包涵体重组蛋白不同纯化方法的比较  

Comparison of various methods for purification of recombinant inclusion body protein

作　　者：常恒祯 常江[1] 战俊澎 杨馨 郭珣 刘益辛 邹德颖[1,2] 任洪林 CHANG Heng-zhen;CHANG Jiang;ZHAN Jun-peng;YANG Xin;GUO Xun;LIU Yi-xin;ZOU De-ying;REN Hong-lin(Key Laboratory of Zoonosis Research,Ministry,of Education,College of Veterinary Medicine,Institute of Zoonosis Jilin University Changchun 130062,Jilin Province,China)

机构地区：[1]吉林大学人兽共患病研究教育部重点实验室/动物医学学院/人兽共患病研究所,吉林长春130062 [2]辽宁省盘锦检验检测中心,辽宁盘锦124000

出　　处：《中国生物制品学杂志》2021年第7期862-867,共6页Chinese Journal of Biologicals

基　　金：国家重点研发计划项目(2018YFD0500900);吉林省科技发展计划项目(20200402059NC)。

摘　　要：目的比较切胶、包涵体复性及Ni-NTA亲和层析3种方法纯化包涵体重组蛋白的效果。方法筛选全长IL-1β与全长IL-1Ra重组质粒(简称IL-1β-1Ra-2重组质粒)及全长IL-1Ra重组质粒的转化感受态菌株[感受态E.coli BL21(DE3)、E.coli BL21(DE3)PLysS及E.coli BL21-Codon Plus(DE3)-RIPL]、IPTG诱导浓度(0.1、0.2、0.4、0.6、0.8、1 mmol/L)、诱导温度(16和37℃)。最佳表达条件诱导的IL-1β-1Ra-2重组质粒,分别用切胶、包涵体复性及Ni-NTA亲和层析法进行纯化,确定最佳纯化方法。采用最佳表达条件分别诱导表达IL-1β-1Ra-2重组质粒及全长IL-1Ra重组质粒后,通过最佳纯化方法进行纯化,纯化产物进行SDS-PAGE及Western blot分析。结果IL-1β-1Ra-2重组质粒及全长IL-1Ra重组质粒最佳转化感受态菌株为E.coli BL21-Codon Plus(DE3)-RIPL,最佳IPTG浓度分别为1和0.4 mmol/L,诱导温度为37℃;最佳纯化方法为包涵体复性纯化法。IL-1β-1Ra-2及全长IL-1Ra重组蛋白包涵体复性纯化产物相对分子质量分别约为10560和19770,纯度可达90%以上,可溶性蛋白回收量分别约为46及63 mg,且均可与小鼠抗His标签单克隆抗体发生特异性结合。结论相同条件下,包涵体复性纯化法获得的可溶性蛋白回收量最大,适用于相对分子质量约10000的非高表达包涵体蛋白的纯化。Objective To compare the purification efficiencies of three methods including gel cutting,inclusion body renaturation and Ni-NTA affinity chromatography for purification of recombinant inclusion body protein.Methods Transformed competent strains[E.coli BL21(DE3),E.coli BL21(DE3)PLysS and E.coli BL21-Codon Plus(DE3)-RIPL]as well as IPTG concentration(0.1,0.2,0.4,0.6,0.8 and 1 mmol/L)and temperature(16 and 37℃)for induction were optimized by using recombinant plasmids with IL-1β-1 Ra-2 and with full-length IL-1 Ra.Recombinant plasmid with IL-1β-1 Ra-2 was induced under the optimal condition,and the expressed product was purified by gel cutting,inclusion body renaturation and Ni-NTA affinity chromatography respectively to optimized the purification method.The recombinant plasmids with IL-1β-1 Ra-2 and with full-length IL-1 Ra were induced under the optimal condition and purified by the optimal method,and the purified products were analyzed by SDS-PAGE and Western blot.Results The optimal transformed competent strain for recombinant plasmids with IL-1β-1 Ra-2 and with full-length IL-1 Ra was E.coli BL21-Codon Plus(DE3)-RIPL,while the optimal IPTG concentration for induction were 1 and 0.4 mmol/L respectively,and the optimal temperature for induction was 37℃.The optimal purification method was renaturation of inclusion body.The relative molecular masses of renaturalized and purified inclusion bodies of IL-1β-1 Ra-2 and full-length IL-1 Ra were about10560 and about 19770 respectively,while the purities were both more than 90%,and the soluble protein recoveries were about 46 mg and about 63 mg respectively,both of which showed specific binding to mouse anti-His-labeled monoclonal antibody.Conclusion Under the same conditions,the recovery of soluble protein obtained by inclusion body renaturation method was the highest,indicating that the method was suitable for the purification of non-highly expressed inclusion body protein with a relative molecular mass of about 10000.

关 键 词：包涵体 纯化 包涵体复性 Ni-NTA亲和层析 切胶 

分 类 号：R373.23[医药卫生—病原生物学] Q75[医药卫生—基础医学]