吉林大学学报(医学版)

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超滤技术去除重组MUC1-MBP融合蛋白中内毒素的效果评价

王娟,谢飞,陈潭琇,孙霞霞,李琼书,台桂香   

  1. 吉林大学基础医学院免疫学教研室,吉林 长春 130021
  • 收稿日期:2013-08-23 出版日期:2014-05-28 发布日期:2014-06-05
  • 通讯作者: 台桂香(Tel:0431-85619471,E-mail:taiguixiang@163.com) E-mail:taiguixiang@163.com
  • 作者简介:王 娟(1988-),女,山东省临沂市人,在读医学硕士,主要从事肿瘤免疫学研究。
  • 基金资助:

    科技部“十二五”重大新药创制专项资助课题(2011ZX09102001-36)

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

WANG Juan,XIE Fei,CHEN Tan-xiu,SUN Xia-xia,LI Qiong-shu,TAI Gui-xiang    

  1. Department of Immunology,School of Basic Medical Sciences,Jilin University,Changchun 130021,China
  • Received:2013-08-23 Online:2014-05-28 Published:2014-06-05

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。

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

Abstract:

bstract: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.

Key words: MUC1-MBP fusion protein, ion exchange chromatography, thin layer chromatography, ultrafiltration technology, endotoxin, chromogenic end-point tachypleus amebocyte lysate

中图分类号: 

  • R392.11