日本七鳃鳗RGD毒素肽Lj-RGD2的克隆、表达及其抗血管新生活性

日本七鳃鳗口腔腺分泌蛋白Lj-RGD2毒素肽分子质量为8 kD,是含有2个RGD（Arg-Gly-Asp）模体序列的多肽.为了探讨其是否具有RGD毒素蛋白的功能,对其基因进行了克隆和表达,并对其重组蛋白进行了抗血管新生活性研究.从日本七鳃鳗口腔腺中提取mRNA,以自行设计的引物进行RT-PCR扩增,以获得Lj-RGD2基因;将获取的目的基因与pET23b载体连接,经转化、克隆、阳性转化子的筛选鉴定;将含组氨酸标签的pET23b-Lj-RGD2转化BL21菌中诱导表达;经亲和层析纯化,获得可溶性重组rLj-RGD2蛋白,并对重组rLj-RGD2蛋白的抗血管新生功能进行了研究.结果显示,rLj-RGD2蛋白的IC50为1.77μmol/L,并以剂量依赖方式抑制人脐静脉内皮细胞ECV304的增殖;rLj-RGD2蛋白还能抑制ECV304细胞对人工基质膜Matrigel的黏附、以BFGF（basic fibroblast growth factor）为趋化剂的迁移及侵袭.鸡胚绒毛尿囊膜CAM（chick chorioallantoic membrane）血管新生实验结果表明,rLj-RGD2蛋白能以剂量依赖方式抑制BFGF诱导的CAM血管新生.由此可见,rLj RGD2蛋白具有RGD毒素肽的活性特征,其有望成为一种抗血管新生基因工程新药. 关键词日本七鳃鳗; RGD毒素蛋白; 细胞黏附; 迁移及侵袭; 血管新生     

重组七鳃鳗富含半胱氨酸RGD蛋白rLj-RGD1对人肝癌HepG2细胞的抑制作用

rLj-RGD1为源于日本七鳃鳗口腔腺的基因重组蛋白,其富含半胱氨酸并具有一个RGD(Arg-Gly-Asp)模体.前期工作表明,rLj-RGD1具有抑制血小板聚集及血管新生的RGD毒素蛋白典型功能.为了研究rLj RGD1是否具有RGD毒素蛋白的另一典型抗肿瘤功能,以人肝癌HepG2细胞为模型,对rLj RGD1进行了活性研究.MTT法结果显示,rLj-RGD1呈剂量依赖方式抑制HepG2细胞的增殖,其半抑制浓度(IC50)为36 μmol/L;细胞迁移与浸润实验结果显示,rLj-RGD1能以剂量依赖方式抑制HepG2细胞碱性成纤维生长因子（bFGF）诱导的迁移与浸润.Hoechst染色和DNA Ladder等细胞凋亡实验表明,rLj-RGD1能够以剂量依赖方式诱导HepG2细胞发生凋亡.细胞黏附实验表明,rLj-RGD1以剂量依赖方式抑制HepG2细胞与玻连蛋白(vitronectin, VN)的黏附.上述结果表明,rLj-RGD1具有抑制人肝癌HepG2细胞增殖、迁移和浸润的功能,并可诱导其发生失巢凋亡. 本研究结果提示,rLj-RGD1具有典型的RGD毒素蛋白抗肿瘤功能,其未来具有成为抗肿瘤药物的潜力.     

白眉蝮蛇去整合素Adinbitor的基因克隆、表达及其部分生物学活性

为了寻找去整合素家族的新成员 ,研究其在抗血栓与抗肿瘤等方面的作用 ,从中国白眉蝮蛇毒腺中提取总RNA进行RT PCR扩增 ,获得了 2 19bp的白眉蝮蛇去整合素基因 .测序结果显示 ,其与韩国的同为蝮蛇去整合素的saxatilin的DNA序列同源性为 95 8% ,蛋白质序列同源性为 91 8% ,且蛋白质中含有去整合素的特征模体 RGD .将去整合素基因进行克隆、转化与诱导后 ,得到了该蛋白的可溶性高效表达 .经组氨酸亲和层析纯化 ,获得了分子量为 9kD的均质蛋白 ,并将其命名为adinbitor .活性测定结果显示 ,adinbitor能明显抑制由碱性成纤维细胞生长因子诱导的人脐静脉血管内皮细胞ECV30 4的增殖 ,诱导ECV30 4细胞发生凋亡 ,并且呈剂量依赖性方式抑制ADP诱导的人血小板聚集     

