超滤技术去除重组MUC1-MBP融合蛋白中内毒素的效果评价

摘要

目的：评价超滤技术去除纯化后重组 MUC1-MBP融合蛋白（MUC1-MBP）溶液中内毒素的效果，阐明超滤技术对去除内毒素的作用。方法：选取表达 MUC1-MBP大肠杆菌基因工程菌，分别通过 CM Sepharose FF离子交换层析（CM）（CM 组）、CM 联合 Phenyl Sepharose 6 FF 疏水层析（C6）（CM＋C6组）、CM 加超滤（CM＋超滤组）以及CM联合C6加超滤（CM＋C6组＋超滤）进行纯化，并去除内毒素；采用 SDS-PAGE 分析蛋白纯度；鲎试剂显色基质法检测内毒素表达水平。结果：SDS-PAGE分析，在预期相对分子质量62000处呈现单一条带， Quantity One分析纯度＞96％；CM组与CM＋C6组内毒素表达水平均较高，但2组比较差异无统计学意义（P＞0.05）；与 CM 组比较， CM＋超滤组内毒素表达水平明显降低（P＜0.01）；与 CM＋C6组比较， CM＋C6超滤组内毒素表达水平明显降低（P＜0.01）；CM＋超滤组与CM＋C6＋超滤组内毒素表达水平比较差异无统计学意义（P＞0.05）。结论：CM或 CM联合 C6均不能有效去除内毒素，其中 C6对去除内毒素几乎无作用；CM加超滤可有效去除内毒素，超滤技术在去除内毒素中发挥重要作用。%Objective To evaluate the effectiveness of ultrafiltration technology in endotoxin removal from purified recombinant MUC1-MBP fusion protein (MUC1-MBP)and to demonstrate the effect of ultrafiltration on endotoxin removal.Methods CM Sepharose FF weak cation exchange (CM)(CM group), CM combined with Phenyl Sepharose 6 FF exchange (C6)(CM+C6 group),CM combined with ultrafiltration (CM+ultrafiltration group), and CM combined with C6 and ultrafiltration (CM+C6+ultrafiltration group)were used to purify the MUC1-MBP from E.coli. and remove endotoxin;the expression level of endotoxin was detected by Chromogenic End-point Tachypleus Amebocyte Lysate.Results There was a single band at the expected molecular weight of 62 000 by SDS-PAGE analysis.and the purity>96% by Quantity One analysis.The endotoxin levels in CM group and CM +C6 group were quite high and there was no significant difference between two groups (P>0.05 );the endotoxin level in CM+ultrafiltration group was significantly lower than that in CM group, and there was significant difference (P<0.01);the endotoxin level in CM+C6+ultrafiltration group was significantly decreased compared with CM+C6 group (P<0.01);there was no significant differences of endotoxin level between CM+ultrafiltration group and CM +C6+ultrafiltration group (P>0.05 ). Conclusion The effects of CM or CM combined with C6 on endotoxin removal are quite poor, especially C6;CM combined with ultrafiltration are quite effective on endotoxin removal,and ultrafiltration plays an important role in endotoxin removal.