| 大豆球蛋白A1a酸性多肽的原核表达及纯化 |
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| 引用本文： | 刘宾,滕达,杨雅麟,王建华.大豆球蛋白A1a酸性多肽的原核表达及纯化[J].食品科学,2011,32(17):278-282. |
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| 作者姓名： | 刘宾 滕达 杨雅麟 王建华 |
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| 作者单位： | 1.农业部饲料生物技术重点开放实验室
2.中国农业科学院饲料研究所基因工程研究室 |
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| 基金项目： | “十一五”国家科技支撑计划项目(2006BAD12B04-06); 中国农业科学院基本科研业务费专项(2009-2010) |
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| 摘 要： | 为建立大豆球蛋白免疫检测方法及致敏机理的深入研究提供基础材料，人工合成了大豆球蛋白A1a酸性多肽基因的cDNA序列，通过聚合酶链式反应(PCR)和Bam HI、Kpn I双酶切构建了原核表达载体pET30a-A1a，将重组质粒转化大肠杆菌(E.coli)BL21后用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达。SDS-PAGE分析表明，A1a酸性多肽融合蛋白能以可溶蛋白形式进行分泌表达，并且诱导5h后，A1a酸性多肽融合蛋白表达量达到最高。经His亲和层析纯化，获得纯度达90%的融合A1a蛋白，分子质量大小约为38kD。Western blotting显示，所获得的A1a融合蛋白能与对豆粕过敏的仔猪血清发生免疫反应，表明所获得融合蛋白具有免疫活性。
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| 关 键 词： | 大豆球蛋白 A1a酸性多肽 原核表达 B细胞表位 |
Prokaryotic Expression and Purification of A1a Acidic Peptide from Glycinin |
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| LIU Bin,TENG Da,YANG Ya-lin,WANG Jian-hua.Prokaryotic Expression and Purification of A1a Acidic Peptide from Glycinin[J].Food Science,2011,32(17):278-282. |
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| Authors: | LIU Bin TENG Da YANG Ya-lin WANG Jian-hua |
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| Affiliation: | (1.Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing 100081, China；
2. Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China) |
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| Abstract: | The cDNA sequence of full-length A1a acidic peptide from glycinin was synthesized and inserted into the prokaryotic expression vector pET-30a.The A1a fusion protein was expressed in Escherichia coli BL21(DE3) with the induction of IPTG.The highest expression level was observed after IPTG induction for 5 h.The recombinant protein was purified by His-tag affinity chromatography.The purity of Ala fusion protein was 90% and its molecular weight was approximately 38 kD.Western blotting analysis revealed that the... |
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| Keywords: | glycinin A1a acidic peptide prokaryotic expression Western blotting |
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