摘要目的 应用pET表达系统原核表达红色荧光蛋白变种mCherry,分析重组mCherry表达与荧光变化的关系,为其纯化及作为原核报告系统应用提供依据.方法 根据GenBank中收录的mCherry氨基酸序列合成其编码基因,构建pET-mCherry表达质粒,转化入大肠埃希菌BL21 (DE3)表达宿主.在不同诱导时间点,考察菌株荧光强度、mCherry表达量、培养上清液mCherry泄漏情况.诱导10h后提取重组mCherry,利用荧光强度估算提取收率,并通过凝胶电泳分析提取液的蛋白纯度.结果 成功构建pET-mCherry表达载体,并在BL21(DE3)中得到高效诱导表达.荧光检出滞后于mCherry的表达,随诱导时间延长重组mCherry不断泄漏至培养液中.初步提取的重组mCherry纯度达到95％以上.结论 应用pET表达系统可高效表达mCherry,过表达mCherry可泄漏至胞外,方便其纯化.mCherry作为原核报告系统应用时,合理的诱导时间可能是关键影响因素.

abstractsObjective To express red fluorescent protein variant mCherry with pET plasmid in E.coli,and analyze the relationship of mCherry expression with its fluorescent characteristics,providing basis for mCherry purification and its application as prokaryotic report system.Methods The coding sequence was chemically synthesized according to the amino acid sequence of mCherry from GenBank.The constructed expression vector pET-mCherry was transformed into E.coli BL21 (DE3).Fluorescent intensity,expression amount and leakage into medium were analyzed at different inducing time points.Bacteria were collected and resuspended in PBS after 10 h induction.Recombinant mCherry was extracted by continuous stirring process.The recovery rate of mCherry was estimated by fluorescence assay,and the purity was analyzed in gel electrophoresis.Results The expression vector pET-mCherry was successfully constructed and recombinant mCherry was expressed efficiently upon induction.There was a time lag between fluorescence detection and mCherry expression.Recombinant mCherry continuously leaked into medium.After on,step extraction,the product was obtained with purity above 95％.Conclusions mCherry can be efficiently expressed in pET system,and overexpressed mCherry can leak into medium following induction,favoring purification.Culture time would be the key factor when mCherry is used as prokaryotic report gene.