串联亲和纯化标记的HMGB1细胞因子诱导片段1融合表达载体构建和蛋白纯化及其功能初步探讨

Construction and purification of fusion expression plasmid of TAP tagged cytokine-inducing fragment 1 from high mobility group box 1 and preliminary study of its function

目的:构建高迁移率族蛋白B1(HMGB1)中细胞因子诱导片段1(CIF1)与串联亲和纯化(TAP)系统中纯化标签链霉亲和素结合肽(SBP)和钙调蛋白结合肽(CBP)序列相融合的原核表达载体,观察重组蛋白对小鼠单核/巨噬细胞白血病细胞释放肿瘤坏死因子-α(TNF-α)的影响,为HMGB1参与炎症反应作用机制的研究以及CIF1结构域细胞膜表面受体蛋白的获得和鉴定奠定基础。方法:采用PCR方法分别扩增出CBP-SBP和CIF1的编码序列,构建表达质粒pET-14b/CBP-SBP-CIF。利用Ni-NTA亲和树脂纯化融合蛋白,纯度达85%以上。采用纯化后的蛋白诱导小鼠单核/巨噬细胞白血病细胞RAW264.7,利用LiquiChip液相蛋白芯片系统检测RAW264.7细胞释放TNF-α的水平变化。结果:表达载体构建正确,CIF1重组蛋白表达量高,纯度好。当蛋白浓度大于0.5ng/μl时,CIF1重组蛋白诱导细胞TNF-α的分泌量与对照组相比差异具有显著性(P<0.05),并且在CIF1蛋白浓度0～2.5ng/μl范围内,TNF-α的分泌量与CIF1重组蛋白的浓度呈显著的剂量依赖关系。结论:原核表达纯化了His-CBP-SBP-CIF1融合蛋白,该融合蛋白可以有效地作用于巨噬样细胞内的信号转导过程,介导了炎症因子TNF-α的分泌表达。

Objective： To construct the prokaryotic expression plasmid of cytokine-inducing fragment 1 （CIF1） of high mobility group box 1 （HMGB1） and two tandem affinity tags： a streptavidin binding peptide （SBP） and a calmodulin binding peptide （CBP）, to observe the effects of recombinant protein on the release of tumor necrosis factor-α （TNF-α） in mouse leukaemic monocyte macrophage line cells. Methods： The CBP-SBP and CIF1 gene were amplified by polymerase chain reaction （PCR） to construct and express the plasmid pET-14b/CBP-SBP- CIF1. After purification by Ni^2- affinity chromatography, the purity of fusion protein was up to 85%. Using LiquiChip system, the levels of TNF-α released by mouse leukaemic monocyte macrophage cells RAW 264.7 were assayed after the addition of purified protein. Results： The pET-14b/CBP SBP-CIF1 plasmid was correctly constructed. The recombinant protein had high yield and purity. Compared to control group, it was able to induce mouse leukaemic monocyte macrophage cells to release TNF-α in much higher quantity （P〈0.05） when its con- centration ≥0. 5 ng/μl. The relationship between TNF-α production and protein concentration was in dose-dependent manner. Conclusion： His-CBP-SBP-CIF1 protein is obtained and purified, which can act on the signal transduction in mouse leukaemic monocyte macrophage cells and induce the release of inflammatory factor, such as TNF-α. This study may provide the basis to further elucidate the mechanism of HMGB1 involved in the inflammatory response and identify cell membrane receptor proteins of CIF1 domain.