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人丙氨酸氨基转移酶基因(ALT1)的克隆、表达、纯化及酶活性的鉴定 被引量:1

Cloning,expression,purification and determination of enzymatic activity of homan alanine aminotransferase(ALT1)
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摘要 目的克隆、表达、纯化重组人丙氨酸氨基转移酶(ALT1),并测定该酶活性,为建立特异性的ALT分型检测方法奠定基础。方法通过RT-PCR从肝癌细胞中扩增丙氨酸氨基转移酶(ALT1)基因,并将其克隆至pET-28 a表达载体中。重组表达质粒pET28 a-ALT1,转化大肠杆菌BL21,经1.0mmol.L-1IPTG诱导,表达可溶性重组融合蛋白,通过硫酸铵沉淀、镍离子柱亲和色谱及QHP柱色谱3步纯化,并检测融合蛋白活性。结果经过表达条件的优化增加了可溶性蛋白的表达,纯化后目的蛋白纯度达到85%,经测定重组蛋白具有很高的丙氨酸氨基转移酶活性(200 U.mg-1)。结论通过基因克隆及大肠杆菌表达,能得到活性较高的丙氨酸氨基转移酶蛋白。 Objective To clone,express and purify human alanine aminotransferase(ALT1)and determine its activity.To lay the basis for the establishment of a sub-type-specific detection method of ALT.Methods The gene encoded alanine aminotransferase 1(ALT1)was cloned from hepatoma cell by RT-PCR,and then inserted into pET28a vector.Recombination plasmids(pET28a-ALT1)were transformed into Escherichia coli BL21.The recombinant strains induced by IPTG(1.0 mmol·L-1)expressed the soluble fusion protein.Through these steps including(NH4)2SO4 precipitating,Ni Sepharose high performance chromatography and Q Sepharose high performance chromatography,the soluble fusion protein was purified and the activity of purified protein was detected.Results After modifying the expression conditions,the quantity of soluble recombinant protein was increased.Through purification,the purity of interest protein was about 85% and the recombinant protein performed high enzymatic activity(200 U·mg-1).Conclusions High enzymatic activity fusion protein of alanine aminotransferase is made by gene cloning and expressing in Escherichia coli.
出处 《沈阳药科大学学报》 CAS CSCD 北大核心 2010年第6期489-494,共6页 Journal of Shenyang Pharmaceutical University
基金 国家科技支撑计划课题资助项目(2007BAI07A22)
关键词 丙氨酸氨基转移酶 克隆 融合蛋白 alanine aminotransferase clone fusion protein
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