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目的阐明小鼠PTA1(mPTA1)/mCD226对杀伤性T淋巴细胞(CTL)分化的影响。方法构建小鼠PTA1-hIgFc真核表达载体,表达并纯化mPTA1融合蛋白,免疫家兔,获得抗mPTA1的多克隆抗体,并用偶联mPTA1-Fc融合蛋白的Sepharose-4B亲和层析柱纯化多克隆抗体。间接免疫荧光染色后经流式细胞术鉴定多克隆抗体的特异性。通过混合淋巴细胞培养(MLC)诱导产生CTL效应细胞,以51Cr释放试验检测mPTA1多克隆抗体在MLC中对T淋巴细胞分化及杀伤功能的影响。结果获得了mPTA1-hIgFc融合蛋白和针对mPTA1胞膜外区的多克隆抗体,该抗体能与转染细胞表面天然mP-TA1分子结合。mPTA1多克隆抗体对MLC诱导的CTL的杀伤有明显的抑制作用,并呈剂量依赖关系,mPTA1多抗加入的时间愈早,抑制杀伤作用的效果愈明显。结论抗小鼠PTA1的多克隆抗体可在小鼠混合淋巴细胞培养中明显抑制CTL的分化。
Objective To elucidate the effect of mouse PTA1 (mPTA1) / mCD226 on the differentiation of killer T lymphocytes (CTL). Methods The mouse pTA1-hIgFc eukaryotic expression vector was constructed, the mPTA1 fusion protein was expressed and purified, and the rabbit was immunized to obtain the polyclonal antibody against mPTA1. The mPTA1-Fc fusion protein was purified by Sepharose-4B affinity chromatography Clone antibody. Indirect immunofluorescence staining by flow cytometry to identify the specificity of polyclonal antibodies. CTL effector cells were induced by mixed lymphocyte culture (MLC), and the effect of mPTA1 polyclonal antibody on T lymphocyte differentiation and cytotoxicity in MLC was tested by 51Cr release assay. Results The mPTA1-hIgFc fusion protein and the polyclonal antibody to the extracellular membrane region of mPTA1 were obtained and this antibody could bind to the native mP-TA1 molecule on the surface of transfected cells. The mPTA1 polyclonal antibody significantly inhibited the killing of CTL induced by MLC in a dose-dependent manner. The earlier the mPTA1 polyclonal antibody was added, the more obvious the effect of inhibiting the killing effect was. Conclusion The anti-mouse PTA1 polyclonal antibody can markedly inhibit CTL differentiation in mouse mixed lymphocyte culture.