FILIP1L重组蛋白及抗体研制
发布时间：2018-10-08 14:35
【摘要】： 背景 血管发生是肿瘤的生长、侵袭和转移所必须的。对肿瘤血管生成的抑制被认为是一种重要的抗肿瘤治疗方法。现在已经发现的众多血管生成抑制剂中,有的已经投入临床使用。它们的抗肿瘤作用机制表现在抑制内皮细胞的增殖和移行或诱导血管内皮细胞凋亡。 Filamin A interacting protein 1-like (FILIP1L)是在检测内皮细胞在对血管生成抑制剂的反应时基因表达的情况时,所鉴定出来的一个上调表达的基因。研究表明过表达的FILIP1L导致了内皮细胞增殖和迁移的抑制,并且细胞的凋亡也增加。对FILIP1L的研究提示我们:它可能是血管生成抑制剂作用的调节因子;并且是潜在的抗肿瘤血管生成的靶标。 本研究首先从食管鳞癌细胞系EC9706中克隆FILIP1L胞外段编码基因,经测序验证后,构建pET22b- FILIP1L原核表达载体并转入大肠杆菌表达感受态,采用亲和纯化技术纯化重组融合蛋白,并制备以FILIP1L蛋白为抗原的多克隆抗体,进一步探讨FILIP1L相互作用蛋白的表达情况。 目的 通过构建pET22b- FILIP1L原核表达载体并诱导蛋白表达纯化后,用所得蛋白制备多克隆抗体,研究在肿瘤细胞和正常细胞中与FILIP1L蛋白结合的相互作用蛋白组分差异,阐明FILIP1L在抗肿瘤血管生成中发挥抑制肿瘤血管生成作用的分子机制,为进一步明晰恶性肿瘤多分子、多阶段演进分子机制及癌症治疗新靶点奠定重要基础。 方法 从食管上皮鳞癌细胞EC9706中提取mRNA并反转录成cDNA,利用RT-PCR技术扩增含有全长FILIP1L基因(963bp左右)的cDNA,将其定向插入原核表达载体pET22b,经测序确定载体构建成功后,诱导重组质粒蛋白表达;Western blotting检测表达蛋白成功,并利用亲和层析技术纯化得到诱导蛋白;用诱导蛋白免疫动物制备多克隆抗体,进一步运用免疫共沉淀技术检测FILIP1L的相互作用蛋白表达差异。 结果 1.从食管上皮鳞癌细胞EC9706中得到了FILIP1L胞外段基因,验证序列准确无误; 2.构建了FILIP1L原核表达载体并诱导诱导蛋白表达,纯化得到高纯度蛋白。 3.运用纯化得到的蛋白制备得到效价为1:1×106的高效价多克隆抗体。 4.利用免疫共沉淀技术筛选正常食管上皮细胞NEC与食管上皮鳞癌细胞EC9706中的与FILIP1L相关蛋白的表达差异情况。 结论 本研究从食管上皮鳞癌细胞9706中得到了FILIP1L胞外段基因,构建了FILIP1L原核表达载体,利用FILIP1L重组质粒表达融合蛋白,并制备多克隆抗体,检测正常组织与肿瘤组织中相互作用蛋白的差异表达,发现6个与FILIP1L相关的蛋白条带在食管癌细胞与正常食管上皮细胞中的表达有差异,对这些蛋白条带的进一步鉴定,将为今后进一步阐明FILIP1L蛋白在肿瘤发生发展过程中抑制肿瘤血管生成作用的分子机制奠定重要的工作基础,并为探索癌症治疗新靶点开辟新途径。
[Abstract]:Background Angiogenesis is necessary for tumor growth, invasion and metastasis. Inhibition of tumor angiogenesis is considered to be an important antitumor therapy. Some of the many angiogenic inhibitors that have been discovered have been put to clinical use. Their antitumor mechanism is to inhibit the proliferation and migration of endothelial cells or induce apoptosis of vascular endothelial cells. Filamin A interacting protein 1-like (FILIP1L) is an up-regulated gene identified by detecting the expression of genes in endothelial cells in response to angiogenesis inhibitors. Studies have shown that overexpression of FILIP1L leads to inhibition of endothelial cell proliferation and migration and increased apoptosis. The study of FILIP1L suggests that it may be a regulatory factor for angiogenesis inhibitors and a potential target for anti-angiogenesis. In this study, the extracellular coding gene of FILIP1L was cloned from esophageal squamous cell carcinoma cell line EC9706. After sequencing, the prokaryotic expression vector of pET22b- FILIP1L was constructed and transformed into Escherichia coli expression receptive state. The recombinant fusion protein was purified by affinity purification. The polyclonal antibody with FILIP1L protein as antigen was prepared to further investigate the expression of FILIP1L interaction protein. Objective to construct a prokaryotic expression vector of pET22b- FILIP1L and induce the expression and purification of FILIP1L protein, then to prepare the polyclonal antibody with the obtained protein, and to study the difference of the interaction protein components between tumor cells and normal cells. To elucidate the molecular mechanism of inhibiting tumor angiogenesis by FILIP1L in tumor angiogenesis, and to lay an important foundation for further understanding the molecular mechanism of multimolecule, multi-stage evolution of malignant tumor and the new target of cancer therapy. Methods mRNA was extracted from esophageal squamous carcinoma cell EC9706 and reverse transcribed into cDNA,. CDNA, containing the full-length FILIP1L gene (963bp or so) was amplified by RT-PCR technique and inserted into the prokaryotic expression vector pET22b,. The expression of recombinant plasmid protein was successfully detected by Western blotting and purified by affinity chromatography, and the polyclonal antibody was prepared by immunizing animals with induced protein. Furthermore, the differential expression of FILIP1L interacting protein was detected by immunoprecipitation technique. Result 1. The extracellular segment of FILIP1L gene was obtained from esophageal squamous carcinoma cell EC9706, and the sequence was proved to be accurate. 2. The prokaryotic expression vector of FILIP1L was constructed and the protein was induced to express. 3. A high titer polyclonal antibody with a titer of 1:1 脳 106 was obtained from the purified protein. 4. The differential expression of FILIP1L related proteins in normal esophageal epithelial cells (NEC) and esophageal squamous carcinoma cells (EC9706) was screened by immunoprecipitation technique. Conclusion in this study, the extracellular segment of FILIP1L gene was obtained from esophageal epithelial squamous carcinoma cell line 9706, the prokaryotic expression vector of FILIP1L was constructed, the fusion protein was expressed by FILIP1L recombinant plasmid, and the polyclonal antibody was prepared. The differential expression of interacting proteins in normal tissues and tumor tissues was detected. Six protein bands associated with FILIP1L were found to be differentially expressed in esophageal cancer cells and normal esophageal epithelial cells. It will lay an important foundation for further elucidation of the molecular mechanism of FILIP1L protein inhibiting tumor angiogenesis in the process of tumorigenesis and development, and open up a new way to explore new targets for cancer treatment.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2257133