日本七鳃鳗Arg-Gly-Asp毒素蛋白Lj-RGD3野生型与RGD全缺失突变体Lj-112的抗血管新生作用

七鳃鳗Arg-Gly-Asp (RGD) 毒素肽Lj-RGD3与富含组氨酸糖蛋白HRG具有序列同源性,而RGD毒素蛋白及HRG都具有抑制血管新生的活性,但作用靶点不同。为研究Lj-RGD3结构与功能的关系,对野生型Lj-RGD3及其RGD全缺失突变体Lj-112进行了抗血管新生功能研究。将3个RGD模体的全缺失突变体基因Lj-112全序列合成后构建于pET-23b载体,对野生型Lj-RGD3及突变体 Lj-112蛋白进行IPTG诱导表达,重组蛋白经组氨酸亲和层析纯化;采用MTT法测定野生型和突变体蛋白对人     

重组七鳃鳗细胞毒素蛋白rLj-RGD3表达及对Hela细胞活性的影响

为研究重组七鳃鳗细胞毒素蛋白rLj-RGD3的抗肿瘤活性,确定其生物学地位及意义,本研究提取成体七鳃鳗(Lampetra japonica)口腔腺组织总RNA,经RT-PCR获得cDNA序列长度为357bp的目的基因片段,将其克隆于pET23b载体,获得带有组氨酸标签的分子量为15kD的基因重组蛋白rLj-RGD3的高效可溶性表达,通过组氨酸亲和层析纯化蛋白。采用MTT法检测不同浓度rLj-RGD3对bFGF诱导下的Hela细胞增殖的抑制作用,结果表明rLj-RGD3能显著抑制Hela细胞的增殖,IC50为2.6μmol/L。rLj-RGD3作用后的Hela细胞经Hoechst染色及DNAladder检测结果显示,细胞均发生凋亡。rLj-RGD3对Hela细胞黏附玻连蛋白(VN)作用的实验结果为有效抑制。采用Transwell细胞培养板对bFGF诱导下的Hela细胞迁移实验表明,rLj-RGD3能够抑制Hela细胞的迁移,且抑制率达60%。采用人工基质膜基质Matrigel及Transwell模仿体内环境研究rLj-RGD3对Hela细胞浸润行为实验显示,以bFGF为趋化剂的Hela细胞穿透Matrigel的...     

hVEGF165 和嵌合水蛭肽融合基因的构建、表达和活性检测

根据抗 PTCA 或支架后再狭窄的基因治疗需要多基因治疗的特点,用基因重组技术构建了 hVEGF165 和嵌合水蛭肽 (fused hirudin , FH) 融合基因,并克隆到真核表达载体 pcDNA3.0 中,通过脂质体介导将 pcDNA3.0/hVEGF165 - FH 转染到人内皮细胞株 (ECV304) 中, RT-PCR 及蛋白质印迹证明融合基因 hVEGF165 - FH 在 ECV304 细胞中得到表达 ( 分子质量为 24 ku 左右 ). 通过体外活性检测——— MTT 法检测 hVEGF165 - FH 对 ECV304 细胞增殖的影响,通过体外血管生成分析 hVEGF165 - FH 对内皮细胞株 ECV304 增殖的影响 . 通过体外抗栓活性检测,表明表达产物具有促进内皮细胞株增殖及加快血管生成的作用,同时显著抑制了 ADP 诱导的血小板聚集率 (P < 0.05) 并显著延长 APTT 和 TT (P < 0.05) . 实验结果表明,融合基因在内皮细胞株中得到表达,表达的融合蛋白具有 hVEGF165 和嵌合水蛭肽 (FH) 的双重活性,这为以后的融合基因治疗再狭窄的动物实验打下了良好基础 .     

