食品与发酵工业2024，Vol.50Issue(9)：92-98,7.DOI:10.13995/j.cnki.11-1802/ts.035476
重组荔枝类甜蛋白的高效表达、纯化及活性鉴定
Efficient soluble expression,purification,and activity identification of recombinant litchi thaumatin-like protein
摘要
Abstract
Litchi thaumatin-like protein(LcTLP)is one of the main causes of the inflammatory response to excessive consumption of litchi in some people.It was proved in the early stage of this study that it had the ability to activate mouse RAW264.7 cells for the activity of inflammatory response to deeply explore the cellular mechanism of inflammation,reveal its crystal structure and biological characteristics such as pro-inflammatory active sites,it was necessary to obtain a large amount of high-purity LcTLP.However,there was no extraction method that could obtain high-purity soluble LcTLP in large quantities in the existing reports.Therefore,the expression vector pCold-Nu-sA-LcTLP was constructed after codon optimization of the LcTLP gene in this study,and the soluble recombinant protein NusA-LcTLP was successfully expressed.Through the two-step purification method of affinity chromatography and gel filtration chromatography,the final yield of recombinant protein could reach 36.26 mg/L,and the purity could reach more than 90％,which showed that this purification scheme could efficiently produce high-purity recombinant LcTLP.After verification of the inflammatory activity of RAW264.7 cells,the re-combinant LcTLP stimulated the secretion of nitric oxide to 12.75 μmol/L and 14.68 μmol/L at 1 µg/mL and 2 μg/mL,respectively,which were 2.91 and 3.36 times that of the blank group.It was verified that the recombinant LcTLP had certain pro-inflammatory activity.The expression vector and purification scheme were highly expressed to obtain soluble recombinant LcTLP,which laid the foundation for the subsequent in-depth study of the functional mechanism of LcTLP in cell life activities.关键词
荔枝/重组类甜蛋白/诱导表达/纯化/活性鉴定Key words
litchi/recombinant thaumatin-like protein/induced expression/purification/activity identification引用本文复制引用
曹琳彩,文舜华,王凯,刘旭炜,胡卓炎,赵雷,沈兴..重组荔枝类甜蛋白的高效表达、纯化及活性鉴定[J].食品与发酵工业,2024,50(9):92-98,7.基金项目
广东省自然科学基金项目(2023A1515012599) （2023A1515012599）
国家自然科学基金项目(31771980) （31771980）
