人心肌肌酸激酶MB同工酶的原核表达及纯化 |
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引用本文: | 李子颖,贾娟娟,刘一兵,韩世泉.人心肌肌酸激酶MB同工酶的原核表达及纯化[J].粉末涂料与涂装,2013,26(7):931-934. |
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作者姓名: | 李子颖 贾娟娟 刘一兵 韩世泉 |
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作者单位: | 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 |
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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。
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关 键 词: | 心肌 肌酸激酶 同工酶 原核细胞 基因表达 纯化 |
Prokaryotic expression and purification of humn creatine kinase-MB isoenzyme |
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Abstract: | 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. |
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Keywords: | Cardiac muscle Creatine kinase Isoenzyme Prokaryotic cells Gene expression Purification |
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