大豆球蛋白A1a酸性多肽原核表达及纯化.pdfVIP

2 78 2011, Vol. 32, No. 17 食品科学 ※生物工程 大豆球蛋白A1a 酸性多肽的原核表达及纯化 1,2 1,2 , 1,2 1,2 , 刘 宾 ，滕 达 * ，杨雅麟 ，王建华 * ( 1.农业部饲料生物技术重点开放实验室，北京 1000 8 1； 2.中国农业科学院饲料研究所基因工程研究室，北京 10008 1) 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A 1a 酸性多 肽基因的cDNA 序列，通过聚合酶链式反应(PCR)和Bam HI 、Kp n I 双酶切构建了原核表达载体pET30a-A 1a ，将 重组质粒转化大肠杆菌(E.coli)BL2 1 后用异丙基硫代- β-D - 半乳糖苷(IPTG)诱导表达。SDS分析表明，A 1a 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，A 1a 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A 1a 蛋白，分子质量大小约为38kD 。Western blotting 显示，所获得的A 1a 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。 关键词：大豆球蛋白；A 1a 酸性多肽；原核表达；Western b lott in g Prokaryotic Expression and Purification of A 1a Acidic Peptide from Glycinin LIU Bin 1,2 ，TENG Da1,2, * ，YANG Ya-lin 1,2 ，WANG Jian-hu a1,2, * (1.Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beij ing 10008 1, China； 2. Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beij ing 100081, China) Abstract：The cDNA sequence of full-length A 1a acidic peptide from glycinin was synthesized and inserted into the prokaryotic expression vector pET-30a. The A 1a fusion protein was expressed in Escherichia coli BL2 1 (DE3) with the induction of IPTG. The highest expression level was ob served 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 recombinant protein had immunologica