Eukaryotic expression analysis and calibration materials evaluation of IGFBP-3 antigen
Objective:To observe the expression differences of IGFBP-3 antigen in different eukaryotic cells,and the optimal antigen was screened for calibration materials evaluation to develop IGFBP-3 antigen diagnostic kit.Methods:Human IGFBP-3 gene was respectively cloned into Pichia pastoris expression vector pPIC9K and mammalian expression vector pCMV,and then transformed into Pichia pastoris GS115,HEK293 cell and CHO cell for gene expression.Activity analysis and purification of the expressed prod-ucts were performed with Ni-NTA and SDS-PAGE,and calibrating performance of the purified antigen was analyzed with IGFBP-3 test kit.Results:IGFBP-3 antigen was easily degradable during the expression process,and only the HEK293 cells enabled to obtain the full-length antigen,and reactivity and stability of the calibration products using antigen expressed in HEK293 cells could meet the calibration material requirements for diagnostic kits,laying a solid foundation for further development of IGFBP-3 diagnostic kits.Conclusion:IGFBP-3 expressed by HEK293 cells is more suitable for calibration materials preparation.
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