重组人B淋巴细胞刺激因子受体－抗体融合蛋白的肽图分析

目的: 建立重组人B淋巴细胞刺激因子受体－抗体融合蛋白(TACI-Fc)的肽图分析方法。 方法: 将TACI-Fc蛋白用胰蛋白酶酶解、用尿素将蛋白分子变性、用二硫苏糖醇打断二硫键、用碘乙酰胺封闭巯基、用胰蛋白酶再次酶解等方法处理后,采用高效液相色谱分析,色谱柱为Vydac C₁₈(250 mm×4.6 mm,5 μm),流动相A液为含0.1%三氟乙酸的水溶液,流动相B液为含0.1%三氟乙酸的乙腈溶液,梯度洗脱,流速为1.0 mL· min^-1,检测波长为214 nm。 结果: TACI-Fc蛋白被有效酶解,酶解后的各个片段能够很好的分离。 结论: 本法准确性高,重复性好,是重组人B淋巴细胞刺激因子受体－抗体融合蛋白结构确认的有效方法。

Objective: To establish a peptide mapping analysis method in recombinant human B lymphocyte stimulator receptor:IgG Fc fusion protein. Method: The TACI-Fc protein was digested with trypsin and then was denatured by urea.Next,the disulfide bonds were interrupted by dithiothreitol and the thiol groups were closed with iodoacetamide.Finally,the treated TACI-Fc protein was digested with trypsin again and assayed by HPLC.Vydac C₁₈(250 mm×4.6 mm,5 μm) was used as analysis column,0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile were used as mobile phase A and mobile phase B,respectively.The gradient elution process was performed.The detection wavelength was 214 nm and flow rate was 1.0 mL·min^-1. Results: The fragments of TACI-Fc were well digested and separated. Conclusion: This method is accurate,stable,and reliable for testing of human B lymphocyte stimulator receptor:IgG Fc fusion protein peptide mapping.