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钠离子依赖的中性氨基酸转运蛋白2(SNAT2)多克隆抗体的制备
刘丽1, 闵伟勇1, 张舟2
1.上海师范大学;2.沈阳药科大学
摘要:
钠离子依赖的中性氨基酸转运蛋白2(SNAT2)属于SLC38家族,参与小的中性氨基酸跨膜转运,在哺乳动物组织中广泛表达.SNAT2的功能紊乱可以导致许多神经性疾病,如阿尔茨海默症、帕金森症等.采用PCR方法扩增得到SNAT2氨基端75个氨基酸的编码序列,构建重组质粒pET28a-SNAT2-75aa,并将其转入大肠杆菌BL21(DE3)中,通过IPTG诱导表达重组蛋白.利用组氨酸标签蛋白经镍柱亲和纯化后得到了纯度在95%以上的SNAT2氨基端蛋白SNAT2-75aa,将其作为免疫原免疫新西兰大白兔,从而获得 SNAT2多克隆抗体.采用巯丙基琼脂糖凝胶6B (Thiopropyl sepharose ) 固定抗原亲和纯化法进一步对该抗体进行纯化,纯化后的抗体效价至少提高10倍.蛋白免疫印迹确定该抗体对SNAT2具有高度的特异性,表明SNAT2多克隆抗体制备的成功,为进一步研究SNAT2蛋白的结构和功能奠定了生化基础.
关键词:  转运蛋白  中性氨基酸  抗体  蛋白纯化
DOI:
分类号:
基金项目:国家自然科学基金项目(31270883);上海市教委科研创新项目(13ZZ103)
Preparation of polyclonal antibody of sodium dependent neutral amino acid transporter-2 (SNAT2)
LIU Li1, MIN Weiyong1, ZHANG Zhou2
1.College of Life and Environment Sciences,Shanghai Normal University;2.School of Life Science and Biopharmaceutics,Shenyang Pharmaceutical University
Abstract:
Sodium-dependent neutral amino acid transporter 2 (SNAT2),transport of small neutral amino acids,is the second member of the SLC38 family and widely expresses in mammals.SNAT2 dysfunction can lead to many neurological diseases,such as Alzheimer disease and Parkinson's disease.In order to prepare SNAT2 special antibody,the gene of 75 amino acids of the N terminal of SNAT2 was amplied by PCR from pBK-CMVΔ-SNAT2-HA plasmid.The expression plasmid pET28a-SNAT2-75aa was constructed and transformed into BL21 (DE3).SNAT2-75aa protein was induced to express by IPTG.After purification by Ni-column,the recombinant protein SNAT2-75aa was used to immunize New Zealand white rabbit in order to obtain SNAT2 polyclonal antibody.In order to further purify the antibody,Thiopropyl sepharose 6B column was used.Western blotting results show that the purified antibody can be used specially to detect SNAT2 transient expression in HEK293 cells.Successful preparation of SNAT2 polyclonal antibody will be benefit to further studying structure and function of SNAT2.
Key words:  transporter  neutral amino acid  antibody  protein purification