斑马鱼心脏发育标记基因Wnt11的多克隆抗体制备  

Preparation of Polyclonal Antibody of Heart Development Marker Gene Wnt11 in Zebrafish

作　　者：雷孝锋[1] 江志钢[1] 李志[1] 王华[1] 万永奇[1] 袁婺洲[1] 吴秀山[1] 

机构地区：[1]湖南师范大学蛋白质化学及鱼类发育生物学教育部重点实验室心脏发育研究中心,中国湖南长沙410081

出　　处：《生命科学研究》2011年第2期107-110,共4页Life Science Research

基　　金：国家自然科学基金资助项目(30671053;30871340;30930054;30900851);国家重点基础研究发展计划资助项目(2005CB522505)

摘　　要：Wnt11为Wnt家族的成员,是参与Wnt信号途径调控的转录因子,对心脏的正常发育起着非常重要的作用.根据已报道的Wnt11基因序列,通过RT-PCR从斑马鱼心脏组织中得到Wnt11部分编码区序列,将其连接到pGEX-4T-1原核表达载体上.经酶切及测序鉴定后,质粒构建成功,将重组质粒(pGEX-4T-Wnt11)转化E.coli BL2l,通过IPTG诱导表达出融合蛋白,采用谷胱甘肽琼脂糖珠亲和纯化.将纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western Blot检测抗体的效价和特异性.结果显示,获得了Wnt11原核表达重组融合蛋白及高效价的特异性兔抗Wnt11多克隆抗体,为Wnt11功能的进一步研究奠定了基础.Wnt11 is one of the members of the Wnt family,which is a transcription factor involved in regulation of Wnt signaling pathway and plays a very important role in development of heart.The part of encoding sequence of zebrafish Wnt11 was amplified with RT-PCR from zebrafish heart,and inserted into pGEX-4T-1 vector in frame.The recombinant plasmid was identified with enzyme digestion and sequencing.The competent cells of host strain of BL21 were transformed by the recombinant plasmid（pGEX-4T-Wnt11）.Expression of the target protein was induced with IPTG and assayed by SDS-PAGE.The recombinant products were purified by Glutathione Sepharose.Then the New Zealand white rabbits was immuned with purified recombinant protein to generate antibody.The antibody titer and specificity was identified by Western blot.All these results showed that GST-Wnt11 fusion protein was successfully purified and the high sensitivity and high specificity anti-Wnt11 polyclonal antibody was generated,which provided a powerful tool for the further studies of Wnt11 function.

关 键 词：Wnt11 融合蛋白 斑马鱼 多克隆抗体 

分 类 号：Q71[生物学—分子生物学]