小鼠朊蛋白相关蛋白Shadoo的表达、纯化及多克隆抗体的制备

Expression and purification of mouse PrP-like protein Shadoo and preparation of polyclonal antibody against Shadoo

构建SPRN重组原核表达载体,并进行目的蛋白的表达、纯化及多克隆抗体的制备。提取BALB/c小鼠全血基因组,PCR扩增SPRN基因,克隆至融合表达载体,在大肠埃希菌中表达鼠Shadoo蛋白并纯化;以纯化蛋白为抗原免疫新西兰白兔制备抗血清,并检测抗体效价,用Western blot方法分别检测与重组及内源性Shadoo蛋白的反应性。结果表明,成功制备了小鼠Shadoo蛋白兔源多克隆抗体,为研究Shadoo蛋白与PrP蛋白之间的功能调节关系、自身的生理学作用以及Shadoo蛋白在传染性海绵状脑病发病机制过程中的生物学功能奠定了良好的基础。

To construct the SPRN recombinant prokaryotic expression vector and to express and purify Shadoo protein, then prepare the rabbit polyclonal antibody against Shadoo protein.BALB/c mouse genomic was extracted,and Shadoo SPRN gene was amplified by PCR,then cloned into fusion expression vector.The recombinant plasmid was trans- formed into E.coli and the Shadoo protein was expressed and purified.New Zealand white rabbits were immunized with the purified protein to prepare Shadoo antiserum and its titer was characterized,and then the specificity of the polyclonal antibody with recombinant and endogenous Shadoo protein was,detected by Western blot respectively. The results demonstrated that rabbit polyclonal antibody against mouse Shadoo protein was successfully prepared, which makes a good foundation of studying the functional regulating relationship between Shadoo protein and PrP protein,and the physiological role of Shadoo protein,as well as the biological function of Shadoo protein in pathogenesis of transmissible spongiform encephalopathy.