【摘要】：成纖維細(xì)胞生長因子受體3(FGFR3)是一個(gè)被熟知的原癌基因，F(xiàn)GFR3的信號通路對細(xì)胞的發(fā)育起著至關(guān)重要的作用，它跟許多腫瘤的發(fā)生有密切的關(guān)系。因此，越來越多的研究人員開始意識到受體激酶抑制劑的潛在應(yīng)用價(jià)值，以及把研究重點(diǎn)聚焦到能夠靶向抑制FGFR3信號傳導(dǎo)的單克隆抗體或單鏈抗體上。在本論文研究中，我們設(shè)計(jì)了一種新的融合蛋白（ScFv-9R），這個(gè)蛋白含有能夠靶向FGFR3的單鏈抗體又含有能夠結(jié)合siRNA的9個(gè)精氨酸的短肽。 單鏈抗體(single chain antibody fragment，ScFv)，是通過十五個(gè)左右氨基酸的短肽(linker)將抗體的重鏈可變區(qū)和輕鏈的可變區(qū)連接而成，它不僅保留了特異性識別抗原的特性，而且具有自身體積小、穿透力強(qiáng)、免疫原性低等優(yōu)點(diǎn)。精氨酸為堿性氨基酸，在中性條件下帶正電荷，能夠與帶負(fù)電荷的核酸結(jié)合。因此由9個(gè)精氨酸形成的短肽能夠結(jié)合siRNA。 傳統(tǒng)的原核表達(dá)系統(tǒng)得到的蛋白大都不能夠正確的折疊而以包涵體的形式存在于沉淀中，為后續(xù)實(shí)驗(yàn)帶來了很多不便。為了讓目的蛋白能夠可溶性表達(dá)并且得到更高的產(chǎn)量，我們建立了一種可溶性表達(dá)系統(tǒng)，通過PCR反應(yīng)在基因水平上將ScFv和ScFv-9R融合了SUMO（小泛素修飾復(fù)合物）并且在大腸桿菌BL21中表達(dá)。重組細(xì)菌經(jīng)過0.5mM的IPTG（isopropyl-β-D-thiogalactopyranoside）20°C誘導(dǎo)20小時(shí)，收集目的蛋白Sumo-ScFv和Sumo-ScFv-9R的上清液依次通過陰離子交換層析DEAE和親和層析（Ni-NTA）得到純度較高的目的蛋白。將得到的Sumo-ScFv和Sumo-ScFv-9R經(jīng)過Sumo蛋白酶消化，將融合蛋白SUMO與目的蛋白ScFv和ScFv-9R分離，從而得到ScFv和ScFv-9R。ScFv和ScFv-9R的純度高于95%，濃度大約為4mg/L。體外實(shí)驗(yàn)表明，在存在或者缺少FGF9的情況下ScFv-9R能夠使FGFR3和ERK的磷酸化水平降低，抑制或者減弱FGFR3的信號通路。通過凝膠阻滯實(shí)驗(yàn)，我們可以看出目的蛋白ScFv-9R在結(jié)合siRNA的情況下呈現(xiàn)明顯拖帶的現(xiàn)象，表明ScFv-9R能夠結(jié)合siRNA，1μg的ScFv-9R能夠結(jié)合4pmol siRNA。通過熒光顯微鏡技術(shù)可以發(fā)現(xiàn)ScFv-9R能夠有效地?cái)y帶siRNA進(jìn)入FGFR3高表達(dá)的RT112（膀胱癌細(xì)胞系）細(xì)胞中，但是卻不能結(jié)合以及進(jìn)入到FGFR3陰性的細(xì)胞如THP1細(xì)胞（人急性白血病細(xì)胞系）。 綜上所述，我們利用Sumo融合表達(dá)系統(tǒng)在大腸桿菌中能夠獲得具有高產(chǎn)量的、可溶性的融合蛋白ScFv-9R。該蛋白具有雙重功能，在攜帶藥物的同時(shí)能夠靶向結(jié)合FGFR3陽性的腫瘤細(xì)胞，，是一個(gè)高效的藥物傳遞系統(tǒng)。我們的結(jié)果顯示ScFv-9R作為一個(gè)新型的藥物載體，在治療腫瘤以及藥物的開發(fā)方面具有很好的潛在應(yīng)用價(jià)值。
[Abstract]:Fibroblasts growth factor receptor 3 (FGFR3) is a well-known proto-oncogene. FGFR3 signaling pathway plays an important role in cell development and is closely related to the occurrence of many tumors. As a result, more and more researchers are beginning to realize the potential application value of receptor kinase inhibitors and focus on monoclonal antibodies or single chain antibodies that can target FGFR3 signal transduction. In this thesis, we designed a new fusion protein (ScFv-9R), which contains single chain antibodies targeting FGFR3 and 9 arginine peptides that bind to siRNA. Single chain antibody (single chain antibody fragment,ScFv) is formed by connecting the variable region of the heavy chain and the variable region of the light chain through the short peptide (linker) of about fifteen amino acids. It not only retains the characteristic of specific recognition antigen, but also has the advantages of small volume, strong penetration and low immunogenicity. Arginine is an basic amino acid, which is positively charged under neutral conditions and can bind to negatively charged nucleic acid. Therefore, short peptides formed by nine arginine can bind to siRNA.. Most of the proteins obtained by the traditional prokaryotic expression system can not be folded correctly and exist in the form of inclusion bodies, which brings a lot of inconvenience to the subsequent experiments. In order to enable the target protein to express soluble and obtain higher yield, we established a soluble expression system, which merges ScFv and ScFv-9R at the gene level by PCR reaction and expresses it in E. coli BL21. The recombinant bacteria were induced by 0.5mM IPTG (isopropyl- 尾-D-thiogalactopyranoside) 20 擄C for 20 hours. The target proteins Sumo-ScFv and Sumo-ScFv-9R were collected and purified by anion exchange chromatography (DEAE) and affinity chromatography (Ni-NTA). The obtained Sumo-ScFv and Sumo-ScFv-9R were digested by Sumo protease and the fusion protein SUMO was separated from the target proteins ScFv and ScFv-9R. The purity of ScFv and ScFv-9R.ScFv and ScFv-9R was more than 95% and the concentration was about 4 mg 路L ~ (- 1). In vitro experiments showed that ScFv-9R could decrease the phosphorylation level of FGFR3 and ERK and inhibit or weaken the signal pathway of FGFR3 in the presence or absence of FGF9. Through gel block test, we can see that the target protein ScFv-9R shows obvious towing phenomenon when it binds to siRNA, which indicates that ScFv-9R can bind to siRNA, 1 渭 g ScFv-9R and bind 4pmol siRNA.. By fluorescence microscope, it can be found that ScFv-9R can effectively carry siRNA into RT112 (bladder cancer cell line) with high expression of FGFR3, but it can not bind to and enter FGFR3 negative cells such as THP1 cells (human acute leukemia cell line). To sum up, we can obtain high yield and soluble fusion protein ScFv-9R. in E. coli by using Sumo fusion expression system. The protein has dual functions and can target FGFR3 positive tumor cells while carrying drugs. It is an efficient drug delivery system. Our results show that ScFv-9R, as a new drug carrier, has a good potential application value in the treatment of tumor and the development of drugs.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R3411

【參考文獻(xiàn)】

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1 鄭海學(xué);靳野;尹雙輝;郭慧琛;尚佑軍;白興文;劉湘濤;謝慶閣;;T7 RNA聚合酶表達(dá)系統(tǒng)的真核化及其偶聯(lián)表達(dá)系統(tǒng)的建立[J];生物工程學(xué)報(bào);2007年05期

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