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人工设计合成芋螺毒素基因Mr VIB来构建表达载体p ET32a/Trx-EK-Mr VIB,将其转化大肠杆菌BL21(DE3)plys S进行诱导表达。菌体经超声破碎后利用Ni-NTA琼脂糖柱进行亲和层析纯化融合蛋白,SDS-PAGE电泳分析融合蛋白表达。结果表明融合表达载体p ET32a/Trx-EK-Mr VIB经PCR扩增和测序鉴定具有正确的开放阅读框。SDS-PAGE电泳显示融合蛋白在大肠杆菌中获得高效可溶性表达,经一步亲和层析获得纯度大于90%的融合芋螺毒素达73.6 mg/L。本文成功构建了融合表达载体p ET32a/Trx-EK-Mr VIB,融合芋螺毒素Trx-EK-Mr VIB在大肠杆菌中获得高效可溶性表达。
Abstract：Artificial designed conotoxin Mr VIB gene was synthesized to construct expression vector p ET32a/TrxEK-Mr VIB, which was transformed into E. coli BL21(DE3) p Lys S and induced expression by IPTG. Recombinant fusion proteins were purified by affinity chromatography using Ni-NTA agarose column, the expression and purification of fusion proteins to be analyzed by SDS-PAGE. Fusion expression vector p ET32a/Trx-EK-Mr VIB was confirmed correctly by PCR and sequence analysis. The result of SDS-PAGE showed that fusion protein was high effectively expressed in E. coli, which was purified by one-step affinity chromatography and the purity of fusion conotoxin was greater than 90% and its yield was 73.6 mg/L. Fusion expression vector p ET32a/Trx-EK-Mr VIB was successfully constructed, and fusion conotoxin Trx-EK-Mr VIB was high effectively expressed in E. coli.
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基本信息:
DOI：10.13417/j.gab.034.000326
中图分类号:Q78
引用信息:
[1]高炳淼,刘云海,彭超,等.融合表达载体pET32a/Trx-EK-MrVIB的构建及在大肠杆菌中表达[J].基因组学与应用生物学,2015,34(02):326-331.DOI:10.13417/j.gab.034.000326.
基金信息:
海南医学院培育基金(HY2013-11);; 海南医学院大学生创新课题(HYCX201327);; 海南省自然科学基金(814291);; 海南医学院引进人才科研启动经费;; 国家高技术研究发展计划(863计划)课题(2014AA093501)共同资助
2015-02-25
2015-02-25