摘要:目的构建针对Smad家族相关系列原核表达载体,观察其相应蛋白诱导表达纯化情况。方法 PCR扩增Smad2/3/4全长及截短片段,定向插入p GEX-6P-1载体中,构建原核表达重组质粒pGEX-6P-1-Smad2/3/4。经酶切和测序正确后,重组质粒转化表达菌株BL21(DE3),异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达;上清经GST-Sepharose4B亲和纯化,SDS-PAGE考马斯亮蓝染色鉴定。结果原核表达载体p GEX-6P-1-Smad2/3/4构建成功,SDS-PAGE证实Smad3A和Smad4存在包涵体,而上清中GST-Smad3(B-D)和GST-Smad2可被纯化。结论构建的融合蛋白原核表达载体通过诱导和纯化后得到GST-Smad3截断融合蛋白(B-D)和GST-Smad2。
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