Kruppel在果蝇发育过程中起着重要的调控作用。为了进一步研究Kruppel的功能, 需要制备Kruppel蛋白及其抗体.对已有的Kruppel序列进行分析, 选取适当区域进行引物设计, 从果蝇心脏cDNA文库中PCR扩增得到Kruppel部分编码区序列, 并其连接到pET-28a载体上。将重组质粒(pET-28a-Kruppel)转化rosetta受体菌, 通过IPTG(Isopropyl β-D-thiogalactoside)诱导表达融合蛋白, 用镍柱进行亲和纯化。将纯化得到的融合蛋白免疫新西兰大白兔制备多克隆抗体, 并用Western blot检测抗体的效价。

Kruppel plays a very important role in development of heart. Obtianing the Kruppel protein and it's antibody is the first step to detect the function of Kruppel. After the sequence of Kruppel was analyzed and the primer was designed, the part of encoding sequence of drosophila Kruppel was amplified with PCR from cDNA of drosophila heart, and inserted into pET-28a vector in frame. The competent cells of host strain of rosetta were transformed by the recombinant plasmid (pET-28a-Kruppel). Expression of the target protein was induced with IPTG and purified by Ni-NTA affinity chromatography under denaturing conditions. Then the New Zealand white rabbits was immuned with purified recombinant protein to generate antibody. The antibody titer was identified by Western blot.