基础医学与临床 ›› 2008, Vol. 28 ›› Issue (11): 1192-1196.

• 技术与方法 • 上一篇    下一篇

人Polycomb家族成员NSPc1蛋白的表达纯化及多克隆抗体制备

龚燕华 王旭(北京) 吴旭东 强伯勤 彭小忠 袁建刚   

  1. 中国医学科学院基础医学研究所 北京协和医学院 医学分子生物学国家重点实验室 中国医学科学院基础医学研究所 北京协和医学院 医学分子生物学国家重点实验室 中国医学科学院基础医学研究所 北京协和医学院 医学分子生物学国家重点实验室 中国医学科学院基础医学研究所 北京协和医学院 医学分子生物学国家重点实验室 中国医学科学院基础医学研究所 北京协和医学院 医学分子生物学国家重点实验室 中国医学科学院基础医学研究所 北京协和医学院 医学分子生物学国家重点实验室
  • 收稿日期:2007-12-28 修回日期:2008-02-28 出版日期:2008-11-25 发布日期:2008-11-25
  • 通讯作者: 袁建刚

Prokaryotic Expression of Human nervous system polycomb 1(NSPc1) and Preparation of Anti-NSPc1 polyclonal Antibody

Yan-hua GONG, Xu WANG, Xu-dong WU, Bo-qin QIANG, Xiao-zhong PENG, Jian-gang YUAN   

  1. National Laboratory of Medical Molecular Biology, IBMS, CAMS&PUMC National Laboratory of Medical Molecular Biology, IBMS, CAMS&PUMC National Laboratory of Medical Molecular Biology, IBMS, CAMS&PUMC National Laboratory of Medical Molecular Biology, IBMS, CAMS&PUMC National Laboratory of Medical Molecular Biology, IBMS, CAMS&PUMC National Laboratory of Medical Molecular Biology, IBMS, CAMS&PUMC
  • Received:2007-12-28 Revised:2008-02-28 Online:2008-11-25 Published:2008-11-25
  • Contact: Jian-gang YUAN

摘要: 目的 Polycomb家族成员NSPc1多克隆抗体的制备与检测。方法 应用PCR技术扩增人NSPc1编码区全长序列并插入到pET-43.1a(+)载体中,在大肠杆菌BL21中表达融合蛋白HIS-NSPc1。利用所表达的融合蛋白中含有的6×His 标签进行亲和纯化。用所获得的纯化蛋白免疫新西兰大白兔,获得兔抗人NSPc1多克隆抗体。用Western blot检测抗体免疫反应的特异性。结果 在大肠杆菌中表达并纯化了人NSPc1重组蛋白,纯度达95%,用其免疫新西兰大白兔,成功制备了相应兔源多克隆抗体,Western blot实验中该抗体可以特异识别内源及外源性NSPc1蛋白。结论 原核表达的NSPc1融合蛋白可以被纯化,并具有足够的蛋白免疫原性,所制备的相应多克隆抗体具有良好的特异性,可以运用于NSPc1基因的功能研究。

Abstract: Objective To express the human recombinant NSPc1 protein and prepare specific polyclonal antibody against it. Methods The recombinant expression plasmid pET43.1a-HIS-NSPc1 was made and transformed into E.coli.(BL21), and then the recombinant fusion protein HIS-NSPc1 was expressed and purified. Four New Zealand rabbits were immuniged with purified recombinant HIS-NSPc1 protein as antigen and polyclonal antibodies against NSPc1 were prepared. The specificity of the anti-sera were analyzed by Western Blot. Results The purity of the recombinant NSPc1 protein is up to about 95%. Rabbit against NSPc1 antibodies were obtained. Western blot showed that the antibodies were able to detect both endogenous and/or exogenous NSPc1. Conclusion The NSPc1 polyclonal antibodies prepared by using recombinant NSPc1 protein as antigen has better specificity to NSPc1.It can be used for functional analysis of NSPc1 in vivo and in vitro.