论文部分内容阅读
目的:用中国仓鼠卵巢(CHO)细胞表达抗人CD86双价抗体(CD86-diabody),鉴定该抗体对表达CD86分子肿瘤细胞的识别及介导的生物学效应。方法:从分泌抗人CD86小鼠源抗体的杂交瘤1D1中克隆抗体重、轻链可变区基因VH及VL。SOE-PCR构建VH-(GGGGS)-VL双价抗体基因,整合到真核表达载体pIRES2-EGFP中,脂质体法转染CHO细胞,FCM筛选G418加压培养的阳性克隆。IMAC纯化并定量抗体。免疫荧光法分析抗体对人B系淋巴瘤细胞株Raji及Daudi膜型CD86分子的阳性结合率,将抗体加入到Raji细胞的培养体系中,培养72 h,MTT法分析抗体对细胞体外增殖的影响。结果:获得了稳定分泌抗人CD86-diabody的CHO细胞株。培养上清中抗体纯品的得率为5.24 mg/L。抗体与Raji及Daudi细胞的阳性结合率分别为77.2%及70.6%。经抗体孵育72 h,Raji细胞体外增殖抑制率为37%。结论:成功制备了抗人CD86-diabody,可特异性结合细胞膜型CD86分子,并对表达该分子的肿瘤细胞体外增殖具有抑制效应。
OBJECTIVE: To express the anti-human CD86 diabody using Chinese hamster ovary (CHO) cells and identify the biological effects of the antibody on the recognition and mediation of CD86-expressing tumor cells. METHODS: The antibody heavy and light chain variable region genes VH and VL were cloned from hybridoma 1D1 secreting anti-human CD86 mouse antibody. The VH- (GGGGS) -VL bivalent antibody gene was constructed by SOE-PCR and integrated into the eukaryotic expression vector pIRES2-EGFP. CHO cells were transfected by lipofectamine and the positive clones were selected by FCM. IMAC purified and quantified antibody. The antibody positive rate of Raji and Daudi membrane type CD86 molecules was analyzed by immunofluorescence. The antibody was added into Raji cell culture system and cultured for 72 h. The effect of antibody on cell proliferation was analyzed by MTT assay . Results: A CHO cell line stably secreting anti-human CD86-diabody was obtained. The yield of pure antibody in the culture supernatant was 5.24 mg / L. The positive binding rates of antibodies to Raji and Daudi cells were 77.2% and 70.6%, respectively. After 72 h of antibody incubation, the proliferation inhibition rate of Raji cells in vitro was 37%. Conclusion: The anti-human CD86-diabody was successfully prepared and could specifically bind to the membrane-type CD86 molecule and inhibit the proliferation of tumor cells expressing this molecule in vitro.