单链抗体融合蛋白ScFv-9R在大肠杆菌中的高效表达和介导siRNA靶向RT112细胞的研究
发布时间：2019-06-29 14:43
【摘要】：成纤维细胞生长因子受体3(FGFR3)是一个被熟知的原癌基因，FGFR3的信号通路对细胞的发育起着至关重要的作用，它跟许多肿瘤的发生有密切的关系。因此，越来越多的研究人员开始意识到受体激酶抑制剂的潜在应用价值，以及把研究重点聚焦到能够靶向抑制FGFR3信号传导的单克隆抗体或单链抗体上。在本论文研究中，我们设计了一种新的融合蛋白（ScFv-9R），这个蛋白含有能够靶向FGFR3的单链抗体又含有能够结合siRNA的9个精氨酸的短肽。 单链抗体(single chain antibody fragment，ScFv)，是通过十五个左右氨基酸的短肽(linker)将抗体的重链可变区和轻链的可变区连接而成，它不仅保留了特异性识别抗原的特性，而且具有自身体积小、穿透力强、免疫原性低等优点。精氨酸为碱性氨基酸，在中性条件下带正电荷，能够与带负电荷的核酸结合。因此由9个精氨酸形成的短肽能够结合siRNA。 传统的原核表达系统得到的蛋白大都不能够正确的折叠而以包涵体的形式存在于沉淀中，为后续实验带来了很多不便。为了让目的蛋白能够可溶性表达并且得到更高的产量，我们建立了一种可溶性表达系统，通过PCR反应在基因水平上将ScFv和ScFv-9R融合了SUMO（小泛素修饰复合物）并且在大肠杆菌BL21中表达。重组细菌经过0.5mM的IPTG（isopropyl-β-D-thiogalactopyranoside）20°C诱导20小时，收集目的蛋白Sumo-ScFv和Sumo-ScFv-9R的上清液依次通过阴离子交换层析DEAE和亲和层析（Ni-NTA）得到纯度较高的目的蛋白。将得到的Sumo-ScFv和Sumo-ScFv-9R经过Sumo蛋白酶消化，将融合蛋白SUMO与目的蛋白ScFv和ScFv-9R分离，从而得到ScFv和ScFv-9R。ScFv和ScFv-9R的纯度高于95%，浓度大约为4mg/L。体外实验表明，在存在或者缺少FGF9的情况下ScFv-9R能够使FGFR3和ERK的磷酸化水平降低，抑制或者减弱FGFR3的信号通路。通过凝胶阻滞实验，我们可以看出目的蛋白ScFv-9R在结合siRNA的情况下呈现明显拖带的现象，表明ScFv-9R能够结合siRNA，1μg的ScFv-9R能够结合4pmol siRNA。通过荧光显微镜技术可以发现ScFv-9R能够有效地携带siRNA进入FGFR3高表达的RT112（膀胱癌细胞系）细胞中，但是却不能结合以及进入到FGFR3阴性的细胞如THP1细胞（人急性白血病细胞系）。 综上所述，我们利用Sumo融合表达系统在大肠杆菌中能够获得具有高产量的、可溶性的融合蛋白ScFv-9R。该蛋白具有双重功能，在携带药物的同时能够靶向结合FGFR3阳性的肿瘤细胞，，是一个高效的药物传递系统。我们的结果显示ScFv-9R作为一个新型的药物载体，在治疗肿瘤以及药物的开发方面具有很好的潜在应用价值。
[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 掳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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R3411
本文编号：2507900
[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 掳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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R3411
【参考文献】
相关期刊论文 前1条
1 郑海学;靳野;尹双辉;郭慧琛;尚佑军;白兴文;刘湘涛;谢庆阁;;T7 RNA聚合酶表达系统的真核化及其偶联表达系统的建立[J];生物工程学报;2007年05期
本文编号：2507900
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