蜂毒明肽的原核表达及纯化

Prokaryotic Expression and Purification of Apamin in E.coli

目的原核表达并纯化蜂毒明肽。方法人工合成蜂毒明肽基因,与肠激酶识别序列基因串联,插入原核表达载体pGEX-2T中,构建蜂毒明肽与谷胱甘肽硫转移酶的融合表达载体pGEX-APAM。将重组质粒转化E. coli BL21(DE3),IPTG诱导表达,表达产物经SDS-PAGE和Western blot鉴定,并进行纯化和肠激酶切割。结果经Western blot鉴定,表达产物具有良好的反应原性。在优化的表达条件下,融合蛋白的表达量约占菌体总蛋白的25.8%,且大部分以可溶性形式存在。纯化的融合蛋白纯度大于95%,每毫克融合蛋白经肠激酶切割后,可得蜂毒明肽58.5μg,回收率为81.9%。结论已成功地原核表达并纯化了蜂毒明肽。

Objective To express apamin in prokaryotic cells and purify the expressed product.Methods Apamin gene with enterokinase recognition sequence was synthesized and inserted into prokaryotic expression vector pGEX-2T to construct the fusion expression vector pGEX-APAM for apamin and GST,which was transformed to E.coli BL21（DE3）for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot,then purified and digested with enterokinase.Results Western blot showed good reactogenicity of expressed product.Under the optimal condition for expression,the fusion protein contained 25.8% of total somatic protein,most of which existed in a soluble form.The purified fusion protein reached a purity of more than 95%.A portion of 58.5 μg of apamin was obtained from 1 mg of fusion protein after digestion with enterokinase,indicating a recovery rate of 81.9%.Conclusion Apamin was successfully expressed in prokaryotic cells and purified.