摘要
odd是一个新发现的心脏特异表达基因。为了进一步研究odd在心脏发育中的功能,根据已报道的odd基因序列,以果蝇cDNA文库为模板进行PCR扩增,将所得的odd部分编码区序列连接到pET-28a载体上构建原核表达载体;重组子经酶切测序鉴定后,转化到大肠杆菌BL21菌株,IPTG诱导融合蛋白表达,Ni-IDA凝胶柱亲和纯化,纯化后的His-odd融合蛋白免疫新西兰大白兔以制备多克隆抗体,Western Blot检测抗体活性。结果表明,获得了odd原核表达重组融合蛋白以及高效价的、特异性兔抗Odd多克隆抗体,为后续的odd功能研究奠定了基础。
odd is a novel heart specific expression gene.Obtaining the odd protein and it＇s antibody is the first step to study its function in heart development.The cDNA fragment of part coding sequence of odd was amplified by PCR and the obtained fragment was cloned into PET-28a vector.After identified by restriction enzyme and sequenced,the recombinant which containing odd gene was transformed into Escherichia coli BL21 strain and the fusion protein was induced by IPTG.A New Zealand white rabbit was immunized with the obtained protein which was purified by immobilized Glutathione Sepharose to prepare polyclonal antibody.The antibody was identified by Western blot.We had successfully obtained GST-tag fusion protein of odd and anti-odd polyclonal antibody with high sensitivity and specificity.Its lays a solid foundation for the further studies of odd function.
出处
《激光生物学报》
CAS
CSCD
2011年第6期778-781,共4页
Acta Laser Biology Sinica
基金
国家自然科学基金项目(81170088
30871340
81170229
30930054
30970425)
湖南省普通高校学科带头人培养计划(湘教通2008-315)
国家精品课程(教高函2009-21)
关键词
ODD
融合蛋白
原核表达
多克隆抗体
odd
fusion protein
prokaiyotic expression
polyclonal antibody
