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目的筛选出TLR7 TIR区对其信号传递起关键作用的位点,并解释该位点的作用机制。方法针对TLR7野生型序列,设计构建一系列TLR7缺失突变与点突变质粒。将这些质粒转染入HEK293T细胞,利用双荧光素酶报告基因检测这些质粒对NF-κB信号通路的影响。利用免疫共沉淀与蛋白质印迹实验验证TLR7突变体与其下游接头蛋白MyD88结合能力的变化。结果通过对TLR7胞内区截短突变分析,发现在其TIR区存在一个由16个氨基酸残基组成的区域对其信号传递至关重要。进一步的研究发现,该区域存在一个RXR信号基序。该基序位于1004位与1006位的两个保守精氨酸残基,为TLR7正常信号传递所必需。通过免疫共沉淀与蛋白质印迹实验,可以发现TLR7位于1004位的精氨酸对其与下游蛋白MyD88的结合至关重要。结论成功鉴定出TLR7 TIR区1004位的精氨酸对其信号传递起重要作用,该位点是通过影响TLR7与MyD88结合的方式发挥功能。
Objective To screen the site of TLR7 TIR signaling that plays a key role and to elucidate the mechanism of action of this site. Methods Aiming at TLR7 wild type sequence, a series of TLR7 deletion mutation and point mutation plasmids were designed and constructed. These plasmids were transfected into HEK293T cells and the effect of these plasmids on the NF-κB signaling pathway was examined using a dual luciferase reporter gene. Immunoprecipitation and Western blotting were used to verify the binding capacity of TLR7 mutant to its downstream adapter protein MyD88. Results By analyzing the truncation mutation in TLR7 intracellular region, it was found that there was a region of 16 amino acid residues in its TIR region which was crucial for its signal transmission. Further study found that there is a RXR signal motif in this region. This motif is located at two conserved arginine residues at positions 1004 and 1006 and is required for normal TLR7 signaling. By co-immunoprecipitation and western blotting, it was found that arginine at position 1004 of TLR7 is essential for its binding to the downstream protein MyD88. Conclusion Arginine at position 1004 in the TIR region of TLR7 was successfully identified as an important signaling pathway and this site functions by affecting the binding of TLR7 to MyD88.