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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 a method for efficiently expressing, purifying and identifying the biological activity of specific anti-HCC single chain antibody dimer in Pichia pastoris and to observe its effect on the proliferation of HCC cells. Methods: The yeast expression vector pGAPZαA-BDM was constructed and transformed into competent E. coli DH5α. The positive clones with the correct sequencing were selected and amplified. The yeast cells were electrotransformed into yeast cells to express the antibody dimer. The expressed antibody was purified and the dimer Relative molecular mass, ratio of formation and concentration, and its binding activity to hepatoma cells. Immunohistochemistry was used to detect the specificity of the dimer to the hepatoma antigen. The effect of dimer antibody on the proliferation of hepatocellular carcinoma cell line was observed by MTT assay. Results: Sequencing and SDS-PAGE showed that BDM was successfully constructed and expressed in yeast. The purified antibody was detected by gel filtration chromatography. The relative molecular mass of the dimer was 62 × 103, and the dimer formation ratio 92%; expression amount of 30mg / L bacteria solution. The binding titer of BDM to 3 hepatoma cells was 1: 128, higher than that of its parental single chain antibody. Immunohistochemistry showed that the positive rates of BDM in liver cancer, cirrhosis, gastric cancer, colorectal cancer and normal liver tissues were 40.0%, 11.0%, 6.7%, 5.0% and 0, respectively, P < Polybodies BDM showed good tumor inhibition, inhibition rate of liver cancer cell lines was 30%, and a concentration-dependent. CONCLUSION: BDM, an anti-HCC single chain antibody with high expression, high specificity, good activity and high stability, has been successfully prepared and has a significant inhibitory effect on the proliferation of HCC cell lines. In the diagnosis of liver cancer radioimmunotherapy and targeted therapy laid the foundation for the study.