摘要
pitx2基因是一类bicoid相关的同源异型框因子成员,介导心脏左右不对称发育和内脏器官的偏侧化.pitx2基因位于Nodal信号途径的下游,直接由Nodal信号分子控制.用PCR技术扩增斑马鱼pitx2基因的部分编码区,并将其插入到原核表达载体pGEX-4T-1中,经过酶切和测序鉴定后,将重组质粒(pGEX-4T-1-pitx2)转入BL21感受态细胞,通过IPTG诱导表达融合蛋白,用GST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.获得了pitx2原核表达重组融合蛋白及高效价的特异性兔抗pitx2多克隆抗体.提取斑马鱼不同器官和组织蛋白,用自制的多克隆抗体检测pitx2基因在斑马鱼中的表达情况,发现pitx2基因特异性地在心脏、脑、肌肉、小肠和肝脏表达,尤其在心脏中有很高的表达,表明制备的抗体特异性非常好.
The pitx2 gene is a member of the bicoid-homeodomain class of transcription factors,which mediates differentiation of heart left-right asymmetry and laterality of internal organs.Pitx2 has been proposed to act downstream of Nodal signaling pathway and to be controlled by Nodal signaling molecule directly.In this paper,pitx2 gene was amplified by PCR and then was cloned into pGEX-4T-1 vector.After confirmed by restriction enzyme and sequencing,the recombinant expression plasmid containing pitx2 gene was transformed into BL21 cells and the pitx2 fusion protein was induced by IPTG.The purified protein was obtained by Glulalhione Sepharose and then was immuned the New Zealand white rabbits to prepare antibody.The antibody titer and specification was identified by Western Blot.GST fusion protein of pitx2 have been successfully obtained and preparated high sensitivity and specificity anti-pitx2 polyclonal antibody.Different zebrafish tissues and organs were used to extract proteins to detect pitx2 antibody specification.The result showed that pitx2 antibody specifically expressed in heart,brain,muscle,intestine and liver,suggesting the pitx2 antibody was specific.
出处
《晓庄学院自然科学学报》
CAS
北大核心
2010年第2期98-101,共4页
Journal of Natural Science of Hunan Normal University
基金
湖南省普通高校学科带头人培养计划资助项目(湘教通2008-315)
国家精品课程资助项目(教高函2009-21)
