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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,5μm),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 map analysis method of recombinant human B lymphocyte stimulating factor receptor-antibody fusion protein (TACI-Fc). METHODS: The TACI-Fc protein was enzymatically digested with trypsin, denatured with urea, the disulfide bond was blocked with dithiothreitol, the thiol group was blocked with iodoacetamide, enzymatic hydrolysis with trypsin, High performance liquid chromatography (HPLC) was performed on a Vydac C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid and the mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid , Gradient elution at a flow rate of 1.0 mL.min-1 with a detection wavelength of 214 nm. Results: The TACI-Fc protein was effectively digested, and the digested fragments could be well separated. Conclusion: This method has high accuracy and good repeatability, and is an effective method to confirm the structure of recombinant human B lymphocyte stimulating factor receptor - antibody fusion protein.