抗肝癌单链抗体二聚体的制备及其对肝癌细胞株增殖的影响

Preparation of humanized single-chain Fv dimers for hepatocellular carcinoma and investigation of their effects on HCC cells

目的:探讨特异性抗肝癌单链抗体二聚体在毕赤酵母中高效表达、纯化和鉴定其生物学活性的方法,并观察其对肝癌细胞增殖的影响。方法:构建酵母表达载体pGAPZαA-BDM,转化感受态大肠埃希菌DH5α,选择测序正确的阳性克隆进行扩增后,电转化酵母细胞,表达抗体二聚体,对表达抗体进行纯化、二聚体相对分子质量及形成比例和浓度检测,鉴定其对肝癌细胞的结合活性,免疫组化检测二聚体对肝癌组织抗原的特异性,MTT法观察二聚体抗体对肝癌细胞株增殖的影响。结果:测序及SDS-PAGE显示,成功构建及在酵母中表达抗体二聚体BDM,纯化后的抗体经凝胶过滤色谱检测,二聚体的相对分子质量为62×103,二聚体形成比例为92%;表达量为30mg/L菌液。BDM与3株肝癌细胞结合效价为1:128,高于其亲本单链抗体。免疫组化显示,BDM与肝癌、肝硬化、胃癌、肠癌及正常肝组织反应的阳性率分别为40.0%、11.0%、6.7%、5.0%和0,P值均<0.05;MTT显示,二聚抗体BDM显示较好的抑瘤性,对肝癌细胞株的抑制率为30%,且有浓度依赖性。结论:成功制备高表达量、高特异性、较好的活性及较高稳定性的抗肝癌单链抗体二聚体BDM,其对肝癌细胞株的增殖有明显抑制作用,为后续将该抗体用于肝癌的放射免疫诊断和靶向治疗研究奠定了基础。

OBJECTIVE：To explore the methods of expressing humanized single-chain Fv dimers for hepatocellular carcinoma BDM in yeast pichia pastoresa and a ssaying its biological activities,and investigate the effects of BDM on HCC cells.METHODS：pGAPZαA-BDM was contructed and transformed it into DH5α.The recombinant vector plasmids were sequenced and then transformed to yeast pichlia pastores GS115.BDM was expressed in yeast pichlia pastores.Expression products were identified by SDS-PAGE,Western blotting,size exclusion gel chromatography（SEC）,ELISA and immunohistochemistry.The effect of BDM on HCC cells was investigated by MTT.RESULTS：Sequence analyzing and SDS-PAGE showed that the diabodies BDM was successfully expressed in yeast pichlia pastores.The SEC results suggested the molecular weight of the expressed products was 62×103 with 92% dimerization.The concentration of BDM was 30 mg/L.The expression products showed significantly stronger binding to hepatocellular carcinoma cells than the parental scFv,with the binding valence was 1：128;The purified diabodies were specially binding to tumor antigen of HCC,with the binding-positive rate 40.0% versus those of cirrhosis,gastric cancer,intestine cancer and normal liver tissue （binding-positive rate were 11.0%,6.7%,5.0%,0 respectively）.There was a significant inhibition of BDM on the growth of HCC cells.The inhibition ratio was 30% and dependent by antibody concentration.CONCLUSION：The functional humanized diabodies that has inhibitory efficiency on HCC cells are achieved,and it is essential of using this diabodies in hepatocellular carcinoma targeted diagnosis and therapy.