天蚕素抗菌肽(Cecropin A)融合蛋白的表达及其分离纯化工艺研究  

Expression and Purification of Cecropin A Fusion Protein

作　　者：何成霞 邹强[1] 刘达玉[1] 苟兴华[1] 陶雪菊 詹茂 黄海韬 HE Cheng-xia;ZOU Qiang;LIU Da-yu;GOU Xing-hua;TAO Xue-ju;ZHAN Mao;HUANG Hai-tao(School of Food and Biological Engineering,Chengdu University,Chengdu 610106,China)

机构地区：[1]成都大学食品与生物工程学院,成都610106

出　　处：《中国调味品》2021年第10期38-42,58,共6页China Condiment

基　　金：四川省重点研发计划项目(2019YFS0525)。

摘　　要：通过单因素实验确定了天蚕素抗菌肽融合蛋白表达的最佳条件,即温度为31℃,种子菌接种量为2.0%,培养基pH值为6.5,诱导时间为10 h,此时融合蛋白的表达量占细胞总蛋白的30.6%;开发融合蛋白纯化工艺,将工程菌细胞超声破碎后,所得包涵体使用8 mol/L尿素溶液溶解,泵入2 mol/L尿素溶液中搅拌使蛋白质复性,复性完成后将2 mol/L尿素溶液通过超滤置换成含0.5 mol/L NaCl、20 mmol/L的PB缓冲液,pH 7.2,并浓缩溶液体积至总体积的30%~40%,采用镍离子亲和层析法纯化Cecropin A融合蛋白,SDS-PAGE凝胶电泳结果显示目标融合蛋白纯度达到87%以上。The optimal expression conditions of Cecropin A fusion protein are determined by single factor experiments,that is,the temperature is 31℃,the inoculation amount of seed bacteria is 2.0%,the pH of culture medium is 6.5,the induction time is 10 h,and the expression level of fusion protein accounts for 30.6%of the total protein of cell.The fusion protein purification process is developed.After the engineering bacteria cells are ultrasonically broken,the resulting inclusion bodies are dissolved in 8 mol/L urea solution and pumped into 2 mol/L urea solution for stirring to make the protein refolding.After renaturation,2 mol/L urea solution is replaced by ultrafiltration into 0.5 mol/L NaCl,20 mmol/L PB buffer solution,pH is 7.2,and the volume of the solution is concentrated to 30%~40%of the total volume.The fusion protein of Cecropin A is purified by nickel ion affinity chromatography.SDS-PAGE gel electrophoresis shows that the purity of the target fusion protein is above 87%.

关 键 词：天蚕素抗菌肽 融合蛋白表达 包涵体 亲和层析 分离纯化 

分 类 号：TS202.3[轻工技术与工程—食品科学]