Construction, Expression And Refolding Of FeSOD-ScFv And Its Co-effect On Lung Adenocarcinoma Tumor With Retinoic Acid

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.