超滤技术去除重组MUC1-MBP融合蛋白中内毒素的效果评价  

Evaluation on effect of ultrafiltration technology on endotoxin removal from recombinant MUC1-MBP fusion protein

作　　者：王娟[1] 谢飞[1] 陈潭琇 孙霞霞[1] 李琼书[1] 台桂香[1] 

机构地区：[1]吉林大学基础医学院免疫学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2014年第3期539-542,共4页Journal of Jilin University：Medicine Edition

基　　金：科技部"十二五"重大新药创制专项资助课题(2011ZX09102001-36)

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 6FF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62 000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.

关 键 词：MUC1-MBP融合蛋白 离子交换层析 疏水层析 超滤技术 内毒素 显色基质法鲎试剂 

分 类 号：R392.11[医药卫生—免疫学]