FILIP1L重組蛋白及抗體研制
發(fā)布時(shí)間：2018-10-08 14:35
【摘要】： 背景 血管發(fā)生是腫瘤的生長(zhǎng)、侵襲和轉(zhuǎn)移所必須的。對(duì)腫瘤血管生成的抑制被認(rèn)為是一種重要的抗腫瘤治療方法。現(xiàn)在已經(jīng)發(fā)現(xiàn)的眾多血管生成抑制劑中,有的已經(jīng)投入臨床使用。它們的抗腫瘤作用機(jī)制表現(xiàn)在抑制內(nèi)皮細(xì)胞的增殖和移行或誘導(dǎo)血管內(nèi)皮細(xì)胞凋亡。 Filamin A interacting protein 1-like (FILIP1L)是在檢測(cè)內(nèi)皮細(xì)胞在對(duì)血管生成抑制劑的反應(yīng)時(shí)基因表達(dá)的情況時(shí),所鑒定出來(lái)的一個(gè)上調(diào)表達(dá)的基因。研究表明過(guò)表達(dá)的FILIP1L導(dǎo)致了內(nèi)皮細(xì)胞增殖和遷移的抑制,并且細(xì)胞的凋亡也增加。對(duì)FILIP1L的研究提示我們:它可能是血管生成抑制劑作用的調(diào)節(jié)因子;并且是潛在的抗腫瘤血管生成的靶標(biāo)。 本研究首先從食管鱗癌細(xì)胞系EC9706中克隆FILIP1L胞外段編碼基因,經(jīng)測(cè)序驗(yàn)證后,構(gòu)建pET22b- FILIP1L原核表達(dá)載體并轉(zhuǎn)入大腸桿菌表達(dá)感受態(tài),采用親和純化技術(shù)純化重組融合蛋白,并制備以FILIP1L蛋白為抗原的多克隆抗體,進(jìn)一步探討FILIP1L相互作用蛋白的表達(dá)情況。 目的 通過(guò)構(gòu)建pET22b- FILIP1L原核表達(dá)載體并誘導(dǎo)蛋白表達(dá)純化后,用所得蛋白制備多克隆抗體,研究在腫瘤細(xì)胞和正常細(xì)胞中與FILIP1L蛋白結(jié)合的相互作用蛋白組分差異,闡明FILIP1L在抗腫瘤血管生成中發(fā)揮抑制腫瘤血管生成作用的分子機(jī)制,為進(jìn)一步明晰惡性腫瘤多分子、多階段演進(jìn)分子機(jī)制及癌癥治療新靶點(diǎn)奠定重要基礎(chǔ)。 方法 從食管上皮鱗癌細(xì)胞EC9706中提取mRNA并反轉(zhuǎn)錄成cDNA,利用RT-PCR技術(shù)擴(kuò)增含有全長(zhǎng)FILIP1L基因(963bp左右)的cDNA,將其定向插入原核表達(dá)載體pET22b,經(jīng)測(cè)序確定載體構(gòu)建成功后,誘導(dǎo)重組質(zhì)粒蛋白表達(dá);Western blotting檢測(cè)表達(dá)蛋白成功,并利用親和層析技術(shù)純化得到誘導(dǎo)蛋白;用誘導(dǎo)蛋白免疫動(dòng)物制備多克隆抗體,進(jìn)一步運(yùn)用免疫共沉淀技術(shù)檢測(cè)FILIP1L的相互作用蛋白表達(dá)差異。 結(jié)果 1.從食管上皮鱗癌細(xì)胞EC9706中得到了FILIP1L胞外段基因,驗(yàn)證序列準(zhǔn)確無(wú)誤; 2.構(gòu)建了FILIP1L原核表達(dá)載體并誘導(dǎo)誘導(dǎo)蛋白表達(dá),純化得到高純度蛋白。 3.運(yùn)用純化得到的蛋白制備得到效價(jià)為1:1×106的高效價(jià)多克隆抗體。 4.利用免疫共沉淀技術(shù)篩選正常食管上皮細(xì)胞NEC與食管上皮鱗癌細(xì)胞EC9706中的與FILIP1L相關(guān)蛋白的表達(dá)差異情況。 結(jié)論 本研究從食管上皮鱗癌細(xì)胞9706中得到了FILIP1L胞外段基因,構(gòu)建了FILIP1L原核表達(dá)載體,利用FILIP1L重組質(zhì)粒表達(dá)融合蛋白,并制備多克隆抗體,檢測(cè)正常組織與腫瘤組織中相互作用蛋白的差異表達(dá),發(fā)現(xiàn)6個(gè)與FILIP1L相關(guān)的蛋白條帶在食管癌細(xì)胞與正常食管上皮細(xì)胞中的表達(dá)有差異,對(duì)這些蛋白條帶的進(jìn)一步鑒定,將為今后進(jìn)一步闡明FILIP1L蛋白在腫瘤發(fā)生發(fā)展過(guò)程中抑制腫瘤血管生成作用的分子機(jī)制奠定重要的工作基礎(chǔ),并為探索癌癥治療新靶點(diǎn)開(kāi)辟新途徑。
[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.
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
本文編號(hào)：2257134
[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.
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
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，本文編號(hào)：2257134
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