人血管內(nèi)皮生長因子受體3結(jié)合肽的篩選和鑒定
發(fā)布時間：2018-10-18 14:42
【摘要】： 多肽介導(dǎo)的靶向性藥物的設(shè)計(jì)策略是近年來提高藥物在病灶組織的富集度,增加藥物療效、降低藥物用量和毒副作用的有效方法。一個良好的多肽導(dǎo)向性藥物的發(fā)現(xiàn)需要兩方面條件,首先是特異性的藥物靶點(diǎn)。血管內(nèi)皮生長因子受體3(vascular epithelia growth factor receptor 3, VEGFR-3)正是這樣一個藥物靶點(diǎn)。VEGFR-3是III型酪氨酸激酶受體家族的新成員,作為第一個被鑒定出的淋巴管標(biāo)記物,它對于胚胎期心血管和淋巴管的發(fā)育具有重要意義;在成年人體中,VEGFR-3主要分布于淋巴管內(nèi)皮細(xì)胞上,它對于淋巴管形態(tài)完整性的維持和淋巴管正常功能的發(fā)揮具有重要作用。VEGFR-3與多種疾病的發(fā)生和發(fā)展密切相關(guān),特別是在腫瘤的診斷、治療和預(yù)后評估方面。大量的研究結(jié)果一致地認(rèn)為VEGFR-3在多種腫瘤中表達(dá)量增高,并且對于判斷腫瘤是否發(fā)生轉(zhuǎn)移以及預(yù)后是否良好具有重要意義。一些動物實(shí)驗(yàn)還表明抑制VEGFR-3和其配體的結(jié)合可以有效抑制腫瘤的生長和轉(zhuǎn)移。這些研究結(jié)果提示VEGFR-3是腫瘤診斷和治療中一個具有良好前景的藥物靶標(biāo)分子。 有了良好的藥物靶點(diǎn),多肽導(dǎo)向性藥物設(shè)計(jì)的第二個條件就是需要有一個能夠特異性識別該靶點(diǎn)的分子作為導(dǎo)向性分子。噬菌體呈現(xiàn)肽庫技術(shù)是近20年來發(fā)展很快的一項(xiàng)技術(shù),它已成為尋找導(dǎo)向性分子的有效工具。 本實(shí)驗(yàn)中,我們以VEGFR-3為靶蛋白,從噬菌體呈現(xiàn)隨機(jī)環(huán)7肽庫中篩選與之結(jié)合的肽,經(jīng)過三輪生物淘篩和噬菌體ELISA,我們獲得了29個陽性重組噬菌體克隆。DNA測序及根據(jù)DNA序列推導(dǎo)出的短肽的序列表明29個克隆中含有1個共同的短肽序列CSDxxHxWC。BLAST未發(fā)現(xiàn)該肽序列與任何已知蛋白具有有意義的同源性(包括VEGFR-3的兩個天然配體VEGF-C和VEGF-D)。在噬菌體ELISA實(shí)驗(yàn)中,表現(xiàn)最優(yōu)的噬菌體被命名為Phage1。體外實(shí)驗(yàn)表明Phage1可以特異性的和VEGFR-3結(jié)合而不與VEGFR-1或VEGFR-2結(jié)合,并且與VEGFR-3的結(jié)合呈現(xiàn)劑量依賴性。在用流式細(xì)胞術(shù)篩選出了VEGFR-3陽性的細(xì)胞后,我們選擇其中的一株陽性細(xì)胞-人結(jié)腸癌細(xì)胞HT29細(xì)胞皮下接種裸鼠,構(gòu)建荷瘤動物模型。噬菌體在荷瘤小鼠體內(nèi)的分布實(shí)驗(yàn)表明,歸巢于腫瘤組織的噬菌體Phage1是對照噬菌體的2.38倍,說明Phage1在腫瘤組織中有一定程度的特異性富集。 為了進(jìn)一步證明Phage1呈現(xiàn)的陽性多肽(命名為P1)在脫離了噬菌體之后仍具有特異性識別VEGFR-3的性質(zhì),我們用化學(xué)合成的方法合成了P1以及對照多肽P5,同時合成了兩種多肽對應(yīng)的FITC標(biāo)記的產(chǎn)物。ELISA實(shí)驗(yàn)表明P1可以特異性的與VEGFR-3結(jié)合而不與VEGFR-1或VEGFR-2結(jié)合,并且與VEGFR-3的結(jié)合呈現(xiàn)劑量依賴性。這與前面Phage1的實(shí)驗(yàn)結(jié)果是一致的。細(xì)胞實(shí)驗(yàn)表明FTIC-P1可以與VEGFR-3陽性的細(xì)胞(HT29細(xì)胞、Y79細(xì)胞)結(jié)合,和VEGFR-3弱陽性的細(xì)胞(A549細(xì)胞)微弱的結(jié)合,而和VEGFR-3陰性(Wish細(xì)胞、EVC304細(xì)胞和HepG2細(xì)胞)不結(jié)合,其行為與抗VEGFR-3單克隆抗體的行為一致。激光共聚焦結(jié)果顯示FTIC-P1結(jié)合于VEGFR-3陽性細(xì)胞的細(xì)胞膜上,符合VEGFR-3在細(xì)胞上的分布特點(diǎn)。在競爭實(shí)驗(yàn)中呈現(xiàn)P1的噬菌體Phage1可以特異性的抑制P1與VEGFR-3的結(jié)合,而且P1亦可以特異性的阻斷FTIC-P1與VEGFR-3陽性細(xì)胞的結(jié)合。這些實(shí)驗(yàn)結(jié)果強(qiáng)烈提示P1在脫離了噬菌體之后仍然具有特異性識別VEGFR-3的性質(zhì)。同樣的,用VEGFR-3陽性的細(xì)胞構(gòu)建荷瘤小鼠模型,體內(nèi)分布實(shí)驗(yàn)表明相對于對照多肽FTIC-P5,FTIC-P1在腫瘤組織中有特異性匯集。 在上述實(shí)驗(yàn)的基礎(chǔ)上,我們還初步研究了P1介導(dǎo)其它分子與VEGFR-3結(jié)合的能力。我們用基因工程的方法構(gòu)建了表達(dá)hIFN-α2a-P1的融合基因。經(jīng)大腸桿菌表達(dá),hIFN-α2a-P1融合蛋白大部分以可溶形式表達(dá)。親和層析純化后測定其體外活性與天然的hIFN-α2a相當(dāng),ELISA實(shí)驗(yàn)也表明hIFN-α2a-P1可以特異性的與VEGFR-3結(jié)合。在細(xì)胞實(shí)驗(yàn)中,hIFN-α2a-P1也表現(xiàn)出了和VEGFR-3陽性細(xì)胞HT29細(xì)胞的結(jié)合能力,這提示P1具有介導(dǎo)hIFN-α2a與VEGFR-3結(jié)合的能力,動物實(shí)驗(yàn)表明,IFN-α2a-P1對荷瘤小鼠體內(nèi)的腫瘤生長具有抑制作用,總體抑瘤率達(dá)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R341
本文編號：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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R341
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