人PD-L2基因克隆及其在大肠杆菌中的表达

目的 克隆人PD-L2基因并构建PD-L2胞外区的原核表达载体,在大肠杆菌中进行表达。方法 以RT-PCR方法从活化的人外周血单个核细胞总RNA中克隆PD-L2基因的cDNA,构建PD-L2胞外区的原核表达载体,在大肠杆菌BL21(ED3)中进行表达并鉴定。结果 克隆到PD-L2基因cDNA编码区全长序列,经DNA测序证明其与已报道的序列一致。进而构建了PD-L2胞外区的原核表达载体,并在大肠杆菌表达,免疫印迹分析表明在IPTG诱导后表达PD-L2胞外区蛋白,相对分子质量Mr为22000,与理论值大小相符。结论 成功克隆PD-L2基因,其胞外区蛋白在大肠杆菌中获得表达,为进一步研究PD-L2功能提供了条件。

Gene cloning of human PD-L2 gene and its expression in Escherichia coli

Objective To clone the human PD-L2 gene and to construct a prokaryotic expression vector for the extracellular domain of PD-L2. Methods The cDNA of human PD-L2 gene was cloned from the total RNA of activated human peripheral blood mononuclear cells by RT-PCR. The prokaryotic expression vector for the extracellular domain of PD-L2 was constructed and its expression in Escherichia coli (E. coli) was determined. Results The full-length coding sequence of PD-L2 gene was cloned and confirmed by DNA sequencing, which was identical to the published gene. The prokaryotic expression vector for the extracellular domain of PD-L2 was then constructed. The recombinant protein was expressed in E. coli after the IPTG induction and identified by Western blotting. The recombinant protein had a molecular weight of 22 000, which was consistent with theoretical calculation. Conclusion The gene of PD-L2 is successfully cloned and the extracellular domain protein of the PD-L2 is expressed in E. coli, which provides a basis for the further study of the function of PD-L2.