C-GPC3蛋白的表达及人源GPC3抗体的筛选

Expression of C-GPC3 protein and screening of human GPC3 antibody

目的构建C-磷脂酰肌醇蛋白聚糖3(glypican-3,GPC3)基因真核表达质粒,表达C-GPC3蛋白,并通过哺乳动物展示型全人源sc Fv抗体库筛选GPC3抗体。方法通过PCR从含有全长GPC3的质粒中扩增C-GPC3基因,将其克隆至p3457-his载体中,构建重组真核表达质粒p3457-C-GPC3,转染293T细胞,对C-GPC3蛋白的表达进行Western blot鉴定。将质粒p3457-C-GPC3瞬时转染293E细胞,获得C-GPC3蛋白,采用Ni亲和柱纯化后,进行FITC标记。以本课题组构建的哺乳动物展示型全人源sc Fv抗体库为起始文库,FITC-C-GPC3抗原按照10、2和0.8 nmol/L的浓度梯度,通过流式细胞术进行分选,获得荧光强度最强、比例约为0.1%的阳性细胞。结果重组表达质粒p3457-C-GPC3经菌液PCR和测序鉴定证明构建正确;瞬时转染293T和293E细胞均可表达相对分子质量约为25 000的C-GPC3蛋白;获得了可与C-GPC3结合的阳性率约为0.1%的阳性细胞。结论成功构建了重组真核表达质粒p3457-C-GPC3,并从哺乳动物展示型全人源sc Fv抗体库中筛选出了阳性细胞,为后期全人源GPC3抗体的构建和表达奠定了基础。

Objective To construct a eukaryotic expression vector for C-terminal glypican-3（C-GPC3）, and screen human GPC3 antibody from mammalian display human sc Fv antibody library. Methods C-GPC3 gene was amplified from the plasmid containing full-length GPC3 gene by PCR then cloned to p3457-his vector. The constructed recombinant plasmid p3457-C-GPC3 was transfected to 293 T cells, and the expressed C-GPC3 protein was identified by Western blot. 293 E cells were transiently transfected with recombinant plasmid p3457-C-GPC3, and the obtained C-GPC3 protein was purified by nickel affinity chromatography then labeled with FITC. Mammalian display human sc Fv antibody library constructed in our laboratory was used as the starting library with the FITC-C-GPC3 gradient concentration（10, 2 and 0. 8 nmol / L）to obtain the positive cells with the strongest fluorescence intensity（about 0. 1%）by flow sorting. Results Recombinant plasmid p3457-C-GPC3 was successfully constructed as proved by colony PCR and sequencing. The C-GPC3 protein with a relative molecular mass of about 25 000 was expressed in both transfected 293 T and 293 E cells. Positive cells which could combine with C-GPC3 protein were obtained, and the positive rate was about 0. 1%. Conclusion Eukaryotic expression vector p3457-C-GPC3 was successfully constructed, and positive cells were screened from mammalian display human sc Fv antibody library, which laid a foundation of the construction and expression of human GPC3 antibody.