靶向性抗肿瘤VEGF-α-Gal融合蛋白的原核表达及其体外活性

发布时间：2018-02-23 19:04

本文关键词： 肿瘤靶向性 VEGF α-Gal 抗肿瘤活性　出处：《重庆医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的: 为了将肺癌的靶向治疗和免疫治疗相结合,本研究扩增了VEGF-α-Gal融合基因,构建原核表达质粒pQE30-VEGF-α-Gal,在大肠杆菌M15内诱导表达rVEGF-α-Gal融合蛋白,并在体外细胞水平上研究重组蛋白的肺癌细胞靶向性和α-Gal表位介导的肿瘤细胞杀伤效应,为探索肺癌及其他肿瘤的新型靶向生物治疗途径奠定基础。方法: 1.体外培养A549细胞,提取总RNA,以RT-PCR法扩增VEGF-α-Gal基因片段作为目的基因,双酶切后定向克隆入pQE30质粒中,构建并鉴定pQE30-VEGF-α-Gal原核表达质粒。 2.将pQE30-VEGF-α-Gal质粒转化进E.coli M15菌,用IPTG诱导目的蛋白表达,用SDS-PAGE法分析重组蛋白的相对分子质量及表达形式,用Western-blot法分析重组蛋白的抗原性,用Ni-Agarose His标签蛋白纯化试剂盒纯化重组VEGF-α-Gal融合蛋白,然后采取逐步降低尿素浓度的方法使重组蛋白复性。 3.通过细胞黏附试验评价重组蛋白中VEGF对肿瘤细胞A549的靶向性,通过血清杀伤试验分析重组蛋白中α-Gal模拟表位介导的体外溶肿瘤细胞效应。结果: 1.经双酶切鉴定及DNA序列测定,证实pQE30-VEGF-α-Gal原核表达质粒构建成功。 2.将pQE30-VEGF-α-Gal质粒成功转化进E.coli M15菌,并实现目的蛋白的诱导表达。经SDS-PAGE法分析,目的蛋白分子量为18KDa,主要以包涵体形式为主。Western-blot结果表明,重组蛋白能分别与抗VEGF、抗α-Gal特异性结合。纯化的重组蛋白纯度约为90%。 3.细胞黏附试验和血清杀伤试验结果证实,纯化复性后的重组蛋白不但能黏附VEGFR+的A549细胞,而且其α-Gal模拟表位在人新鲜血清的参与下,对A549细胞产生了明显的溶细胞效应。结论: 1.成功构建了原核表达重组质粒pQE30-VEGF-α-Gal,并成功表达及纯化了重组VEGF-α-Gal融合蛋白,为探索肿瘤生物治疗新途径奠定了基础。 2.重组VEGF-α-Gal融合蛋白不但具有VEGFR靶向性,还兼具α-Gal表位功能,为研究和开发新型靶向肿瘤生物制剂奠定了基础。
[Abstract]:Objective:. In order to combine the targeted therapy with immunotherapy for lung cancer, the VEGF- 伪 -Gal fusion gene was amplified and the prokaryotic expression plasmid pQE30-VEGF- 伪 -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 伪 -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- 伪 -Gal gene fragment was amplified by RT-PCR as the target gene, and then cloned into pQE30 plasmid. The pQE30-VEGF- 伪 -Gal prokaryotic expression plasmid was constructed and identified. 2. The pQE30-VEGF- 伪 -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- 伪 -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 伪 -Gal mimic epitope on tumor cell lysis in vitro was analyzed by serum cytotoxicity test. Results:. 1. The construction of pQE30-VEGF- 伪 -Gal prokaryotic expression plasmid was confirmed by double enzyme digestion and DNA sequencing. 2. The plasmid pQE30-VEGF- 伪 -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 伪 -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 伪 -Gal mimic epitope with the participation of human fresh serum. Conclusion:. 1. The prokaryotic expression plasmid pQE30-VEGF- 伪 -Galal was successfully constructed, and the recombinant VEGF- 伪 -Gal fusion protein was successfully expressed and purified. 2.Recombinant VEGF- 伪 -Gal fusion protein not only has VEGFR targeting ability, but also has 伪 -Gal epitope function, which lays a foundation for the research and development of novel targeted tumor biological agents.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392-33

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