人Polycomb家族成员NSPc1蛋白的原核表达纯化及多克隆抗体制备  

Prokaryotic expression of human nervous system polycomb 1(NSPc1) and preparation of anti-NSPc1 polyclonal antibody

作　　者：龚燕华[1] 王旭[1] 吴旭东[1] 强伯勤[1] 彭小忠[1] 袁建刚[1] 

机构地区：[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,北京100005

出　　处：《基础医学与临床》2008年第11期1192-1196,共5页Basic and Clinical Medicine

基　　金：国家自然科学基金(30600166;30721063);国家高技术研究发展计划项目(2006AA02Z137)

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。Objective To express the human recombinant NSPcl protein and prepare specific polyclonal antibody against it. Methods The recombinant expression plasmid pET43, la-HIS-NSPcl was constructed and then transformed into E. coll. （ BI21 ） , the recombinant fusion protein HIS-NSPcl was expressed and purified. Four New Zealand rabbits were immunized with purified recombinant HIS-NSPel protein and polyclonal antibody against NSPcl was prepared. The specificity of the anti-sera was analyzed by Western Blot. Results The purity of the recombinant NSPcl protein is up to about 95%. Rabbit against NSPcl antibody was obtained. Western blot result showed that the antibody was able to detect both endogenous and/or exogenous NSPcl. Conclusion The NSPcl polyclonal antibodies prepared by using recombinant NSPcl protein as antigen has high specificity to NSPcl. It can be used for functional analysis of NSPcl in vivo and in vitro.

关 键 词：多梳蛋白 神经系统多梳蛋白1 蛋白表达纯化 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]