人血管内皮生长因子受体3结合肽的筛选和鉴定
发布时间：2018-10-18 14:42
【摘要】： 多肽介导的靶向性药物的设计策略是近年来提高药物在病灶组织的富集度,增加药物疗效、降低药物用量和毒副作用的有效方法。一个良好的多肽导向性药物的发现需要两方面条件,首先是特异性的药物靶点。血管内皮生长因子受体3(vascular epithelia growth factor receptor 3, VEGFR-3)正是这样一个药物靶点。VEGFR-3是III型酪氨酸激酶受体家族的新成员,作为第一个被鉴定出的淋巴管标记物,它对于胚胎期心血管和淋巴管的发育具有重要意义;在成年人体中,VEGFR-3主要分布于淋巴管内皮细胞上,它对于淋巴管形态完整性的维持和淋巴管正常功能的发挥具有重要作用。VEGFR-3与多种疾病的发生和发展密切相关,特别是在肿瘤的诊断、治疗和预后评估方面。大量的研究结果一致地认为VEGFR-3在多种肿瘤中表达量增高,并且对于判断肿瘤是否发生转移以及预后是否良好具有重要意义。一些动物实验还表明抑制VEGFR-3和其配体的结合可以有效抑制肿瘤的生长和转移。这些研究结果提示VEGFR-3是肿瘤诊断和治疗中一个具有良好前景的药物靶标分子。 有了良好的药物靶点,多肽导向性药物设计的第二个条件就是需要有一个能够特异性识别该靶点的分子作为导向性分子。噬菌体呈现肽库技术是近20年来发展很快的一项技术,它已成为寻找导向性分子的有效工具。 本实验中,我们以VEGFR-3为靶蛋白,从噬菌体呈现随机环7肽库中筛选与之结合的肽,经过三轮生物淘筛和噬菌体ELISA,我们获得了29个阳性重组噬菌体克隆。DNA测序及根据DNA序列推导出的短肽的序列表明29个克隆中含有1个共同的短肽序列CSDxxHxWC。BLAST未发现该肽序列与任何已知蛋白具有有意义的同源性(包括VEGFR-3的两个天然配体VEGF-C和VEGF-D)。在噬菌体ELISA实验中,表现最优的噬菌体被命名为Phage1。体外实验表明Phage1可以特异性的和VEGFR-3结合而不与VEGFR-1或VEGFR-2结合,并且与VEGFR-3的结合呈现剂量依赖性。在用流式细胞术筛选出了VEGFR-3阳性的细胞后,我们选择其中的一株阳性细胞-人结肠癌细胞HT29细胞皮下接种裸鼠,构建荷瘤动物模型。噬菌体在荷瘤小鼠体内的分布实验表明,归巢于肿瘤组织的噬菌体Phage1是对照噬菌体的2.38倍,说明Phage1在肿瘤组织中有一定程度的特异性富集。 为了进一步证明Phage1呈现的阳性多肽(命名为P1)在脱离了噬菌体之后仍具有特异性识别VEGFR-3的性质,我们用化学合成的方法合成了P1以及对照多肽P5,同时合成了两种多肽对应的FITC标记的产物。ELISA实验表明P1可以特异性的与VEGFR-3结合而不与VEGFR-1或VEGFR-2结合,并且与VEGFR-3的结合呈现剂量依赖性。这与前面Phage1的实验结果是一致的。细胞实验表明FTIC-P1可以与VEGFR-3阳性的细胞(HT29细胞、Y79细胞)结合,和VEGFR-3弱阳性的细胞(A549细胞)微弱的结合,而和VEGFR-3阴性(Wish细胞、EVC304细胞和HepG2细胞)不结合,其行为与抗VEGFR-3单克隆抗体的行为一致。激光共聚焦结果显示FTIC-P1结合于VEGFR-3阳性细胞的细胞膜上,符合VEGFR-3在细胞上的分布特点。在竞争实验中呈现P1的噬菌体Phage1可以特异性的抑制P1与VEGFR-3的结合,而且P1亦可以特异性的阻断FTIC-P1与VEGFR-3阳性细胞的结合。这些实验结果强烈提示P1在脱离了噬菌体之后仍然具有特异性识别VEGFR-3的性质。同样的,用VEGFR-3阳性的细胞构建荷瘤小鼠模型,体内分布实验表明相对于对照多肽FTIC-P5,FTIC-P1在肿瘤组织中有特异性汇集。 在上述实验的基础上,我们还初步研究了P1介导其它分子与VEGFR-3结合的能力。我们用基因工程的方法构建了表达hIFN-α2a-P1的融合基因。经大肠杆菌表达,hIFN-α2a-P1融合蛋白大部分以可溶形式表达。亲和层析纯化后测定其体外活性与天然的hIFN-α2a相当,ELISA实验也表明hIFN-α2a-P1可以特异性的与VEGFR-3结合。在细胞实验中,hIFN-α2a-P1也表现出了和VEGFR-3阳性细胞HT29细胞的结合能力,这提示P1具有介导hIFN-α2a与VEGFR-3结合的能力,动物实验表明,IFN-α2a-P1对荷瘤小鼠体内的肿瘤生长具有抑制作用,总体抑瘤率达57.1%。
[Abstract]:The design strategy of the polypeptide-mediated targeting drug is an effective method for improving the enrichment degree of the drug in the focus tissue in recent years, increasing the curative effect of the medicament, reducing the dosage of the medicament and the toxic and side effects. The discovery of a good polypeptide-guided drug requires two conditions, first of all, drug targets. Vascular endothelial growth factor receptor 3 (VEGF-3) is a drug target. OPG-3 is a new member of the type III tyrosine kinase receptor family as the first identified lymphatic marker, which has an important significance for the development of cardiovascular and lymphatic vessels in the embryo phase; in the adult body, IL-3 is mainly distributed on lymphatic endothelial cells, It plays an important role in the maintenance of lymphatic morphology and the functioning of lymphatic vessels. BCI-3 is closely related to the occurrence and development of various diseases, especially in the diagnosis, treatment and prognosis evaluation of tumors. The results of a large number of studies consistently suggest that the expression of OPG-3 in multiple tumors is increased, and it is important to determine whether metastasis and prognosis of the tumor are good. Some animal experiments also show that inhibition of the binding of OPG-3 and its ligands can effectively inhibit tumor growth and metastasis. These results suggest that GSK-3 is a promising drug target molecule in the diagnosis and treatment of tumors. With a good drug target, the second condition for the design of a polypeptide-guided drug is that there is a need for a molecule capable of specifically identifying the target. Phage-presenting peptide library technology is a technology that has developed very fast in recent 20 years, and it has become a guiding principle. In this experiment, we screened the peptide from phage-presenting random ring 7 peptide library from phage-presenting random ring 7 peptide library, through three-wheel biofilter screen and phage ELISA. 