目的:采用高效液相色谱法(HPLC)建立雪莲培养物指纹图谱。方法:以紫丁香苷为参照物,采用梯度洗脱HPLC法建立雪莲培养物指纹图谱,并进行相似度评价和聚类分析。色谱柱为CAPCELL PAK C18(4.6 mm×250mm,5μm),流动相A为0.1%TFA/水,流动相B为0.1%TFA/乙腈,梯度洗脱,检测波长260 nm。结果:建立了雪莲培养物指纹图谱,方法的精密度、稳定性、重现性较好,共标示出9个共有峰。结论:该法为雪莲培养物的鉴定提供了依据,其指纹图谱可用于雪莲培养物的质量控制。
The only difference of primary structure between single-chain prourokinase (pro-UK or scu-PA) and two-chain urokinase (UK or tcu-PA) is the cleavage of a single peptide bond (Lys158-Ile159) and transform scu-PA into its active two-chain form. A 13-peptide (Thr-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile-Gly- Gly-Glu-Cys), which spans the cleavage peptide bond, was synthesized and linked to KLH (Keyhole limpet hemocyanin). The Balb/c mice were immunized by the conjugated protein with proper adjuvant. According to the Kohler and Milstein’s methods, a hybridoma cell line G7 secreting monoclonal antibody specific for scu-PA was obtained. The anti-scu-PA McAb, purified from the supernatant of porous microcarrier hybridoma cell culture, was conjugated to CNBr-activated Sepharose 4B to prepare an immuno-affinity chromatography column. The u-PA was purified only by this affinity column from the supernatant of cultivating the u-PA-producing recombinant CHO cell, the u-PA recovery ratio is 90.4%, the purification factor was about 50, with the specific activity of 1.2×10 5IU/mg, the scu-PA ratio in the u-PA product was 96.3%. Compared to immuno-affinity chromatography, the 3-step process for purifying u-PA (cation-exchange co-lumn, gel filtration column and benzamidine affinity column)has a u-PA recovery ratio of about 65%, with a specific activity of 1.0×10 5IU/mg, and an scu-PA ratio of about 90%. These results showed that immuno-affinity chromatography is simple to recover u-PA and effective to separate scu-PA from tcu-PA.
