Glypican-3的原核表达及其单克隆抗体的制备和初步应用

Prokaryote expression of GPC3 C terminal protein and prepration of monoclonal antibody

磷脂酰肌醇蛋白聚糖3(Glypican-3)是一类主要表达在原发性肝细胞癌表面的硫酸乙酰肝素聚糖蛋白,是一种重要的肝癌标志物。本实验通过构建含有Glypican-3C端原核载体pIGH3-GPC3并诱导表达,以之免疫BALB/c小鼠后筛选到两株分泌特异性抗Glypican-3抗体的杂交瘤细胞株GPC3-9和GPC3-11,抗体亲和力常数分别达到1.24×109 mol/L和1.668×109 mol/L;经ELISA、Western blot以及免疫荧光试验进一步验证了GPC3-9和GPC3-11均能特异性识别原核表达或肝癌细胞内源性Glypican-3分子,具有潜在的开发及应用前景。

Glypican-3, a kind of heparan sulfate protein glycan has been found highly expressed in hepatocellular carcinoma （HCC） and some other human cancers which could be a potential molecular marker for HCC. In this research, we aim to get monoclonal anibodies （McAb） against Glypican-3 with hight affinity. Using the technique of genetic engineering, the C termi- nal of Glypican-3 （1 020-1 686bp）was obtained by PCR amplification and the recombinant plasmid pIGH3-GPC3 was constructed by insert the PCR product into pIGH3. The recombinant plasmid was transfected into E. coli BL21 and the C terminal of recombinant human Glypican-3 was expressed and purified. The BALB/c mouse was immunized with the recombinant prot- ern to get hybridoma cells. The cell clones named GPC3-9, GPC3-11 which secret monoclonal antibodies against Glypican-3 were identified by ELISA. The results of indirect ELISA, cell ELISA, Western blot and immunofluorescence showed that McAb GPC3-9, GPC3-11 could recognize the prokaryotical expressed or endogenous Glypican-3 in liver cancer cells. Mean- while, the titers of ascitic fluids assessed by ELISA assay were 1 ： 2 048 and the affinity constants were 1.24× 10^9mol/L, 1. 668×10^9mol/L respectively. Taken together, these two McAb could have the potential in detecting Glypican-3 and in the treatment of HCC.