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目的:制备兔抗鼠SKA1(Spindle and Kinetochore Associated1)多克隆抗体。方法:利用PCR的方法得到小鼠SKA1基因并克隆至pGEX-2T原核表达载体中,转化入大肠杆菌BL21表达,经IPTG诱导表达GST-SKA1融合蛋白,经亲和层析纯化浓缩后免疫新西兰大耳白兔制备多克隆抗体,通过ELISA测定抗鼠SKA1多克隆抗体效价,用Western blot测定其特异性。结果:构建了pGEX-2T-SKA1原核表达重组质粒,并诱导表达出GST-SKA1融合蛋白,免疫新西兰大耳白兔5周后获得多克隆抗体。ELISA测定表明SKA1多克隆抗体效价为1∶25600,Western blot分析表明SKA1多克隆抗体具有良好的特异性。通过免疫组织化学方法发现,SKA1表达于高分化前列腺癌细胞中。结论:成功地制备了效价及特异性良好的兔抗鼠SKA1多克隆抗体,为SKA1基因功能的研究奠定基础。
Objective: To prepare polyclonal anti-mouse SKA1 (Spindle and Kinetochore Associated1) antibody. Methods: The mouse SKA1 gene was obtained by PCR and cloned into prokaryotic expression vector pGEX-2T. The recombinant plasmid was transformed into E. coli BL21 and expressed by IPTG. The purified fusion protein was purified by affinity chromatography and immunized into New Zealand large Rabbit rabbits were prepared polyclonal antibody, anti-mouse SKA1 polyclonal antibody titers by ELISA, specificity was determined by Western blot. Results: The prokaryotic expression plasmid pGEX-2T-SKA1 was constructed and the GST-SKA1 fusion protein was induced. The New Zealand white rabbits were immunized for 5 weeks to obtain polyclonal antibodies. ELISA assay showed that the titer of the SKA1 polyclonal antibody was 1:25600, Western blot analysis showed that the SKA1 polyclonal antibody has good specificity. It was found by immunohistochemistry that SKA1 is expressed in well-differentiated prostate cancer cells. CONCLUSION: The rabbit anti-mouse SKA1 polyclonal antibody with good titer and specificity was successfully prepared, which laid the foundation for the study of SKA1 gene function.