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构建以乙肝病毒核心抗原(HBcAg)为载体的带有1个EGFR和2个HER2的模拟B细胞表位多肽的融合表达质粒pET28a/HBcAg-△n,表达出融合蛋白并纯化,通过免疫BALB/c小鼠获得抗目的蛋白的抗体。采用PCR法将3个模拟表位插入HBc序列的第78～79位,再将该序列克隆入pET28a载体中,构建重组表达质粒,用大肠杆菌BL21(DE3)作宿主菌表达出融合蛋白HBHE,纯化后免疫BALB/c小鼠,检测小鼠的体液免疫应答。测序结果表明,重组质粒构建成功,SDS-PAGE电泳显示融合蛋白表达正确,ELISA检测到高滴度抗体。以HBcAg为载体的B表位被成功表达和纯化,获得高滴度的与3个B表位结合的抗体,为进一步研究HER家族成员联合多肽表位疫苗的广谱抗肿瘤作用奠定了基础。
To construct the fusion expression plasmid pET28a / HBcAg-△ n with one EGFR and two HER2-mimicking B-cell epitopes with hepatitis B virus core antigen (HBcAg) as carrier, the fusion protein was expressed and purified, c mice to obtain antibodies against the protein of interest. Three mimotopes were inserted into the 78th to 79th positions of the HBc sequence by PCR, and then cloned into pET28a vector to construct a recombinant expression plasmid. The recombinant protein HBHE was expressed by using E. coli BL21 (DE3) BALB / c mice were immunized after purification to detect the humoral immune response in mice. The sequencing results showed that the recombinant plasmid was successfully constructed, the SDS-PAGE electrophoresis showed that the fusion protein was correctly expressed, and the ELISA detected high titer antibody. The HBcAg-loaded B epitope was successfully expressed and purified to obtain high titers of antibodies binding to the three B epitopes, which laid the foundation for further study on the broad-spectrum anti-tumor effect of HER-family member polypeptide epitope vaccine.