信号蛋白质14-3-3ζ的高效融合表达和鉴定

Expression and Detection of Signal Protein 14-3-3 Zeta in Prokayotic Cell

目的 利用分子克隆技术在原核细胞中研究 1 4-3 -3ζ蛋白的表达。方法 1 4-3 -3ζ c DNA经测序后 ,亚克隆至 p BK-CMV表达载体 ,转化大肠杆菌 BL2 1菌株 ,筛选阳性克隆 ,经异丙基硫代 -β-D-半乳糖苷 (IPTG)诱导表达。结果 经变性聚丙烯酰胺凝胶电泳 (SDS-PAGE)和蛋白印迹 (Western blot)分析 ,表达的融合蛋白相对分子质量为 3 2 0 0 0左右 ,能与 1 4-3 -3ζ蛋白特异性多克隆抗体发生免疫结合反应。结论 人 1 4-3 -3ζ蛋白在原核细胞中有大量表达 。

Objective To study the expression of signal protein 14 3 3 zeta(ζ) in prokaryotic cell using genetic engineering technique. Methods After being confirmed by DNA sequence analysis,the full length 14 3 3ζ cDNA was sub cloned into a β galactosidase fusion protein expression vetor pBK CMV,and the expressed β galactosidase 14 3 3 ζ fusion protein was analyzed. Results SDS PAGE assays yielded a roughly Mr 32 000 expressed protein,which could be further purified by a commercially supplied purification technique for β galactosidase protein Western blot tests revealed that the expressed protein could react with a specific polyclonal antibody for the 14 3 3ζ signal protein. Conclusions Human signal protein 14 3 3ζ could be expressed in escherichia coli,with β galactosidase fusion protein expression system with high yield and good immunoreactivity.