Lrrc10蛋白的表达、纯化及多克隆抗体的制备  

Expression,Purification of Lrrc10 Fusion Protein and Preparation of Its Polyclonal Antibody

作　　者：闵璐[1] 周云雷[1] 周煌[1] 吴秀山[1] 袁婺洲[1] 

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

出　　处：《湖南师范大学自然科学学报》2011年第6期68-71,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(湘教通2008-315);国家精品课程(教高函2009-21)

摘　　要：Lrrc10是一个心脏特异表达基因.为了深入研究Lrrc10在心脏发育中的功能,需要获得Lrrc10蛋白并制备其抗体.以小鼠心脏cDNA文库为模板进行PCR扩增得到Lrrc10部分编码区序列,然后将其连接到pGEX-4T-1载体上获得原核表达载体.重组子经酶切及测序鉴定后,转化大肠杆菌(E.coli)BL21,并用IPTG诱导融合蛋白表达,GST亲和层析法亲和纯化,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Lrrc10原核表达重组融合蛋白及高效价的特异性兔抗Lrrc10多克隆抗体,为Lrrc10功能的进一步研究奠定了基础.Lrrc10 is a novel heart specific expression gene. heart development, prepararion of polyclonal antibody of Lrrc10 In order to study the function of Lrrc10 gene in protein was necessary. In this paper, the coding fragment of Lrrc10 gene was amplified by PCR using mouse heart cDNA library as templates. The fragment was then cloned into pGEX -4T - 1 vector. After identified by restriction enzyme analysis and sequencing, the recombinant was transformed into BL21 host bacteria and was induced to express LrrclO fusion protein by IPTG. The fusion protein was purified by GST Glutathione SepharoseTM 4B and was immuned the New Zealand white rabbits to prepare polyclonal antibody. The antibody was identified by Western blot in the end. The result shows that GST - tag fusion protein and polyclonal antibody of LrrclO is successfully obtained, which lies a solid foundation for further functional studies on Lrrc10 gene.

关 键 词：Lrrc10 融合蛋白 多克隆抗体 

分 类 号：Q78[生物学—分子生物学]