摘要
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 an eukaryotic expression vector that expresses MHC- Ⅰ - fluorescin tetramer and then induce the protein to express and be purified in order to detect antigenic specificity T cell more conveniently. Methods The eukaryotic expression vector of a fusion protein was constructed by linking the target gene fragment with the sequence of the sKb-ZsGreen-6 His Tag by molecular cloning technique, and then the recombinant was transfeeted into 293 FT to express the protein. Ni-affinity chromatography was used to purify the objective protein from the transfected cell lysis. Flow eytometry was used to detect the existence of B3Z cells pretreated with the mixture of our obtained protein, β2 mieroglobulin and peptide OVA （4 μg：4 μg：2.77 μg in 100 μl PBS）. Results An eukaryotic expression vector of MHC- Ⅰ - fluorescin tetramer was constructed successfully, and the protein was expressed in 293FT cells and purified. The mixture treated B3Z cells showed 88.3% green fluorescence in compared with those untreated cells. Conclusion The MHC- Ⅰ- fluorescin tetramer is produced and purified. The tetramer is recognized by B3Z cells after bounding to 132 microglobulin and peptide OVA, and it can be used as a convenient method for detecting antigenic specificity T cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2009年第6期484-486,共3页
Journal of Third Military Medical University
基金
国家高技术研究发展计划(863计划)(2006AA02Z416)~~
