特异性HPV16E7 CTL表位肽-MHC I复合物单抗的制备及鉴定

目的制备抗HPV16E7CTL表位（49-57）-MHC Ⅰ类分子复合物的特异性单克隆抗体，并对其敏感性、特异性及亲和力进行鉴定，为今后研究HPV16感染后机体对病毒蛋白的免疫呈递途径研究奠定基础。方法制备高纯度的HPV16E7 CTL表位（49．57）肽RAHYNIVTF，将其与TAP缺陷的RMA-S细胞孵育后免疫BALB／c小鼠，常规收集小鼠脾细胞与SP2／0细胞融合，筛选杂交瘤细胞株的阳性克隆，并对其进行一系列鉴定。结果筛选出的杂交瘤细胞株能稳定分泌抗HPV16E7（49．57）-MHC Ⅰ类分子复合物的单抗，不仅能与装载有RAHYNIVTF的TAP缺陷的RMA-S细胞结合，也能与具有内源性抗原处理能力的RMA细胞结合，但不能与装载有类似变异肽（进行氨基置换后的RAHYNIVTF）或乙肝病毒对照肽的RMA-S或RMA细胞反应，具有良好的敏感性、特异性及亲和力。结论本实验制备表位肽．MHC Ⅰ类复合物单抗的方法，对今后研究包括HPV16感染在内的病毒感染免疫有很大的临床应用价值。

Production and identification of the monoclonal-antibodies specific for MHC class I complexes bound with HPV16E7 CTL epitopic peptide

Objective To produce specific monoclonal-antibodies (MAbs) for MHC class I complexes bound with HPV16E7 CTL epitopic peptide (49-57),and provide a foundation for the investigation of the present pathway of viral antigen protein after in vitro viral infection. Methods Highly purified HPV16E7CTL epitope(49-57) (RAHYNIVTF) was produced,and then TAP-deficient RMA-S cells incubated with RAHYNIVTF were used to immunize the BALB/c mouse.The spleen cells of the mice were regularly harvested and fused with the SP2/0 cells.The growing fusion wells were screened and the abstracted Mabs were identified in terms of sensitivity,specificity and affinity. Results The screened hybriroma cells could steadily secret the MAbs specific for MHC class I complexes bound with HPV16E7CTL epitopic peptide.The MAbs showed high reactivity with TAP-deficient RMA-S cells loaded with RAHYNIVTF and RMA-S cells which have the ability to process the endogenous MHC class I complexes,while minimally bound to class I molecules bearing other peptides,the results indicated excellent sensitivity, specificity and affinity of the MAbs. Conclusion The experiments provide a method for producing MAbs for epitopic peptide bound MHC class I complexes.