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将质粒p ET30a(+)-Cry1C转入大肠杆菌进行表达,对其表达条件进行了优化,并评价了蛋白的表达和纯化效果。结果显示:最佳诱导时间、IPTG的终浓度和诱导温度分别为5 h、0.6 mmol/L和25°C。大多数的靶蛋白以包涵体形式表达,通过纯化和复性,最终获得高纯度有活性的靶蛋白。通过SDS-PAGE Western Blot、糖蛋白测定、LC MS/MS鉴定和杀虫试验表明:大肠杆菌中表达的Cry1C蛋白和转基因水稻的Cry1C蛋白具有实质等同性。
The plasmid p ET30a (+) - Cry1C was transformed into E. coli for expression, and its expression conditions were optimized, and the protein expression and purification effects were evaluated. The results showed that the optimal induction time, IPTG final concentration and induction temperature were 5 h, 0.6 mmol / L and 25 ℃ respectively. Most target proteins are expressed as inclusion bodies, resulting in high purity and active target proteins by purification and refolding. By SDS-PAGE Western Blot, glycoprotein determination, LC MS / MS identification and insecticidal tests showed that: Cry1C protein expressed in E. coli and Cry1C protein of transgenic rice have substantial identity.