29 positive recombinant phage clones. DNA sequencing and the sequence of short peptides derived from the DNA sequence showed that one common short peptide sequence, CSDxxHxWC, was contained in 29 clones. BLAST has not found that the peptide sequence has significant homology to any known protein (including two native ligands V of IL-3). EGF-C and VEGF-D. Phage 1 was designated as Phage1. In vitro experiments showed that Phage1 could bind specifically to caspase-3 and not bind to IL-1 or IL-2, and was associated with VEGF The combination of R-3 presents dose-dependent. After screening the cells with IL-3 positive by flow cytometry, we select one of the positive cells-human colon cancer cells HT29 cells. A nude mouse was inoculated to construct a tumor-bearing animal model. The distribution of phage in tumor-bearing mice showed that Phage1, which belongs to the tumor tissue, was 2.38 times that of the control phage, indicating that Phage1 was in the tumor group. In order to further demonstrate that the positive polypeptide (named P1) presented by Phage1 still has the properties of specifically recognizing IL-3 after separation from the phage, we synthesize P1 and control polypeptide P5 by chemical synthesis. The results of the ELISA show that P1 can be specifically bound to the IL-3 without binding to the IL-1 or IL-2. and exhibits a dose-dependent relationship with the binding of levodopa-3. This was consistent with the experimental results of the previous Phage1. Cell experiments showed that FLIC-P1 could be combined with Jurkat-3-positive cells (HT29 cells, Y79 cells), and weak binding to caspase-3-weakly positive cells (A549 cells), without binding to caspase-3-negative (Wish cells, EVC304 cells, and HepG2 cells), The results of confocal laser confocal showed that FLIC-P1 was bound to the cell membrane of caspase-3 positive cells. In the competition experiment, P1 phage Phage1 can specifically inhibit the binding of P1 to IL-3, and P1 also can specifically block FT. The combination of IC-P1 with TUNEL-3 positive cells strongly suggests that P1 is isolated from phage After that, the tumor-bearing mouse model was constructed with IL-3 positive cells, and in vivo distribution experiments showed that FLIC-P5 relative to the control polypeptide FFIC-P1 has a specific collection in tumor tissue. On the basis of the above experiments, we The ability of P1 to mediate the binding of other molecules to IL-3 was also studied. Construction of fusion gene expressing hIFN-Fas2a-P1 was constructed by the method of engineering. The activity of hIFN-V2a-P1 fusion protein was expressed in soluble form, and its activity in vitro was comparable to that of natural hIFN-CD2a after purification. hIFN-Fas2a-P1 may be specific for binding to caspase-3. In cell experiments, hIFN-0142a-P1 also showed the binding ability of HT29 cells in Jurkat-3 positive cells, suggesting that P1 has the ability to mediate the binding of hIFN-0142a to hIL-3, and animal experiments showed that IFN-0142a-P1 was responsible for tumor bearing.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341
本文编号：2279444
[Abstract]:The design strategy of the polypeptide-mediated targeting drug is an effective method for improving the enrichment degree of the drug in the focus tissue in recent years, increasing the curative effect of the medicament, reducing the dosage of the medicament and the toxic and side effects. The discovery of a good polypeptide-guided drug requires two conditions, first of all, drug targets. Vascular endothelial growth factor receptor 3 (VEGF-3) is a drug target. OPG-3 is a new member of the type III tyrosine kinase receptor family as the first identified lymphatic marker, which has an important significance for the development of cardiovascular and lymphatic vessels in the embryo phase; in the adult body, IL-3 is mainly distributed on lymphatic endothelial cells, It plays an important role in the maintenance of lymphatic morphology and the functioning of lymphatic vessels. BCI-3 is closely related to the occurrence and development of various diseases, especially in the diagnosis, treatment and prognosis evaluation of tumors. The results of a large number of studies consistently