激光镊子拉曼光谱检测重组大肠杆菌表达蚕豆14-3-3b可溶性蛋白与包涵体蛋白  

Detection of Expression of Broad Bean 14-3-3b Soluble and Inclusionbody Protein in Single Living Escherichia coli Cell by Laser Tweezers Raman Spectroscopy

作　　者：周冰[1] 卢明倩[1,2] 赵丽伟[1] 黄庶识[1,2] 陈丽梅[1] 

机构地区：[1]昆明理工大学生物工程技术研究中心,昆明650500 [2]广西科学院生物物理实验室,南宁530007

出　　处：《分析化学》2013年第12期1789-1794,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然基金资助项目(No.31260063)资助

摘　　要：采用激光镊子拉曼光谱(LTRS)分析大肠杆菌(Escherichia.coli)表达蚕豆(Vicia faba L.)14-3-3b可溶性蛋白(Soluble protein)与包涵体蛋白(Inclusionbody protein)的结构差异以及两种蛋白在不同温度下在重组菌中的表达水平。结果表明,14-3-3b可溶性蛋白与包涵体蛋白有明显不同的拉曼光谱特征峰,说明LTRS技术可以鉴定14-3-3b的可溶性蛋白和包涵体蛋白。14-3-3b可溶性蛋白特征峰1002,1451和1665 cm!1在16℃下诱导的重组菌中明显增强,在28℃下诱导的重组菌中明显减弱,而反映14-3-3b包涵体蛋白的特征峰900和1446 cm!1在16℃下诱导的重组菌中明显减弱,在28℃下诱导的重组菌中明显增强,这几个峰强的变化反映14-3-3b可溶性蛋白与包涵体蛋白在大肠杆菌细胞中表达水平的变化和SDS-PAGE电泳分析的结果一致。说明LTRS是检测单个大肠杆菌细胞中可溶性重组蛋白和包涵体蛋白表达水平的一种快速有效的方法。To explore a simple and rapid method without cell destruction to detect the real-time expression levels of the soluble and inclusionbody proteins in Escherichia. coli, Raman Laser Tweezers spectroscopy （LTRS） was used to investigate the structural differences and expression levels of the soluble and inclusionbody proteins of broad bean （ Vicia faba L. ） 14-3-3b in E. coli cells induced under different temperatures. The results showed that 14-3-3b soluble and inclusionbody protein had different Raman lines, indicating that LTRS could be used to identify the soluble and inclusionbody protein of 14-3-3b. Raman lines in 1002, 1451 and 1665 cm-1 of the soluble protein was enhanced evidently in the recombinant E. coli induced at 16 ℃ and were reduced significantly in the recombinant E. coli induced at 28 ℃. In contrast, Raman lines in 900 and 1446 cm-1 of the inclusionbody protein were decreased evidently in the recombinant E. coli induced at 16℃ and were increased significantly in the recombinant E. coli induced at 28 ℃. Changes in the soluble and inclusionbody protein of 14-3-3b expression levels reflected by these Raman lines was consistent with those obtained by the analysis of sodium dodecylsulfate polyacrylamide gel electrophoresis （SDS-PAGE）. This evidence confirms that LTRS is an effectively and rapid method for detection of the expression levels of soluble and inclusionbody proteins in the living single E. coli cell.

关 键 词：大肠杆菌 14-3-3b 可溶性蛋白 包涵体蛋白 激光镊子拉曼光谱 

分 类 号：Q78[生物学—分子生物学]