Construction of the eukaryotic expression vector of mouse 24p3 gene and its expression in NIH3T3 cell lines / 第三军医大学学报

ABSTRACT

Objective To construct the eukaryotic expression vector of mouse 24p3 gene and express it in NIH3T3 fibroblast cell line and verify its biological function of delivering iron into cells. Methods 24p3cDNA was cloned by RT-PCR and the eukaryotic expression vector of mouse 24p3 gene was constructed by gene recombination technique and the recombinant plasmid was verified by restriction enzyme digestion analysis and sequenceing, then transfected into NIH3T3 cells using DOTAP transfection reagent. Then the positive clones were selected and amplified. The expression of 24p3 gene in the culture supernatant of positive clones was analyzed by ELISA and Western blotting and its biological activity of delivering iron into cells was tested by atomic absorption spectrometer. Results The eukaryotic expression vector was constructed successfully. The NIH3T3 cells that stably expressed 24p3 were screened out. 24p3/SIP24 protein could express in the culture supernatant of positive clones and deliver iron into NIH3T3 cells. Conclusion The 24p3 gene was expressed successfully in the transfected NIH3T3 cells with good biological activity, which laid the foundation for further studying its biological function and mechanism involved in the iron metabolism.