【摘要】 目的 克隆、表达人Toll-like receptor2(TLR2)胞外段(A26-T588)基因,获得人TLR2胞外段蛋白。方法 RT-PCR扩增TLR2胞外段基因,以pcDNA3.1+质粒为载体在HEK293细胞中表达TLR2胞外段蛋白,同时以pcDNA3.1+/TLR2(A26-T588)重组质粒免疫昆明鼠,制备抗TLR2胞外段蛋白多抗。结果 PCR扩增及重组质粒测序结果表明成功地构建了pcDNA3.1+/TLR2(A26-T588)真核表达质粒,SDS-PAGE分析纯化产物在M_r为68000处出现明显蛋白条带。重组质粒DNA免疫小鼠3次后,血清抗体滴度可达1∶250。TLR2胞外段蛋白可与LPS结合,并在一定的质量浓度范围内呈剂量依赖性。结论 构建的pcDNA3.1+/TLR2(A26-T588)真核表达质粒可在哺乳动物细胞中表达TLR2胞外段蛋白,并与重组质粒DNA免疫小鼠抗血清及TLR2单克隆抗体TL2.1特异性反应,由此证明其表达正确。更多还原

【Abstract】 Objectives Cloning and expression of the extracelhilar domain（A26-T588） of human TLR2, identification and purification the TLR2 protein. Methods The extracellular domain gene of human TLR2 was amplified from the total RNA of human mononuclear cell by RT-PCR, then the gene with six histididine segments was cloned into pcDNA3.1+ and the recombinant plasmid pcDNA3.1+/TLR2 was transfected into HEK293 cell. The recombinant TLR2 protein was purified by histidine affinity chromatography system. In addition, purified pcDNA3.1+/TLR2 plasmid was injected i.m. into Kunming mice with immunization dosage of 100μg/mouse per time. Results The TLR2 gene was proved to be correct by DNA sequencing. Expressed TLR2 protein analyzed by SDS-PAGE showed a correct size and reacted specifically with the antibody from mice immunized with pcDNA3.1+/TLR2 and TL2.1. The extracellular domain of TLR2 could bind with LPS, and there was a dose-response relationship between LPS and OD_490nm. Conclusion The recombinant plasmid pcDNA3.1+/TLR2 could express TLR2 extracellular domain protein correctly in HEK293. The polyclonal antibody from mice immunized with pcDNA3.1+/TLR2 can react with TLR2 protein specifically.更多还原