重组白眉蝮蛇去整合素定点突变对其生物活性的影响

RGD为存在于许多糖蛋白配体中的氨基酸序列,对整合素具有识别作用.此序列也发现于许多蛇毒去整合素分子中.采用基因克隆技术从大连产白眉蝮蛇的毒腺中克隆出的去整合素adinbitor是含73个氨基酸残基的去整合素,分子中含有12个半胱氨酸和RGD模体.实验证明,adinbitor作为去整合素的新成员,具有典型的抗ADP诱导的人血小板聚集作用和抗肿瘤血管新生作用.为了将adinbitor的这2种功能分开,采用PCR基因定点突变的方法,将其cDNA序列中RGD模体改变成KGD.重组adinbitor(KGD)在E.coli BL21得到表达,并通过His&#8226;Bind亲和层析予以纯化.实验发现,adinbitor对ADP诱导的人血小板聚集具有明显抑制作用,其IC50=85 nmol/L,明显优于adinbitor(RGD) （IC50=150 nmol/L）.然而,与adinbitor(KGD)相比,adinbitor(KGD)则丧失了对血管生成的抑制作用.结果说明,adinbitor(KGD)可作为专一的抗人血小板聚集药具有潜在的开发前景.     

七鳃鳗口腔腺蛋白质功能的研究进展

寄生期的七鳃鳗为水中"吸血鬼",依靠吸食宿主血肉为生。近年来,陆续有研究报道从七鳃鳗口腔腺分泌液中鉴定出多种与抗凝、免疫逃逸、抗血管新生、镇痛以及氧化应激等有关的活性蛋白质。明确七鳃鳗口腔腺内活性蛋白质的特性及生物学功能将有助于阐明七鳃鳗独特的寄生机制,并为这些活性蛋白质的应用奠定理论基础。     

将RGD模体改变为RGDNM可减弱Echistatin对血小板聚集的抑制但增强血管新生的抑制作用

为了研究去整合素echistatin RGD(Arg-Gly-Asp) 模体周边氨基酸突变后对其生物学功能的影响,根据echistatin序列设计合成6个片段,利用重叠延伸PCR法合成cDNA,使echistatin RGD模体周边序列变为ARGDNM (D27→N),与载体pTXB1连接后转化E.coli BL21(DE3),建立echistatin (ARGDNM)的表达体系．工程菌经IPTG诱导后融合蛋白表达量占菌体总蛋白约30%,几丁质亲和纯化后,DTT裂解释放目的蛋白分子量约5.4 kD．体外血小板聚集和体内鸡胚绒毛尿囊膜 (chick chorioallantoic membrane, CAM)血管新生实验结果表明,echistatin (ARGDNM)抑制血小板聚集的作用减弱,而其抑制血管新生的作用增强．RGD模体周围氨基酸的改变影响了去整合素的生物学功能,echistatin (ARGDNM)增强了与αⅤβ3结合的特异性,本工作为研究特异性更强的去整合素药物奠定了基础．     

