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目的制备可生物素化的可溶性HLA-A2/HPV6E7单体,进一步组装成PE标记的可溶性HLA-A2/HPV6E7四聚体。方法以原核表达的sHLA-A2BSP为重链,β2m为轻链,与人工合成的HPV6E7(22-30)抗原肽(GLH-CYEQLV)利用稀释法进行共折叠复性得到单体。利用HLAI类分子单抗(W6/3)和抗Bzm抗体进行Westernblot和ELISA鉴定折叠产物的构象。以BirA酶对其进行生物素化,经过超滤离心后得到纯化的sHLA-A2/HPV6E7单体,再与Streptavidin-PE按4:1比例混合形成四聚体。结果该四聚体具有与HLA-A2阳性HPV6感染的CA患者的抗原特异性CTL结合活性。结论成功获得了天然构象的sHLA-A2/HPV6E7单体及PE标记的HLA-A2/HPV6E7四聚体。
Objective To prepare biotinylated soluble HLA-A2 / HPV6E7 monomers and further assemble into PE-labeled soluble HLA-A2 / HPV6E7 tetramers. Methods The prokaryotic expression of sHLA-A2BSP as the heavy chain, β2m as the light chain, and artificial synthetic HPV6E7 (22-30) antigen peptide (GLH-CYEQLV) were diluted by the method of co-folding renaturation monomer. The conformation of the folded product was identified by Western blot and ELISA using HLAI-like monoclonal antibody (W6 / 3) and anti-Bzm antibody. Biotinylated with BirA enzyme, the purified sHLA-A2 / HPV6E7 monomer was purified by ultrafiltration and then mixed with Streptavidin-PE at a 4: 1 ratio to form a tetramer. As a result, the tetramer had antigen-specific CTL-binding activity to HLA-A2-positive CA6-infected CA patients. Conclusion The natural conformation of sHLA-A2 / HPV6E7 and PE-labeled HLA-A2 / HPV6E7 tetramers were successfully obtained.