本文關(guān)鍵詞： 腫瘤靶向性 VEGF α-Gal 抗腫瘤活性　出處：《重慶醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的: 為了將肺癌的靶向治療和免疫治療相結(jié)合,本研究擴增了VEGF-α-Gal融合基因,構(gòu)建原核表達質(zhì)粒pQE30-VEGF-α-Gal,在大腸桿菌M15內(nèi)誘導(dǎo)表達rVEGF-α-Gal融合蛋白,并在體外細胞水平上研究重組蛋白的肺癌細胞靶向性和α-Gal表位介導(dǎo)的腫瘤細胞殺傷效應(yīng),為探索肺癌及其他腫瘤的新型靶向生物治療途徑奠定基礎(chǔ)。 方法: 1.體外培養(yǎng)A549細胞,提取總RNA,以RT-PCR法擴增VEGF-α-Gal基因片段作為目的基因,雙酶切后定向克隆入pQE30質(zhì)粒中,構(gòu)建并鑒定pQE30-VEGF-α-Gal原核表達質(zhì)粒。 2.將pQE30-VEGF-α-Gal質(zhì)粒轉(zhuǎn)化進E.coli M15菌,用IPTG誘導(dǎo)目的蛋白表達,用SDS-PAGE法分析重組蛋白的相對分子質(zhì)量及表達形式,用Western-blot法分析重組蛋白的抗原性,用Ni-Agarose His標簽蛋白純化試劑盒純化重組VEGF-α-Gal融合蛋白,然后采取逐步降低尿素濃度的方法使重組蛋白復(fù)性。 3.通過細胞黏附試驗評價重組蛋白中VEGF對腫瘤細胞A549的靶向性,通過血清殺傷試驗分析重組蛋白中α-Gal模擬表位介導(dǎo)的體外溶腫瘤細胞效應(yīng)。 結(jié)果: 1.經(jīng)雙酶切鑒定及DNA序列測定,證實pQE30-VEGF-α-Gal原核表達質(zhì)粒構(gòu)建成功。 2.將pQE30-VEGF-α-Gal質(zhì)粒成功轉(zhuǎn)化進E.coli M15菌,并實現(xiàn)目的蛋白的誘導(dǎo)表達。經(jīng)SDS-PAGE法分析,目的蛋白分子量為18KDa,主要以包涵體形式為主。Western-blot結(jié)果表明,重組蛋白能分別與抗VEGF、抗α-Gal特異性結(jié)合。純化的重組蛋白純度約為90%。 3.細胞黏附試驗和血清殺傷試驗結(jié)果證實,純化復(fù)性后的重組蛋白不但能黏附VEGFR+的A549細胞,而且其α-Gal模擬表位在人新鮮血清的參與下,對A549細胞產(chǎn)生了明顯的溶細胞效應(yīng)。 結(jié)論: 1.成功構(gòu)建了原核表達重組質(zhì)粒pQE30-VEGF-α-Gal,并成功表達及純化了重組VEGF-α-Gal融合蛋白,為探索腫瘤生物治療新途徑奠定了基礎(chǔ)。 2.重組VEGF-α-Gal融合蛋白不但具有VEGFR靶向性,還兼具α-Gal表位功能,為研究和開發(fā)新型靶向腫瘤生物制劑奠定了基礎(chǔ)。
[Abstract]:Objective:. In order to combine the targeted therapy with immunotherapy for lung cancer, the VEGF- 偽 -Gal fusion gene was amplified and the prokaryotic expression plasmid pQE30-VEGF- 偽 -Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal-Gal@@. The aim of this study was to study the targeting of lung cancer cells by recombinant protein and the cytotoxicity of tumor cells mediated by 偽 -Gal epitope in vitro, which laid a foundation for the exploration of new targeted biotherapy approaches for lung cancer and other tumors. Methods:. 1. A549 cells were cultured in vitro and total RNAs were extracted. The VEGF- 偽 -Gal gene fragment was amplified by RT-PCR as the target gene, and then cloned into pQE30 plasmid. The pQE30-VEGF- 偽 -Gal prokaryotic expression plasmid was constructed and identified. 2. The pQE30-VEGF- 偽 -Gal plasmid was transformed into E. coli M15. The expression of the target protein was induced by IPTG. The relative molecular weight and expression form of the recombinant protein were analyzed by SDS-PAGE method, and the antigenicity of the recombinant protein was analyzed by Western-blot method. The recombinant VEGF- 偽 -Gal fusion protein was purified by using Ni-Agarose His tag protein purification kit, and the recombinant protein was renatured by decreasing urea concentration step by step. 3. The targeting of VEGF in recombinant protein to A549 was evaluated by cell adhesion assay, and the effect of 偽 -Gal mimic epitope on tumor cell lysis in vitro was analyzed by serum cytotoxicity test. Results:. 1. The construction of pQE30-VEGF- 偽 -Gal prokaryotic expression plasmid was confirmed by double enzyme digestion and DNA sequencing. 2. The plasmid pQE30-VEGF- 偽 -Gal was successfully transformed into E. coli M15, and the target protein was induced to express. The molecular weight of the protein was 18KDa.Western-blot showed that the protein was mainly in the form of inclusion body. The recombinant protein could specifically bind to anti VEGF and anti 偽 -Gal, and the purity of purified recombinant protein was about 90%. 3. The results of cell adhesion test and serum cytotoxicity test showed that the purified recombinant protein could not only adhere to A549 cells of VEGFR, but also had a significant cytolytic effect on A549 cells by 偽 -Gal mimic epitope with the participation of human fresh serum. Conclusion:. 1. The prokaryotic expression plasmid pQE30-VEGF- 偽 -Galal was successfully constructed, and the recombinant VEGF- 偽 -Gal fusion protein was successfully expressed and purified. 2.Recombinant VEGF- 偽 -Gal fusion protein not only has VEGFR targeting ability, but also has 偽 -Gal epitope function, which lays a foundation for the research and development of novel targeted tumor biological agents.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392-33

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本文編號：1527257