摘要
目的该研究通过SGK2α基因的克隆、原核蛋白表达以及纯化,制备抗SGK2α抗体,为进一步研究SGK2α蛋白的生物学功能奠定基础。方法利用原核表达载体PGEX-4T-3构建重组质粒,并在大肠杆菌中通过IPTG诱导表达;亲和层析法纯化重组蛋白;以纯化的重组蛋白为抗原免疫新西兰大耳白兔制备抗SGK2α多克隆抗体,ELISA方法检测抗体效价,免疫印迹法检测抗体的特异性。结果构建了PGEX-4T-3-SGK2α原核表达质粒,经原核表达和亲和层析获得GST-SGK2α融合蛋白,蛋白纯度在90%以上;利用GST-SGK2α融合蛋白制备多克隆抗体,抗体效价阳性且具有较强的特异性。结论利用原核表达的GST-SGK2α融合蛋白成功地制备了抗SGK2α多克隆抗体,可用于免疫组织化学和免疫印迹检测以及功能研究。
【Objective】To provide the basis for further investigation of biological functions of SGK2α through cloning and prokaryotic expression of SGK2α gene and purification and preparation of anti -SGK2α antibody. 【Methods】The recombinant expression plasmid was constructed by using prokaryotic PGEX-4T-3 vector, which was inducible expression by IPTG in E.Coli. Recombinant protein was purified by chromatography under nondenaturing conditions. The rabbit was immunized with purified protein to prepare polyclonal antibody of rabbit anti-human SGK2α. The reactivity and specificity of the polyclonal antibody were detected by ELISA assay and Western Blot respectively.【Results】 Recombinant plasmid PGEX-4T-3-SGK2α was constructed. GST-SGK2α fusion protein was obtained through prokaryotic expression and affinity chromatography, its purity was more than 90%. Polyclonal antibody was prepared by using GST-SGK2α fusion protein. The reactivity of antibody was positive and had specificity. 【Conclusions】 Anti-SGK2α polyclonal antibody was successfully prepared by using GST-SGK2α fusion protein, which can be used in immunohistochemistry, Western blot and functional study.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2010年第12期1765-1768,共4页
China Journal of Modern Medicine
基金
国家自然科学基金资助项目(No:30570904)
黑龙江省教育厅资助项目(No:11531431)
关键词
SGK2α
重组蛋白纯化
多克隆抗体
serum and glucocorticoid regulated protein kinase 2α （SGK2α）
recombinant protein purification
polyclonal antibody
