目的 制备1株抗黑猩猩腺病毒68型（chimpanzee adenovirus type 68，AdC68）六邻体（hexon，hex）蛋白重组单克隆抗体（单抗）。方法 表达并纯化AdC68-hex蛋白，免疫BALB/c小鼠，取其脾细胞与骨髓瘤细胞融合，经间接ELISA及细胞免疫染色法筛选得到1株分泌抗AdC68-hex蛋白单抗的杂交瘤细胞。抽提杂交瘤细胞RNA进行测序，获得单抗重链与轻链可变区序列，分别与小鼠重链与轻链恒定区序列拼接得到小鼠全长IgG1κ抗体重链与轻链，并构建至真核细胞高效表达质粒。将重链与轻链质粒共同转染至哺乳动物细胞表达并纯化得到抗AdC68-hex重组单抗。将抗体用于滴定AdC68、重组AdC68及AdC68重组疫苗，并开展方法学验证。结果 表达制备的AdC68-hex蛋白能有效诱导小鼠的病毒产生抗AdC68-hex抗体。抗AdC68-hex重组单抗用于重组AdC68滴定具备良好的专属性、耐用性，在4.56~9.57 lg感染单位/ml范围内具备良好的线性、准确度和精密度。结论 制备的重组抗AdC68-hex单抗适用于AdC68及AdC68重组疫苗载体相关的基因治疗药物及疫苗等的检测。

Objective To generate a recombinant monoclonal antibody (mAb) against the hexon(hex) protein of chimpanzee adenovirus type 68 (AdC68). Methods The AdC68-hex was expressed and purified. BALB/c mice were immunized with the purified AdC68-hex and splenocytes were harvested for fusion with myeloma cells.One hybridoma cell line secreting mAb against AdC68-hex was selected using indirect ELISA and cell immunostaining. RNA was extracted from hybridoma cells for sequence analysis, and the variable regions of the heavy and light chains of the mAb were obtained. The variable regions were then joined with the constant regions of mouse heavy and light chains to generate the full-length mouse IgG1κ heavy and light chain genes, respectively to cloned into high-efficiency eukaryotic expression vectors. The heavy and light chain plasmids were co-transfected into mammalian cells to express and purify the recombinant mAb against AdC68-hex. Using this mAb for viral titration of AdC68, recombinant AdC68, and AdC68-based vaccines, the methodological validation was conducted. Results The expressed AdC68 hex protein successfully induced anti-AdC68-hex mAb production in mice. Recombinant mAb against AdC68-hex was used for viral titration of recombinant AdC68, showing good specificity, robustness,as well as good linearity, accuracy, and precision within the range of 4.56 to 9.57 lg infectious unit/ml. Conclusion The recombinant mAb against AdC68-hex generated is suitable for the detection of gene therapy drugs and vaccines related to AdC68 vectors.