9.1C3/LAIR-1的真核表达、纯化及单克隆抗体的制备和鉴定

目的 表达并纯化9.1C3/LAIR-1胞膜外区与人IgCl Fc段的融合蛋白，轩针对9.1C3/LAIR-1胞膜外区的单克隆抗体（mAb)。方法 构建真核表达载体pIg/3C-9.1C3/LAIR-1,表达并纯化9.1C3/LAIR-1－Fe融合蛋白。以9.1C3/LAIR-1－Fc融合蛋白免疫BALB／c小鼠，制备。以间接免疫荧光染色和流式细胞术鉴定mAb的特异性，以竞争结合实验鉴定mAb识别LAIR－1的表位。结果表达并经9.1C3/LAIR-1－hIgFc融合蛋白，以其免疫BALB／c小鼠，获得1株针对9.1C3/LAIR-1胞膜外区的mAb 4H7。4H7对HL－60细胞和经9.1C3/LAIR-1 cDNA转染的COS7 的表位识别，与已报道的抗9.1C3 mAb体不同。 结论 成功地构建了9.1C3/LAIR-1胞膜外区基因的真核表达载体，并表达纯化了9.1C3/LAIR-1－hIgFc融合蛋白。获得一株针对9.1C3/LAIR-12胞膜外区的mAb，为进一步研究9.1C3/LAIR-1分子的2结构和功能提供了新的手段。

Eukaryotic expression and purification of 9.1C3/LAIR-1 and its preparation of monoclonal antibody

Aim To express and purify the fusion protein of the extracellular region of 9.1C3/LAIR 1 molecule with Fc fragment of human IgG1 and to prepare the monoclonal antibody (mAb) against the cxcellular region of 9.1C3/LAIR 1 expressed in the eukaryotic cells. Methods The eukaryotic cell expression vector pIg/3C 9.1C3LAIR 1 was constructed. BALB/c mice were immunized with affinity column purified fusion protein from the culture supernatant of 9.1C3/LAIR 1 hIgFc vector transfected COS7 cells for preparing mAb. The specificity of mAb was identified by indirect immunofluorescence staining and flow cytometry. The epitopes recognized by mAbs were identified by competitive binding assay. Results The 9.1C3/LAIR 1 hIgFc fusion protein was expressed and purified. One positive hybridoma named 4H7 was obtained, which could react with both the HL 60 cells expressing natural LAIR 1 molecule and COS7 cells transfected with LAIR 1 cDNA. The epitopes recognized by mAbs 9.1C3 and 4H7 were distinct. Conclusion We have successfully constructed the expression vector of recombinant 9.1C3/LAIR 1 hIgFc fusion gene and expressed the 9.1C3/LAIR 1 hIgFc fusion protein. One specific mAb against the extracellular region of 9.1C3/LAIR 1 is raised, which may provide a useful tool for studying the structure and function of 9.1C3/LAIR 1.