超滤-高效液相色谱法检测pHLA复合物中的抗原肽

Detection of the antigenic peptides in pHLA complexes by ultrafiltration-high performance liquid chromatography

摘要:

重组HLA-Ⅰ类分子/抗原肽复合物(pHLA复合物)在研究人类T细胞特异性免疫应答中有重要用途。pHLA复合物的制备以基因工程及蛋白体外稀释折叠复性技术为基础，在体外复性体系中重组HLA-Ⅰ类分子正确折叠，并结合抗原肽形成复合物。本研究建立了一种超滤-高效液相色谱法(超滤-HPLC法)定量检测重组pHLA复合物中的抗原肽，尤其针对少量制备产物中抗原肽的检测。通过将重组HLA-Ⅰ类分子和抗原肽加入到复性缓冲液中，使重组HLA-Ⅰ类分子的重链(heavy chain,HC)与轻链(β2m)复性折叠，与含锚定残基的VYF抗原肽结合形成pHLA复合物，经超滤去除未结合的游离抗原肽VYF而保留复合物，最后将pHLA复合物经酸处理破坏其相互作用从而释放抗原肽，再超滤收集VYF抗原肽并进行HPLC检测，所测得的VYF抗原肽即为重组HLA-Ⅰ类分子与抗原肽相互作用所结合的抗原肽。结果显示，制备的重组pHLA复合物可被HLA-Ⅰ分子构象特异性抗体W6/32识别，这说明重组HLA-Ⅰ类分子折叠构象正确，可鉴定为pHLA复合物；而超滤-HPLC法也可检测到pHLA复合物中含有抗原肽VYF，因此将超滤-HPLC法用于检测pHLA复合物的方法可行。与Western blotting法相比，超滤-HPLC法定量检测抗原肽浓度范围为0–9μg/mL，可根据复合物中结合的抗原肽量来优化不同结合条件，以提高HLA-Ⅰ类分子折叠效率并促进HLA-Ⅰ类分子结合抗原肽，还可根据pHLA复合物结合的抗原肽含量计算复性体系中形成pHLA复合物的制备率。因此文中所建立的超滤-HPLC法可用于pHLA复合物制备过程的质量控制，在T细胞特异性免疫研究、人工抗原呈递细胞以及特异性四聚体探针应用开发方面都具有优势。

Abstract:

Recombinant HLA-Ⅰ molecules/antigenic peptide complexes (pHLA complexes) are applied in the research of human T cell-specific immune responses.The preparation of pHLA complex is based on genetic engineering and protein in vitro dilution and folding-refolding technology.In an in vitro refolding system,recombinant HLA-Ⅰ molecules correctly fold and bind with antigenic peptides to form complexes.In this study,ultrafiltration-high performance liquid chromatography (ultrafiltration-HPLC) was used for quantitative determination of the antigenic peptides in recombinant pHLA complexes,especially for those in a small amount of prepared products.By adding the recombinant HLA-Ⅰ molecules and antigenic peptides into the refolding buffer,the heavy chain (HC) and light chain (β2m) of recombinant HLA-Ⅰ molecules were refolded and bond with the VYF antigenic peptide containing anchor residues to form a pHLA complex.The unbound free antigenic peptide VYF was removed by ultrafiltration to retain the complex.Finally,the pHLA complex was treated by acid to destroy its interaction,thus releasing the antigenic peptide.The results showed that the prepared recombinant pHLA complex was recognized by HLA-Ⅰ molecule specific antibody W6/32,which indicated that the recombinant HLA-Ⅰ class molecule had correct folding and was identified as pHLA complex.The antigen peptide VYF contained in the pHLA complex was also detected by ultrafiltration-HPLC,so it is feasible to apply ultrafiltration-HPLC for determination of pHLA complex.Compared with Western blotting,the concentration of antigenic peptides detected by ultrafiltration-HPLC was 0–9 μg/mL.The binding conditions can be optimized according to the amount of antigenic peptides bound in the complex in order to improve the folding efficiency of HLA-Ⅰ molecules and promote the binding of HLA-Ⅰ molecules to antigenic peptides.The production rate of pHLA complexes in the refolding system can also be calculated according to the content of antigenic peptides bound by pHLA complexes.Therefore,ultrafiltration-HPLC in this study can be used for the quality control of the preparation process of pHLA complexes,and may facilitate the research of T cell-specific immunity,artificial antigen-presenting cells,and development of specific tetramer probe applications.