小鼠Dectin-1胞外区的融合表达及其识别白念珠菌β-葡聚糖的研究

Fusion expression of the extracellular carbohydrate recognition domain of mouse Dectin-1 and its recognition of β-glucans in the cell wall of Candida albicans

目的克隆、表达并纯化小鼠腹腔巨噬细胞Dectin-1基因胞外区，探讨其识别并结合真菌β-葡聚糖的能力。方法应用RT-PCR方法从小鼠腹腔巨噬细胞RNA中扩增Dectin-1基因胞外区，构建原核重组表达载体pET28-CRD，进行融合表达、纯化和复性。将融合蛋白与白念珠菌酵母相共同孵育，检测其识别、结合白念珠菌细胞壁β-葡聚糖的功能。结果成功克隆并构建原核重组表达质粒pET28-CRD，表达并纯化了融合蛋白，该蛋白能识别、结合白念珠菌酵母细胞壁β-葡聚糖。结论构建的原核重组表达载体能够在原核细胞内表达，表达产物有识别、结合真菌胞壁β-葡聚糖的功能，为进一步研究相应的真菌检测方法奠定了基础。

Objective To clone, express and purify the extracellular carbohydrate recognition domain（CRD） of Dectin-1 in mouse peritoneal macrophages and to further investigate its ability to recognize and bind to β-glucans. Methods The Dectin-1 CRD gene was amplified by RT-PCR from RNA of mouse peritoneal macrophages and cloned into prokaryotic expression vector pET28a （ ＋ ） , the constructed pET- CRD recombinant plasmid was transformed into Escherichia coli BL21 （DE3） and the fusion protein was induced to express. After affinity purification and renaturation, the fusion protein was incubated with Candida albicans yeast and its ability to recognize and bind to β-glucans in the cell wall of fungi. Results The fusion protein could recognize β-glucans in the fungal cell wall. Conclusion The recombinant expression plasmid pET28a-CRD was successfully constructed and the fusion protein was induced. The fusion protein is able to recognize and bind to β-glucans in the fungal cell wall, thus laying a good foundation for fungal detection and the exploration of the biological role of β-glucans.