suggest that the expression of OPG-3 in multiple tumors is increased, and it is important to determine whether metastasis and prognosis of the tumor are good. Some animal experiments also show that inhibition of the binding of OPG-3 and its ligands can effectively inhibit tumor growth and metastasis. These results suggest that GSK-3 is a promising drug target molecule in the diagnosis and treatment of tumors. With a good drug target, the second condition for the design of a polypeptide-guided drug is that there is a need for a molecule capable of specifically identifying the target. Phage-presenting peptide library technology is a technology that has developed very fast in recent 20 years, and it has become a guiding principle. In this experiment, we screened the peptide from phage-presenting random ring 7 peptide library from phage-presenting random ring 7 peptide library, through three-wheel biofilter screen and phage ELISA. 29 positive recombinant phage clones. DNA sequencing and the sequence of short peptides derived from the DNA sequence showed that one common short peptide sequence, CSDxxHxWC, was contained in 29 clones. BLAST has not found that the peptide sequence has significant homology to any known protein (including two native ligands V of IL-3). EGF-C and VEGF-D. Phage 1 was designated as Phage1. In vitro experiments showed that Phage1 could bind specifically to caspase-3 and not bind to IL-1 or IL-2, and was associated with VEGF The combination of R-3 presents dose-dependent. After screening the cells with IL-3 positive by flow cytometry, we select one of the positive cells-human colon cancer cells HT29 cells. A nude mouse was inoculated to construct a tumor-bearing animal model. The distribution of phage in tumor-bearing mice showed that Phage1, which belongs to the tumor tissue, was 2.38 times that of the control phage, indicating that Phage1 was in the tumor group. In order to further demonstrate that the positive polypeptide (named P1) presented by Phage1 still has the properties of specifically recognizing IL-3 after separation from the phage, we synthesize P1 and control polypeptide P5 by chemical synthesis. The results of the ELISA show that P1 can be specifically bound to the IL-3 without binding to the IL-1 or IL-2. and exhibits a dose-dependent relationship with the binding of levodopa-3. This was consistent with the experimental results of the previous Phage1. Cell experiments showed that FLIC-P1 could be combined with Jurkat-3-positive cells (HT29 cells, Y79 cells), and weak binding to caspase-3-weakly positive cells (A549 cells), without binding to caspase-3-negative (Wish cells, EVC304 cells, and HepG2 cells), The results of confocal laser confocal showed that FLIC-P1 was bound to the cell membrane of caspase-3 positive cells. In the competition experiment, P1 phage Phage1 can specifically inhibit the binding of P1 to IL-3, and P1 also can specifically block FT. The combination of IC-P1 with TUNEL-3 positive cells strongly suggests that P1 is isolated from phage After that, the tumor-bearing mouse model was constructed with IL-3 positive cells, and in vivo distribution experiments showed that FLIC-P5 relative to the control polypeptide FFIC-P1 has a specific collection in tumor tissue. On the basis of the above experiments, we The ability of P1 to mediate the binding of other molecules to IL-3 was also studied. Construction of fusion gene expressing hIFN-Fas2a-P1 was constructed by the method of engineering. The activity of hIFN-V2a-P1 fusion protein was expressed in soluble form, and its activity in vitro was comparable to that of natural hIFN-CD2a after purification. hIFN-Fas2a-P1 may be specific for binding to caspase-3. In cell experiments, hIFN-0142a-P1 also showed the binding ability of HT29 cells in Jurkat-3 positive cells, suggesting that P1 has the ability to mediate the binding of hIFN-0142a to hIL-3, and animal experiments showed that IFN-0142a-P1 was responsible for tumor bearing.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341
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