蜂毒肽基因在大肠杆菌中的表达、纯化及初步鉴定  

Expression,purification and preliminary identification of Melittin in E. coli

作　　者：朱文赫[1] 田立玲 孙妙囡[1] 孙德军[1] 

机构地区：[1]吉林大学再生医学科学研究所生物技术药物教研室,吉林长春130021 [2]吉林省辉发制药股份有限公司,吉林辉南135100

出　　处：《中国生化药物杂志》2010年第1期1-5,共5页Chinese Journal of Biochemical Pharmaceutics

基　　金：吉林省科技发展计划项目(20060564)

摘　　要：目的通过pGEX-2T原核表达载体表达蜂毒肽。方法将人工合成整合了肠激酶作用位点的蜂毒肽基因,插入载体pGEX-2T,构建蜂毒肽与谷胱甘肽S-转移酶(GST)融合表达载体pGEX-MEL。重组质粒转化E.coliBL21(DE3)诱导表达,超声破碎菌体,SDS-PAGE检测蛋白表达情况,取超声上清,通过GST亲和色谱系统纯化目的融合蛋白,酶切通过溶血实验检测其溶血活性。结果SDS-PAGE结果显示,表达了相对分子质量约为30 000的融合蛋白,目的融合蛋白量约占菌体总蛋白量的29.5%。经Western blot鉴定,表达的GST融合蛋白与GST抗体有强烈的交叉反应,说明成功表达了目的融合蛋白。取超声上清,亲和色谱纯化融合蛋白,纯度为95%。肠激酶切割得到了人工天然蜂毒肽,测定蜂毒肽回收率为80%。结论通过原核表达系统成功表达蜂毒肽,具有和天然蜂毒相同的溶血活性。Purpose Melittin was expressed in prokaryotic vector pGEX-2T for production. Methods Melittin gene synthesized with enterokinase digested sequence, the gene was cloned into vector pGEX-2T, and constructed a recombinant ptasmid of pGEX-MEL. Then the recombinant vector was introduced into E. coli BI21 （DE3）for expression. Fusion protein was purified by affinity chromatography. Hemolytic activity of Melit- tin was detected. Results Analysis result showed that the expression products accumulate in the cells to about 29.5 % of total cell protein. Detection of western blot using ant-GST as the first antibody showed that a special blot was revealed among the expression products. It certified that we have succeeded in expressing the fusion protein. SDS-PAGE showed that most part of the products is resoluble. The purity of obtained protein is 95 %, by through GST affinity chromatography system. Melittin is harvested with a recovery of 80 % by EK digestion. Test results showed melittin has good hemolytic activity. Conclusion We have expressed Melittin successfully by prokaryotie expression system.

关 键 词：蜂毒肽 大肠杆菌 表达 纯化 

分 类 号：Q78[生物学—分子生物学]