【作者】 陈福鼎；

【导师】 龚兴国；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 目前,癌症已经是危害人类生命健康的首要疾病。近年来,全世界癌症发病每年超过1000万人,每年死亡约700万人。鉴于此,有效抗癌药物的研究刻不容缓。FeSOD-ScFv(Superoxide dismutase-single chain variable fragment)是一种双功能蛋白,能靶向性作用于肺腺癌细胞,然而FeSOD-ScFv通过胞吞作用进入细胞后,大部分被溶酶体消化,只有少部分发挥作用,这就减弱了FeSOD的抗肿瘤效果。研究表明,视黄酸(Retinoic acid,RA)通过改变内吞小泡的胞内途径,降低内吞小泡被溶酶体消化的概率,增强某些药物的药效。本论文通过定点突变技术将本实验室保存的已突变的FeSOD-ScFv质粒回复突变,然后重新构建表达载体pET-28a-FeSOD-ScFv,转化到大肠杆菌BL21中。构建好的工程菌经正交试验得到最佳表达条件为:37℃、0.5 mmol/L IPTG浓度下诱导表达7小时;SDS-PAGE分析表明目的蛋白以包涵体形式表达,表达的目的蛋白占全菌蛋白总量的53%。包涵体经提取、纯化,通过尿素线性梯度透析复性后,蛋白的比活约为620U/mg,回收率为84.3%。将复性后的FeSOD-ScFv与视黄酸单独及联合作用肺腺癌细胞A549,结果表明FeSOD-ScFv在体外具有显著的抗肿瘤作用,且视黄酸显著提高FeSOD-ScFv对肺腺癌细胞A549的抑制作用,抑制效果提高87%。本研究回复突变fesod-ScFv基因,成功构建pET-28a-FeSOD-ScFv工程菌,在大肠杆菌中高效表达了FeSOD-ScFv双功能蛋白,表达的目的蛋白在体外对肺腺癌细胞A549有抑制效果,视黄酸能够增强这一抑制效果,这为FeSOD-ScFv临床应用于治疗肿瘤提供理论基础,具有重要的社会和经济效益。更多还原

【Abstract】 At present, cancer is harmful to human life and health, and it has been the primary disease. The number of cancer incidences is more than 10 million now, and the number of death is about 700 million each year. In view of this, the study of effective anti-cancer drugs is hardly urgent. Although the FeSOD-ScFv (Superoxide dismutase-single chain variable fragment) can targetedly kill lung adenocarcinoma cells, most of FeSOD-ScFv that enter into cells by endocytosis are digested by lysosomes, and only a small parts can really work, which greatly reduce the anti-tumor efficacy of FeSOD. Some studies showed that retinoic acid (RA) could change the intemalization pathway of endocytic vesicles, which enormously reduced the probability that the lysosome digested endosome and enhanced the efficacy of some drugs.In this study, our lab’s FeSOD-ScFv plasmid that had 4 mutated bases was re-mutated by site-directed mutagenesis technology, and then the plasmid of pET-28a-FeSOD-ScFv was constructed and transformated to E.coli.. Expression conditions was optimized by orthogonal experiments. The optimum expression conditions: at 37℃, 0.5 mmol/L IPTG induce expression for 7 hours. SDS-PAGE analysis showed that the protein expressed as inclusion bodies, and the amount of protein accounted for 53% of the total bacterial protein. The inclusion bodies were extracted and purificated. The specific activity of FeSOD-ScFv was about 620 U/mg, the yield was about 84.3% by continuous urea gradient dialysis refolding. The anti-tumor characterization of FeSOD-ScFv targeted to lung adenocarcinoma A549 cells alone or in combination with RA was studied. The results showed that FeSOD-ScFv had significant anti-tumor effeciency in vitro, and RA could significantly increase the effeciency of FeSOD-ScFv on A549 cells more than 87%.In this study, fesod-ScFv gene was re-mutated, pET-28a-FeSOD-ScFv was successfully constructed. fesod-ScFv gene was highly expressed in E.coli.. FeSOD-ScFv had effeciency on A549 cells in vitro, and RA can enhance the effeciency, which contribute to the clinical application of FeSOD-ScFv and has important social and economic benefits.更多还原