| 人UNC5CL原核蛋白的表达及多克隆抗体的制备和鉴定 |
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| 引用本文： | 吕丹,钱晓萍,田晓军,张毓,张君.人UNC5CL原核蛋白的表达及多克隆抗体的制备和鉴定[J].细胞与分子免疫学杂志,2010,26(3). |
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| 作者姓名： | 吕丹 钱晓萍 田晓军 张毓 张君 |
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| 作者单位： | 1. 北京大学,医学部免疫学系T细胞室,北京,100191
2. 北京大学,第三医院泌尿外科,北京,100191 |
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| 基金项目： | 国家自然科学基金资助项目，教育部留学回国人员科研启动基金资助项目 |
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| 摘 要： | 目的:构建UNC5CL基因的原核表达载体,纯化GST-UNC5CL融合蛋白,并以其为抗原免疫家兔,制备抗人UNC5CL多克隆抗体,并鉴定其反应性和特异性。方法:用PCR方法扩增UNC5CL基因(表达其280-518位氨基酸)片段并克隆至pGEX-4T-2原核表达载体中,转化大肠杆菌BL21诱导融合蛋白GST-UNC5CL(aa 280-518)表达;所获得的可溶性蛋白经亲和层析纯化,免疫家兔制备抗血清,再采用ELISA、Western blot和免疫组化的方法检测抗体的效价及特异性。结果:测序证实原核重组质粒构建成功;经IPTG诱导,可表达Mr为52 000的GST-UNC5CL(aa280-518)的融合蛋白;谷胱甘肽亲和层析柱纯化的融合蛋白免疫家兔制备的抗血清经Western blot和免疫组化检测证实可识别目的蛋白。结论:制备了兔抗人UNC5CL的多克隆抗体,且抗体对内源性UNC5CL具有较好的反应性和特异性,为进一步研究UNC5CL的功能奠定了基础。
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| 关 键 词： | 融合蛋白 多克隆抗体 |
Prokaryotic expression,purification and preparation of polyclonal antibody for human UNC5CL |
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| L Dan,QIAN Xiao-ping,TIAN Xiao-jun,ZHANG Yu,ZHANG Jun.Prokaryotic expression,purification and preparation of polyclonal antibody for human UNC5CL[J].Journal of Cellular and Molecular Immunology,2010,26(3). |
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| Authors: | L Dan QIAN Xiao-ping TIAN Xiao-jun ZHANG Yu ZHANG Jun |
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| Institution: | L(U) Dan,QIAN Xiao-ping,TIAN Xiao-jun,ZHANG Yu,ZHANG Jun |
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| Abstract: | AIM:To construct the prokaryotic expression vector of UNC5CL,purify the fusion protein of GST-UNC5CL,prepare the polyclonal antibody of UNC5CL and characterize the reactivity and specificity of the polyclonal antibody.METHODS:The human UNC5CL nucleotide sequence encoding amino acid from 280 to 518 was amplified and cloned into pGEX-4T-2 vector,then transformed into E.coli BL21,in which the fusion protein GST-UNC5CL(aa280-518) was induced and purified by Glutathione Sepharose-4B.Then the purified protein imm... |
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| Keywords: | UNC5CL UNC5CL fusion protein polyclonal antibody |
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