果蝇血液发育基因Nfat的多克隆抗体的制备

摘要

Nfat基因在血液发育中具有重要作用.根据已报道的Nfat基因序列,以果蝇胚胎cDNA为模板,通过PCR扩增Nfat部分编码序列,并将其连接到pET-28A原核表达载体上,经过双酶切以及测序鉴定成功后,将重组质粒转化E.coli BL2l.在37℃时,通过IPTG诱导表达融合蛋白,通过尿素洗涤包涵体并切胶回收纯化融合蛋白,然后免疫新西兰大白兔制备多克隆抗体,并用Western Blotting检测抗体的效价以及特异性.结果表明,获得的Nfat多克隆抗体特异性较强,为后期Nfat基因的研究工作奠定了基础.%Nfat plays a very important role in the development of blood.The part of encoding sequence of Drosophila Nfat was amplified with PCR from cDNA of embryo of Drosophila,and inserted into pET-28a vector inframe.The recombinant plasmid was identified with enzyme digestion and sequencing.The competent cells of host strain of BL21 were transformed by the recombinant plasmid（pET28a-Nfat）.Expression of the target protein was induced with IPTG at 37℃.The fusion protein was purified by urea and separated by SDS-PAGE and recovered by gel extraction.Then the New Zealand white rabbits were immuned with purified recombinant protein to generate antibody.The antibody titer and specificity was identified by Western blotting.The results showed that high specificity anti-Nfat polyclonal antibody was generated,which provided a useful tool for the further studies of Nfat function.