重组肽rLj-RGD4对小鼠黑色素瘤B16细胞活性的影响

该研究在前期重组日本七鳃鳗RGD3毒素蛋白(recombinant Lampetra japonica RGD3,rLj-RGD3)工作基础上,设计并人工合成分子克隆,获得去纯化标签的rLj-RGD4。该论文采用小鼠黑色素瘤(B16)为肿瘤细胞模型,对rLj-RGD4是否抑制B16细胞增殖、迁移、浸润与凋亡进行了研究。研究结果表明,采用不同浓度的rLj-RGD4经MTT方法检测发现,rLj-RGD4对B16细胞的增殖有抑制作用,IC50为9.6μmol/L;使用Transwell法和细胞划痕实验结果证实,rLj-RGD4对B16细胞的迁移和浸润都具有抑制作用,且呈剂量依赖方式;B16细胞经鬼笔环肽-FITC染色结果显示,rLj-RGD4肽对B16细胞骨架发生破坏;Hoechst 33258法和TUNEL-FITC法结果显示,rLj-RGD4可诱导B16细胞发生凋亡;运用Western blot对其诱导B16细胞发生凋亡的作用机制研究结果显示,随着rLj-RGD4浓度的增加cleaved-caspase-8和cleaved-caspase-3的水平显著增加,B淋巴细胞瘤-2(B cell lymphoma/leukaemia-2,Bcl-2)基因水平量显著减少。由此可见,rLj-RGD4具有显著促B16细胞凋亡的作用,具有抑制增殖与侵袭的功能,有可能成为一个具有抗肿瘤作用的候选药物。     

A novel RGD-toxin protein,Lj-RGD3, from the buccal gland secretion of Lampetra japonica impacts diverse biological activities

RGD (Arg-Gly-Asp) motif toxin proteins from snake venoms, saliva glands secretion of leech or tick have typical characteristics of inhibiting platelet aggregation, angiogenesis, and tumor growth. Here we report cloning and characterization of a novel RGD-toxin protein from the buccal gland of Lampetra japonica. In an attempt to study the activities of anticoagulant in the buccal gland secretion of L. japonica, we established buccal gland cDNA library and identified a gene encoding a predicted protein of 118 amino acids with 3 RGD motifs. The predicted protein was named Lj-RGD3. We generated the cDNA of Lj-RGD3 and obtained the recombinant protein rLj-RGD3. The polyclonal antibodies against rLj-RGD3 recognized the native Lj-RGD3 protein in buccal gland secretion in Western blot analyses. The biological function studies reveal that rLj-RGD3 inhibited human platelet aggregation in a dose-dependent manner with IC₅₀ value at 5.277 μM. In addition, rLj-RGD3 repressed bFGF-induced angiogenesis in the chick chorioallantoic membrane model. rLj-RGD3 also inhibited the adhesion of ECV304 cells to vitronectin. Furthermore, rLj-RGD3 induced apoptosis and significantly inhibited proliferation, migration, and invasion evoked by bFGF in ECV304 cells. Taken together, these results suggested that rLj-RGD3 is a novel RGD-toxin protein possessing typical functions of the RGD-toxin protein.     

Effects of the recombinant toxin protein rLj-RGD3 in multidrug-resistant human breast carcinoma cells

We have previously reported that Lj-RGD3, a novel RGD-toxin protein, was isolated from the buccal gland of Lampetra japonica. The recombinant protein rLj-RGD3 has anti-invasive and anti-adhesive activity in tumor cells (HeLa cells) and endothelial cells (ECV304 cells) in vitro, and inhibits αvβ3, αvβ5, and β1 integrin-mediated adhesion. In this study, we investigated the bioactivity of rLj-RGD3 in the drug-resistant MCF-7/Adr breast carcinoma cell line and drug-sensitive parental line MCF-7, and found that rLj-RGD3 inhibited the growth of both cell lines. Biological function studies revealed that rLj-RGD3 could induce the apoptosis in MCF-7/Adr, which was more prevalent than that in the drug-sensitive parental line MCF-7. In addition, rLj-RGD3 inhibited the adhesion of MCF-7/Adr cells to fibronectin. Furthermore, rLj-RGD3 prevented invasion of MCF-7/Adr cells through an artificial matrigel basement membrane. In summary, rLj-RGD3 may be used as a potential drug in multidrug-resistant breast cancer therapy.     

