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目的构建并在CHO细胞中表达抗人CD86单链抗体(CD86-scFv),研究该抗体与抗原的结合特性及介导的生物学功能。方法从分泌鼠源CD86抗体的杂交瘤细胞中克隆出VH和VL基因,构建含有前导肽L-VH-Linker-VL抗体编码基因的表达载体并转染CHO细胞。经筛选高分泌株并扩大培养后收集上清进行纯化。流式细胞术分析CD86-scFv对不同细胞膜型CD86分子的识别及与鼠源亲本抗体1D1的竞争抑制效应。MTT法分析CD86-scFv与Raji细胞表达的CD86结合后对细胞生长的影响。结果获得了稳定表达抗人CD86-scFv的CHO细胞株及相应抗体。CD86-scFv与CD86基因转染细胞株L929-CD86、天然表达CD86分子的人B系淋巴瘤细胞Raji和Daudi细胞的阳性结合率分别为67.0%、72.3%和80.5%。CD86-scFv对鼠源亲本抗体1D1具有竞争结合能力,将CD86-scFv(终浓度20μg/mL)加入Raji细胞共同培养72 h时的细胞生长抑制率为28.3%。结论成功构建了稳定分泌抗人CD86-scFv的CHO细胞株(命名为SA-IV)。该抗体能够识别CD86分子并具有良好的生物学活性。
Objective To construct and express anti-human CD86 single chain antibody (CD86-scFv) in CHO cells and study its binding characteristics and biological functions. Methods VH and VL genes were cloned from murine CD86 antibody-secreting hybridoma cells and an expression vector containing the leader gene L-VH-Linker-VL antibody was constructed and transfected into CHO cells. After screening high secretion strains and expanding the culture supernatant was collected for purification. Flow cytometry was used to analyze the CD86-scFv recognition of different cellular membrane-type CD86 molecules and the competitive inhibition with mouse parental antibody 1D1. MTT assay CD86-scFv and Raji cells expressed CD86 binding on cell growth. Results The CHO cell line stably expressing anti-human CD86-scFv and the corresponding antibody were obtained. The positive binding rates of CD86-scFv to Raji and Daudi cells from CD86 gene-transfected cell line L929-CD86 and CD86-expressing human B lymphoma cells were 67.0%, 72.3% and 80.5% respectively. The ability of CD86-scFv to compete with murine parental antibody 1D1 was 28.3% when CD86-scFv (final concentration 20μg / mL) was added to Raji cells for 72 h. Conclusion The CHO cell line stably secreting anti-human CD86-scFv was successfully constructed (named SA-IV). The antibody recognizes CD86 molecules and has good biological activity.