人ARPC2基因的克隆与表达

摘要/Abstract

摘要： 目的构建人His-ARPC2融合蛋白表达载体,并在原核细胞中进行表达与纯化,以研究其功能及鉴定与其相互作用的蛋白。方法采用PCR方法从人肝cDNA文库扩增ARPC2基因编码区,使用常规酶切、连接方法将其重组至pET-14b载体中。对阳性克隆进行酶切、PCR和测序鉴定。转化BL21(DE3)大肠杆菌株,用异丙基β-D硫代半乳糖(IPTG)诱导融合蛋白表达,并利用Ni-NTA亲和层析纯化该融合蛋白。结果所构建的人ARPC2的His融合蛋白表达载体是正确的,该载体可在大肠杆菌内高效表达,用Ni-NTA纯化获得了相对分子质量约36 000的融合蛋白。结论成功构建了人His-ARPC2融合蛋白表达载体,并在原核细胞中获得了高效表达和纯化,为进一步研究ARPC2的生理功能提供了重要的实验材料。

Abstract: Objective To construct the expression vector of His-ARPC2 fusion protein and obtain its expression and purifica-tion in E.coli. Methods ARPC2 cDNA codon region was amplified by PCR from human liver cDNA library and cloned into pET-14b vector following the routine procedures. After identification by enzyme digestion, PCR and sequencing, the positive clones were transformed into BL21 (DE3) competent cells, and the expression of His-ARPC2 fusion protein was induced with IPTG and further purified by Ni-NTA affinity chromatography. Results The constructed His-ARPC2 fusion protein vector was highly efficiently expressed in E.coli. With Ni-NTA affinity chromatography, a purified His fusion protein with relative molecular mass of approximately 36 000 was obtained. Conclusion The expression vector of His-ARPC2 fusion protein is constructed, expressed and purified under non-denaturing conditions, which may significantly facilitate future study of the physiological functions of ARPC2 and characterization of its interaction proteins.

中图分类号:

龚小卫, 彭毅, 刘靖华, 李志杰, 邓鹏, 秦清和, 姜勇. 人ARPC2基因的克隆与表达[J]. 南方医科大学学报, 2004, 24(06): 628-630,635.

GONG Xiao-wei, PENG Yi, LIU Jing-hua, LI Zhi-jie, DENG Peng, QIN Qing-he, JIANG Yong. Construction and expression of the fusion vector of His-tagged human ARPC2 gene[J]. Journal of Southern Medical University, 2004, 24(06): 628-630,635.

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