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微丝相关蛋白hHBRK1突变体的构建、表达及纯化 被引量:1

Construction,expression and purification of F-actin related protein hHBRK1 mutants
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摘要 目的:构建微丝相关蛋白hHBRK1截短体和突变体原核表达载体,并表达及纯化融合蛋白。方法:采用常规PCR并结合定点突变技术,对hHB rk1基因进行缺失和点突变;利用限制性内切酶将PCR产物克隆至原核表达载体pGEX-4T;IPTG诱导融合蛋白表达,通过谷胱甘肽-琼脂糖纯化技术分离纯化GST融合蛋白。结果与结论:构建了包括氨基端缺失截短体hHBRK1-ΔN、羧基端缺失截短体hHBRK1-ΔC和点突变蛋白hHBRK1-S56G57在内的原核表达质粒,分离获得较高纯度的重组hHBRK1突变体融合蛋白,W estern杂交证实纯化蛋白为GST融合蛋白。本实验为进一步研究hHBRK1的相互作用蛋白及其可能的结合位点提供了基础。 Objective :To construct, express and purify the mutant and the truncated hHBRK1 proteins. Methods: Polymerase chain reaction (PCR) and the site-directed mutagenesis technique were used for deletion and the site mutation of hHBrk1 gene. PCR products were cloned into pGEX-4T vector respectively with restrictive endonuclease. Recombination GST fusion proteins were induced with IPTG and these mutant and truncated hHBRK1 proteins were purified by glutathion Sepharose 4B affinity chromatography. Western blot was used to identify the recombination GST fusion proteins. Results and conclusion: Recombinant plasmids including hHBRK1-AN, hHBRK1-AC and hHBRK1-S56 Gs7 were constructed. These mutants and truncated hHBRK1 proteins were induced by 0.8 mmol/L IPTG, purified from supernatant of bacterial lysis and further identified with Western blot. These mutants and truncated hHBRK1 proteins would be useful in studying the interaction proteins and the function domains of hHBRK1.
出处 《军事医学科学院院刊》 CSCD 北大核心 2005年第6期528-530,553,共4页 Bulletin of the Academy of Military Medical Sciences
基金 北京市自然科学基金项目(5042023)
关键词 hHBRK1 肌动蛋白类 基因突变 融合蛋白 微丝蛋白质类 聚合酶链反应 hHBRK1 F-actins gene mutation fusion protein microfilament proteins polymerase chain reaction
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参考文献9

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共引文献7

同被引文献17

  • 1赵非,黄松明,张爱华,费莉,郭梅,陈荣华.CD2AP、F-actin在肾病大鼠中的表达及意义[J].中国病理生理杂志,2006,22(1):182-186. 被引量:7
  • 2徐勤枝,丁新民,颜贤忠,霍艳英,隋建丽,白贝,吴德昌,周平坤.人微丝相关蛋白hHBRK1突变体的亚细胞定位[J].中国生物化学与分子生物学报,2007,23(4):292-297. 被引量:3
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