作　　者： ; ; ; ; ;

机构地区： 南京农业大学生命科学学院

出　　处： 《南京农业大学学报》 2010年第4期25-30,共6页

摘　　要： 为研究耐铜植物海州香薷的金属硫蛋白(EhMT1)的结构与功能,运用分子生物学方法构建了pGEX-2T-EhMT1重组表达载体,经转化大肠杆菌BL21(DE3)后表达得到大小为33×103左右的融合蛋白GST-EhMT1,与预期结果一致。对融合蛋白诱导表达的温度、时间、IPTG诱导浓度等条件进行了优化,成功构建了能大量表达可溶性GST-EhMT1的优良原核表达体系。诱导表达的融合蛋白经GST-Sepharose亲和层析纯化后,每升菌液获得的可溶性融合蛋白高达70mg。用纯化的融合蛋白免疫新西兰雄兔,制备的抗血清经间接ELISA检测到较高的多克隆抗体效价。蛋白质印迹结果显示,纯化的蛋白质与兔抗血清可以特异性结合。 To characterize the function of Elsholtzia haichowensis metallothionein （EhMT1）,the EhMT1 fusion expression vector was constructed with pGEX-2T,and transformed into Escherichia coli BL21.The SDS-PAGE results showed that an IPTG-induced fusion protein,GST-EhMT1 was expressed.The molecular mass of the fusion protein was about 33 × 103,which was similar to the expected.Under normal expression conditions,the expression products existed in soluble and insoluble forms.To obtain as much fusion protein as possible,we optimized the fermentation conditions including temperature,time or IPTG concentration of expression and an efficient prokaryotic expression system was confirmed.70 mg soluble fusion protein from per litre overnight cultures was obtained after purification by GST-tag affinity chromatography.The purified fusion protein was used as antigen to prepare polyclonal antiserum in New Zealand rabbits.The antiserum was extracted after five injections of antigen.The results of ELISA and Westernblot proved that rabbit polyclonal antiserum obtained was a new reagent with high titration and specificity.

关 键 词： 金属硫蛋白 原核表达 融合蛋白 抗血清

领　　域： [生物学] [生物学]