荔枝类甜蛋白(litchi thaumatin-like protein,LcTLP)是导致部分人群过量食用荔枝引起机体炎症反应的主要原因之一,为深入探究其引起的机体炎症机制,揭示其晶体结构与促炎活性位点等生物学特性,需获得大量高纯度LcTLP。然而现有报道中鲜见大量获取高纯度可溶性LcTLP的提取方法,因此该研究对LcTLP基因进行密码子优化后构建了表达载体pCold-NusA-LcTLP,并成功表达了可溶重组蛋白NusA-LcTLP。通过亲和层析与凝胶过滤层析两步纯化法,最终得到的重组蛋白产量可达36.26 mg/L,纯度达90%以上,表明该纯化方案可高效制得高纯度重组LcTLP。经RAW264.7细胞炎症活性验证,重组LcTLP在1、2 μg/mL时刺激细胞NO分泌量分别为12.75、14.68 μmol/L,为空白组的2.91、3.36倍,验证了重组LcTLP具有一定的促炎活性。该表达载体与纯化方案高效表达得到了可溶性重组LcTLP,为后续深入研究LcTLP在细胞生命活动中的功能机制奠定了基础。
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-NusA-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 recombinant 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.
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