人心肌肌酸激酶MB同工酶的原核表达及纯化  

Prokaryotic expression and purification of humn creatine kinase-MB isoenzyme

作　　者：李子颖 贾娟娟[2] 刘一兵 韩世泉[3] 

机构地区：[1]原子高科股份有限公司医学二部,北京102413 [2]中国食品药品检定研究院化学药品检定所抗肿瘤与放射性药品室,北京100050 [3]中国原子能科学研究院同位素研究所,北京102413

出　　处：《中国生物制品学杂志》2013年第7期931-934,共4页Chinese Journal of Biologicals

摘　　要：目的原核表达并纯化人心肌肌酸激酶MB同工酶(creatine kinase MB,CK-MB)。方法分别从含CK-M、CK-B基因片段的质粒SC319293和SC119007中扩增CK-M、CK-B基因,构建重组原核表达质粒pGEX-4T-1-CK-MB,转化大肠埃希菌BL21(DE3),IPTG诱导表达重组蛋白,经GST亲和层析柱纯化后,ELISA法检测其抗原性。结果重组表达质粒pGEX-4T-1-CK-MB经菌落PCR、双酶切及测序鉴定,证明构建正确;表达的重组CK-MB蛋白相对分子质量约110 000,表达量占菌体总蛋白的57%,主要以包涵体形式存在;纯化的CK-MB蛋白纯度大于90%,可与兔抗CK-B、CK-M多克隆抗体特异性结合。结论成功原核表达并纯化了重组CK-MB蛋白,为其大量生产奠定了基础。Objective To express human creatine kinase MB isoenzyme（CK-MB） in prokaryotic cells and purify the expressed product.Methods CK-M and CK-B genes were amplified from plasmids SC319293 and SC119007 respectively and inserted into prokaryotic expression vector pGEX-4T-1 with GST tag.The constructed recombinant plasmid pGEX-4T-1-CK-MB was transformed to E coli.BL21（DE3） for expression under induction of IPTG.The expressed fusion protein was purified by GST affinity chromatography and determined for antigenicity by ELISA.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pGEX-4T-1-CKMB was constructed correctly.The expressed recombinant fusion protein,with a relative molecular mass of about 110 000,contained 57% of total somatic protein,mainly existed in a form of inclusion body,reached a purity of more than 90%,and showed specific binding to rabbit polycolonal antibodies against CK-M and CK-B.Conclusion Recombinant CK-MB protein was successfully expressed in prokaryotic cells and purified,which laid a foundation of large-scale production of the protein.

关 键 词：心肌 肌酸激酶 同工酶 原核细胞 基因表达 纯化 

分 类 号：R446.112[医药卫生—诊断学] Q786[医药卫生—临床医学]