gcm蛋白的表达、纯化及多克隆抗体的制备

Expression,Purification of gcm Fusion Protein and Preparation of its Polyclonal Antibody

gcm(glial cells missing)是调控神经元细胞和神经胶质细胞相互转化的一个基因开关.在gcm功能缺损的突变体中,预期的神经胶质细胞发育成神经元细胞;而在gcm过表达的突变体中,预期的神经元细胞转化为神经胶质细胞.此外,gcm还调控血浆细胞发育.为了进一步研究gcm在发育中的功能,需要获得gcm蛋白并制备其抗体.根据已报道的gcm基因序列,以果蝇cDNA文库为模板进行PCR扩增得到gcm部分编码区序列,然后将其连接到pET-28a载体以获得原核表达载体.重组载体经酶切测序鉴定确认后,转化大肠杆菌(E.coli)BL21,并用IPTG诱导融合蛋白表达.采用Ni-IDA凝胶柱亲和纯化蛋白,将纯化的His-gcm融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western Blot检测抗体效价.获得的gcm原核表达重组融合蛋白及高效价的特异性兔抗gcm多克隆抗体,为gcm功能的进一步研究奠定了基础.

gcm（glial cells missing） appears to function as a binary genetic switch for glia versus neurons. In loss-of-function gcm mutant alleles, presumptive glia become neurons; In gain-of-function gcm conditions, presumptive neurons become glia. Meanwhile, gcm control plasmatocyte development. Obtaining the gem protein and it＇s antibody is the first step to detect the function of gcm in heart development. The cDNA fragment of part coding sequence of gcm was obtained by PCR amplification, then the fragment was cloned into PET-28a vector and identified with restriction enzyme and sequencing. The recombinant expression plasmid containing gcm gene was transformed into BL21 and fusion protein was induced by IPTG. The puri- fied protein was obtained by treating the lysates with immobilized Glutathione Sepharose. The protein prod- uct was used to immune the New Zealand white rabbits to prepare antibody. The antibody titer and specifi- cation was identified by Western blot. We have successfully obtained GST-tag fusion protein of gcm and got high sensitivity and specificity anti-gcm polyclonal antibody, which lays a solid foundation for the further studies of gcm function.