[Abstract]:Background Angiogenesis is necessary for tumor growth, invasion and metastasis. Inhibition of tumor angiogenesis is considered to be an important antitumor therapy. Some of the many angiogenic inhibitors that have been discovered have been put to clinical use. Their antitumor mechanism is to inhibit the proliferation and migration of endothelial cells or induce apoptosis of vascular endothelial cells. Filamin A interacting protein 1-like (FILIP1L) is an up-regulated gene identified by detecting the expression of genes in endothelial cells in response to angiogenesis inhibitors. Studies have shown that overexpression of FILIP1L leads to inhibition of endothelial cell proliferation and migration and increased apoptosis. The study of FILIP1L suggests that it may be a regulatory factor for angiogenesis inhibitors and a potential target for anti-angiogenesis. In this study, the extracellular coding gene of FILIP1L was cloned from esophageal squamous cell carcinoma cell line EC9706. After sequencing, the prokaryotic expression vector of pET22b- FILIP1L was constructed and transformed into Escherichia coli expression receptive state. The recombinant fusion protein was purified by affinity purification. The polyclonal antibody with FILIP1L protein as antigen was prepared to further investigate the expression of FILIP1L interaction protein. Objective to construct a prokaryotic expression vector of pET22b- FILIP1L and induce the expression and purification of FILIP1L protein, then to prepare the polyclonal antibody with the obtained protein, and to study the difference of the interaction protein components between tumor cells and normal cells. To elucidate the molecular mechanism of inhibiting tumor angiogenesis by FILIP1L in tumor angiogenesis, and to lay an important foundation for further understanding the molecular mechanism of multimolecule, multi-stage evolution of malignant tumor and the new target of cancer therapy. Methods mRNA was extracted from esophageal squamous carcinoma cell EC9706 and reverse transcribed into cDNA,. CDNA, containing the full-length FILIP1L gene (963bp or so) was amplified by RT-PCR technique and inserted into the prokaryotic expression vector pET22b,. The expression of recombinant plasmid protein was successfully detected by Western blotting and purified by affinity chromatography, and the polyclonal antibody was prepared by immunizing animals with induced protein. Furthermore, the differential expression of FILIP1L interacting protein was detected by immunoprecipitation technique. Result 1. The extracellular segment of FILIP1L gene was obtained from esophageal squamous carcinoma cell EC9706, and the sequence was proved to be accurate. 2. The prokaryotic expression vector of FILIP1L was constructed and the protein was induced to express. 3. A high titer polyclonal antibody with a titer of 1:1 脳 106 was obtained from the purified protein. 4. The differential expression of FILIP1L related proteins in normal esophageal epithelial cells (NEC) and esophageal squamous carcinoma cells (EC9706) was screened by immunoprecipitation technique. Conclusion in this study, the extracellular segment of FILIP1L gene was obtained from esophageal epithelial squamous carcinoma cell line 9706, the prokaryotic expression vector of FILIP1L was constructed, the fusion protein was expressed by FILIP1L recombinant plasmid, and the polyclonal antibody was prepared. The differential expression of interacting proteins in normal tissues and tumor tissues was detected. Six protein bands associated with FILIP1L were found to be differentially expressed in esophageal cancer cells and normal esophageal epithelial cells. It will lay an important foundation for further elucidation of the molecular mechanism of FILIP1L protein inhibiting tumor angiogenesis in the process of tumorigenesis and development, and open up a new way to explore new targets for cancer treatment.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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