重组SIRPα1的体外表达及其在红细胞CD47-SIRPα信号通路研究中的应用

In vitro expression of the recombinant SIRPα1 and its application in the study of erythrocyte CD47-SIRPα signaling pathway

目的获得野生型信号调节蛋白α1（SIRPα1）功能结构域重组蛋白,以用于研究正常或异常红细胞的吞噬和清除中CD47-SIRPα信号通路的作用。方法全基因合成人源野生型SIRPα1 IgV结构域编码区;将合成片段构建入携带组氨酸（His）标签的pET-21a大肠杆菌载体,获得重组表达载体。使用大肠杆菌BL21 （DE3）细胞体外表达重组SIRPα1,并通过亲和层析法纯化;利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定重组SIRPα1;采用基于ELISA的红细胞结合试验评估2名Rh缺失表型献血者与正常Rh表型个体对照在红细胞结合水平的差异;流式细胞术检测红细胞CD47表达;采用单因素方差分析对数据做统计分析。结果成功构建重组表达载体,纯化获得野生型重组SIRPα1。成功建立了基于ELISA的红细胞结合试验方法并应用于评估重组SIRPα1与红细胞的结合。伴随CD47表达量的明显降低,Rh缺失表型个体的红细胞和正常个体相比,其与重组SIRPα1的结合能力下降。结论 CD47-SIRPα信号通路可能在Rh缺失表型红细胞的清除中发挥了作用。

Objective To obtain the recombinant functional domain of wild-type signal regulatory protein alpha 1（ SIRPα1） so as to study the role of the CD47-SIRPα signaling pathway involved in the phagocytosis and clearance of normal or abnormal RBCs. Methods The coding region of the wild-type human SIRPα1 domain IgV was synthesized,and then the recombinant expression vector was constructed by inserting the synthetic fragments into the pET-21 a Escherichia coli（ E. coli） vector with Histidine（ His） tag added. The recombinant SIRPα1 was in vitro expressed in E. coli BL21（ DE3） cells and purified by affinity chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis（ SDS-PAGE） was used for the identification of the recombinant SIRPα1. The ELISA-based erythrocyte-binding assay（ EBA） was used for evaluating the levels of recombinant SIRPα1 binding with RBCs,by using fresh blood from two Rh-deficient donors and controls with normal Rh phenotypes. The expression of CD47 on erythrocytes was evaluated by flow cytometry. The one-way ANOVA was used for data analysis. Results We obtained the correctly constructed recombinant expression vector and purified recombinant wildtype human SIRPα1. The ELISA-based EBA assay was developed and successfully used in the evaluation of recombinant SIRPα1 binding with RBCs. Along with markedly reduced CD47 expression,RBCs from both Rh-deficient donors showed decreased recombinant SIRPα1 binding ability when compared with normal individuals. Conclusion CD47-SIRPα signaling pathway may play a role in clearance of Rh-deficient RBCs.