【摘要】 为了获得同时识别人类和小鼠MAIT细胞的特异性抗体,我们制备了MAIT细胞TCRα链Jα33融合蛋白及其抗体,为MAIT细胞在炎症性肠道疾病(IBD)中作用的研究提供有利的工具。用Jα33的全长序列为引物经PCR扩增Jα33串联体,扩增的串联体片段连接进入T载体,阳性克隆转入pGEX-4T-1表达载体中原核表达GST-Jα33融合蛋白,纯化后的融合蛋白免疫新西兰大白兔制备anti-Jα33多克隆抗体。并分别用Western blot和RT-PCR检测IBD模型小鼠MAIT细胞Jα33的表达。成功获得分子量约为38 kD的融合蛋白,免疫大白兔后得到抗Jα33的多克隆抗体,Western blot结果显示Jα33多克隆抗体能检测到人外周血单个核淋巴细胞表面Jα33的表达,而HeLa中则无Jα33的表达;MAIT细胞Jα33在IBD小鼠肠组织中表达量明显低于正常对照。成功制备了能同时识别人类和小鼠MAIT细胞Jα33的多克隆抗体,为研究MAIT细胞的定位和功能开启了方便之门。更多还原

【Abstract】 To obtain specific antibodies which can identify human and mouse MAIT cells,we produced a polyclone antibody against TCR Jα33 from mouse MAIT cells.This antibody facilitating the study of MAIT cells in IBD.Jα33 concatemer was am- plified by PCR using the Ja33 full sequence as primers.And amplified concatemers were ligated into the T vector.Then positive clones were transferred to pGEX-4T-l expression vector to produce the GST-Ja33 fusion protein.The purified fusion protein was used to immunize New Zealand white rabbits and produced the anti-Ja33 polyclonal antibody.Respectively,we detected the specificity of the anti-Jα33 antibody with Western blot and immunofluorescence,we successfully obtained the fusion protein of the molecular weight about 38kD.After immunized the white rabbit we got an anti-Jα33 polyclonal antibody.Western blot results showed that this Jα33 polyclonal antibody could detect the expression of Jα33 on the surface of human peripheral blood mononuclear cells.However,HeLa cells have no expression of Ja33.The expression of MAIT cell’s a chain in the intestine of IBD model was lower than that in the normal controls.We successfully produced a polyclonal antibody that can recognize human and mouse MAIT cells,which will open the door for the research of the localization and function of MAIT cells.更多还原