serine protease. Bioorg Med Chem Lett, 1998, 8:2719~24
8 Rancourt J, Cameron DR, Gorys V, et al. Peptide-based inhibitors of the hepatitis C virus NS3 protease: structure-activity relationship at the C-terminal position. J Med Chem, 2004, 47:2511~22
9 Li BC, Zhang SQ, Dan WB, et al. Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori. Biotechnol Lett, 2007, 29:1031~6
10 Li ML, Liao RW, Qiu JW, et al. Antimicrobial activity of synthetic all-D mastoparan M. Int J Antimicrob Agents,2000, 13:203~8
11 Steiner H, Hultmark D, Engstr?m A, et al. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature,1981,292:246~8
12 Charnley M, Moir AJ, Douglas CW, et al. Anti-microbial action of melanocortin peptides and identification of a novel X-Pro-d/l-Val sequence in Gram-positive and Gram-negative bacteria. Peptides,2008,Feb 15 [Epub ahead of print]
13 Nedjar-Arroume N, Dubois-Delval V, Adje EY, et al. Bovine hemoglobin: An attractive source of antibacterial peptides. Peptides, 2008, Jan 31 [Epub ahead of print]
14 Cruz-Gonzalez I, Sanchez-Ledesma M, Baron SJ, et al. Efficacy and safety of argatroban with or without glycoprotein IIb/IIIa inhibitor in patients with heparin induced thrombocytopenia undergoing percutaneous coronary intervention for acute coronary syndrome. J Thromb Thrombolysis, 2007, 15
15 Kaluski E, Uriel N, Hendler A, et al. Interventional cardiology in Israel at 2005 - state of practice. Acute Card Care, 2007, 9:104~10
16 Berkowitz SD. Current knowledge of the platelet glycoprotein Ⅱb/Ⅲa reporter antagonists for the treatment of coronary artery disease. Haemostasis, 2000, 30: 27~43
17 Hackalamannil S. G-protein coupled receptor antagonists-1: protease activated receptor-1 (PAR-1) antagonists as novel cardiovascular therapeutic agents. Curr Top Med Chem, 2003, 3: 1115~1123
18 O'Shea JC, Tcheng JE. Eptifibatide: a potent inhibitor of the platelet receptor integrin glycoprotein IIb/IIIa. Expert Opin Pharmacother, 2002, 3:1199~210
19 Stiernberg J, Norfleet AM, Redin WR, et al. Acceleration of full-thickness wound healing in normal rats by the synthetic thrombin peptide, TP508. Wound Repair Regen, 2000, 8:204~15
20 Pfister RR, Sommers CI.. L-arginine-threonine-arginine (RTR) tetramer peptide inhibits ulceration in the alkali-injured rabbit cornea. Cornea, 2006, 25:1187~92
21 Carron CP, Meyer DM, Engleman VW, et al. Peptidomimetic antagonists of alphavbeta3 inhibit bone resorption by inhibiting osteoclast bone resorptive activity, not osteoclast adhesion to bone. J Endocrinol, 2000, 165:587~98
22 Ebinuma H, Nakamoto N, Li Y, et al. Identification and in-vitro expansion of functional antigen-specific CD25+FoxP3+ regulatory T-cells in hepatitis C virus infection. J Virol, 2008 Mar 12 [Epub ahead of print]
23 Haslin C, Lévêque M, Ozil A, A recombinant humanmonoclonal anti-R7V antibody as a potential therapy for HIV infected patients in failure of HAART. Hum Antibodies,2007,16:73~85
24 《生物多样性公约(1992)》第 2 条,1992
25 Zou Y, Chen Y, Jiang Y, et al. Targeting matrix metalloproteinases and endothelial cells with a fusion peptide against tumor. Cancer Res, 2007, 67:7295~300
26 Groleau PE, Morin P, Gauthier SF, et al. Effect of physicochemical conditions on peptide-peptide interactions in a tryptic hydrolysate of beta-lactoglobulin and identification of aggregating peptides. J Agric Food Chem, 2003, 51:4370~5
27 Leblanc J, Fliss I, Matar C. Induction of a humoral immune response following an Escherichia coli O157:H7 infection with an immunomodulatory peptidic fraction derived from Lactobacillus helveticus-fermented milk. Clin Diagn Lab Immunol,2004, 11:1171~81
28 Symmank H, Franke P, Saenger W, et al. Modification of biologically active peptides: production of a novel lipohexapeptide after engineering of Bacillus subtilis surfactin synthetase. Protein Eng, 2002, 15:913~21
29 Cortese R, Monaci P, Luzzago A, et al. Selection of biologically active peptides by phage display of random peptide libraries. Curr Opin Biotechnol, 1996, 7:616~21
30 Baek H, Suk KH, Kim YH, et al. An improved helper phage system for efficient isolation of specific antibody moleculesin phage display. Nucleic Acids Res, 2002, 30: 8
31 李家大,王克夷. 从噬菌体多肽文库筛选α2葡萄糖苷酶的抑制剂. 生物化学与生物物理学报, 2001, 33 :5132~518
32 Zhan JB , Xia Z , Wang KY. Using phage display to identify peptides binding to human natural antibodies. Chinese Peptide Symposium Program, 2002, 62
33 Livnah O, Stura EA, Johnson DL, et al. Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8 A. Science, 1996, 273:464~71
34 郝文波,徐伟文,陈白虹等。噬菌体展示ICAM-1模拟肽的制备及活性鉴定。细胞与分子免疫学杂志(Hao WB, Xu WW, Chen BH, et al. Preparation and identification of phage displayed ICAM-1-mimic peptide. Chin J cell Mol Immunol),2003,19