IGFBP-3抗原的真核表达分析及校准品制备评估

Eukaryotic expression analysis and calibration materials evaluation of IGFBP-3 antigen

目的:观察IGFBP-3抗原在不同真核细胞中的表达差异,筛选最优的IGFBP-3抗原进行IGFBP-3抗原诊断试剂盒开发的校准品性能分析。方法:将人IGFBP-3基因分别克隆至毕赤酵母表达载体pPIC9K及哺乳动物细胞表达载体pCMV中,分别转化毕赤酵母GS115、HEK293细胞及CHO细胞进行表达。以Ni-NTA和SDS-PAGE技术对表达产物进行活性分析及纯化鉴定,利用IGFBP-3检测试剂盒对纯化抗原进行校准品性能分析。结果:IGFBP-3抗原表达过程中易降解,仅在HEK293细胞中成功得到全长蛋白,且配制成的校准品考核反应性及稳定性均满足需求,为诊断试剂盒的进一步开发奠定了坚实基础。结论:HEK293体系表达的IGFBP-3抗原更适用于试剂盒校准品。

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 products 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.