大肠埃希菌周质抗体细胞释放工艺方法研究

Research for extraction of periplasmic proteins in Escherichia coli

本文应用细胞裂解液提取法、超声波细胞破碎法、细胞冻融法、bugbuster提取法和高压细胞破碎法,对比研究了从大肠埃希菌周质中提取可溶性抗体的相关参数,并通过SDS-PAGE电泳,BCA蛋白浓度测定和ELISA法检测了上述四种方法的有效性。结果表明,对于试验室小试规模的抗CEA抗体的细胞释放,超声法破碎,提取的目的抗体量最多,优于细胞裂解液提取法,冻融法和bugbuster法,最佳条件为:细胞缓冲液与湿菌体的比例为5mL/g,磁力搅拌混匀,冰浴中以250W功率,超声波破碎:4s工作5s间隔140次。另外,对于大规模抗体的制备,高压破碎仪在850Pa下,破碎两次效果最佳。

This article reviews several cell breakage methods, such as extraction buffer method, ultrasonic disruption, repetitive freezing and thawing, bugbuster and high-pressure cracker. Compare the periplasmic protein we get from each mehod, in aspects of weight and activity, by using SDS-PAGE electrophoresis, BCA Protein Assay Kit, and ELISA. The conclusions are as follows： in a small scale laboratory, ultrasonic disruption is the most efficacious method, and the optimal condition is putout power 250W, radiation/intermission time 4s/Ss, total working times is 140, and the amount of cell buffer 5mL/g with ice bath. Besides, for the large-scale periplasmic protein preparation, the optimal method is high-pressure cracker in 850Pa, crushing twice.