rLj-RGD3全RGD缺失基因重组突变体——rLj-112的分子克隆及其广谱抑真菌活性

Lj-112是日本七鳃鳗RGD毒素蛋白野生型Lj-RGD3的RGD全缺失基因突变体,其一级结构具有富组氨酸的特点．富组氨酸糖蛋白具有抑菌功能．为了研究Lj-112是否具有抑菌作用,使用基因合成和原核表达的方法,获得了纯化重组蛋白rLj-112,将其对不同菌种进行抑菌试验.结果表明,以野生型rLj-RGD3、RGD模体与组氨酸全缺失突变体rLj-26为对照,rLj-112有较广谱的抑菌活性,其中对白念球菌的最低抑菌浓度（MIC）为7 μmol/L,对稻瘟病菌的MIC值为7.5 μmol/L,对毛癣菌和禾谷病菌的MIC值分别为11.9 μmol/L和29.9 μmol/L．可见,rLj-112能作为抗真菌药物候选,为提高农作物的生产质量和减轻人类感染疾病的用药压力奠定了基础．     

Purification and characterization of a new RGD/KGD-containing dimeric disintegrin, piscivostatin, from the venom of Agkistrodon piscivorus piscivorus: the unique effect of piscivostatin on platelet aggregation

Piscivostatin, a novel dimeric disintegrin containing Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) sequences, was isolated from the venom of Agkistrodon piscivorus piscivorus. The molecule consisted of two chains designated as the alpha and beta chains, comprising 65 and 68 amino acid residues, respectively. Piscivostatin had two binding motifs recognized by platelet glycoprotein IIb/IIIa (GPIIb/IIIa), and the biological activity of dimeric disintegrin piscivostatin toward platelet aggregation differed from those of other monomeric disintegrins such as trimestatin and echistatin. We measured platelet aggregation by the laser light scattering method during the process of ADP-induced platelet aggregation. Both dimeric and monomeric disintegrins inhibited the formation of small (9 to 25 microm in diameter), medium-sized and large aggregates (25 to 70 microm in diameter) in a dose-dependent manner. The platelet aggregates disaggregated after reaching a maximal number on either treatment with ADP alone or monomeric disintegrin/ADP. However, the small aggregates did not disaggregate on treatment with piscivostatin/ADP even when applied over time. When washed platelets were incubated with an anti-GPIIb/IIIa monoclonal antibody, PT25-2, which induces conformational changes of GPIIb/IIIa to a form accessible to fibrinogen and other adhesion proteins without platelet activation, piscivostatin induced a platelet shape change alone with no aggregate formation. The present study indicated that piscivostatin has two unique contradictory activities; acting as a double inhibitor of platelet aggregation and platelet aggregate dissociation.     

Cloning and characterization of Adinbitor, a novel disintegrin from the snake venom of Agkistrodon halys brevicaudus stejneger

Disintegrin is a family of small proteins mainly derivedfrom snake venoms. Most of the disintegrins containRGD or KGD sequence which is the structural motif re-cognized by the platelet fibrinogen receptor α2bβ3, andthey also act as potent antagonists of several integrinsincluding αvβ3 and α5β1 which are expressed on vascularendothelial cells and some tumor cells. In addition todisintegrins’ potent antiplatelet activity, studies ondisintegrins have revealed their new applications in in…     

Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling.

Recently, a new protein containing a disintegrin domain, alternagin-C (Alt-C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt-C an ECD amino acid mogif takes the place of the RGD sequence. Most disintegrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investigated the effects of Alt-C on human neutrophil chemotaxis in vitro and the activation of integrin-mediated pathways. Alt-C showed a potent chemotactic effect for human neutrophils when compared to N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt-C significantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt-C and fMLP. A significant increase of F-actin content was observed in cells treated with Alt-C, showing that the chemotactic activity of Alt-C on neutrophils is driven by actin cytoskeleton dynamic changes. Furthermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular signal-regulated kinase 2 nuclear translocation. The chemotactic activity of Alt-C was partially inhibited by LY294002, a specific phosphatidylinositol 3-kinase inhibitor, and by PD98056, a Map kinase kinase inhibitor. These findings suggest that Alt-C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin-activated pathways and that these effects could be related to the ECD mogif present in disintegrin-like domain.