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目的构建MHC-Ⅰ荧光四聚体真核表达质粒,并表达、纯化该蛋白,以更简便地检测特异性T细胞。方法运用分子克隆技术,构建sKb-ZsGreen-6× His标签的融合蛋白真核表达质粒,转染293FT细胞表达,并以镍亲和层析法纯化。结果经酶切及测序鉴定证实成功构建了MHC-Ⅰ荧光四聚体真核表达质粒并完成表达、纯化,并经SDS-PAGE电泳证实。结论得到了纯化的MHC-Ⅰ荧光四聚体,并经流式细胞术证实其与β2-微球蛋白、OVA肽结合后能被B3Z细胞识别,为特异性T细胞检测提供了一种更方便的方法。
Objective To construct the eukaryotic expression plasmid of MHC-Ⅰ fluorescent tetramer and express and purify the protein to detect the specific T cells more conveniently. Methods The fusion protein eukaryotic expression vector sKb-ZsGreen-6 × His was constructed by molecular cloning technique and transfected into 293FT cells for expression. The fusion protein was purified by nickel affinity chromatography. Results The digestion and sequencing confirmed that the eukaryotic expression plasmid of MHC-I fluorescent tetramer was successfully constructed, expressed and purified, and confirmed by SDS-PAGE electrophoresis. Conclusions The purified MHC-Ⅰ fluorescent tetramer was obtained and confirmed by flow cytometry to be recognized by B3Z cells after binding to β2-microglobulin and OVA peptide, which provided a more convenient method for the detection of specific T cells Methods.