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目的制备H-2Kd限制性胰岛GAD抗原肽/H-2Kd四聚体并用于检测相应的特异性同种CTL。方法以原核表达的sH-2Kd-BSP为重链,β2m为轻链,与人工合成的GAD抗原肽在体外利用稀释法进行共折叠复性得到单体。然后在BirA酶作用下对其进行生物素化,通过凝胶过滤层析法纯化生物素化的复合物单体,再与Strep-tavidin-FITC按4∶1比例混合形成四聚体;取Balb/c小鼠(H-2Kd)巨噬细胞加载胰岛GAD抗原肽,作为刺激细胞,取C3H小鼠脾细胞(H-2KK)作为效应细胞,在体外进行混合淋巴细胞培养,以获得针对H-2Kd限制性胰岛GAD抗原肽的特异性同种CTL。结果该四聚体能够与长期混合淋巴细胞培养出的同种特异性T细胞结合。结论成功构建胰岛GAD抗原肽/H-2Kd四聚体,并可用于针对H-2Kd限制性胰岛GAD抗原肽特异性同种CTL的检测。
Objective To prepare H-2Kd-restricted pancreatic islet GAD antigen peptide / H-2Kd tetramer and used to detect the corresponding specific allo-CTLs. Methods The prokaryotic expression of sH-2Kd-BSP as a heavy chain, β2m light chain, and synthetic GAD antigen peptide in vitro using the dilution method for the co-folding renaturation monomer. Then biotinylated with BirA enzyme, biotinylated complex monomers were purified by gel filtration chromatography and then mixed with Strep-tavidin-FITC at a 4: 1 ratio to form tetramers; Balb / c mouse (H-2Kd) macrophages were loaded with islet GAD antigen peptide, as stimulator cells, C3H mouse splenocytes (H-2KK) were used as effector cells and mixed lymphocyte culture in vitro to obtain H- 2Kd-specific islet GAD antigen peptide. As a result, the tetramer was able to bind to allospecific T cells cultured by long-term mixed lymphocytes. Conclusion The islet GAD antigen peptide / H-2Kd tetramer was successfully constructed and could be used for the detection of specific allo-CTL against H-2Kd-restricted islet GAD antigen peptide.