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为了提高hCGβ的免疫原性 ,构建含新型分子佐剂C3d的hCGβ真核细胞表达质粒pcDNA3 hCGβ C3d3,使其转染稳定表达系统中国仓鼠卵细胞 (CHO )后 ,检测融合蛋白分泌情况。CHO细胞转染了pcDNA3 hCGβ C3d3质粒后 ,经 4 8h培养 ,1 0× 10 6个CHO细胞可分泌 6 6 0ng的重组融合hCGβ C3d3蛋白。细胞免疫化学技术分析显示 ,表达产物既有hCGβ的组分也有C3d的成分。经SDS PAGE及免疫印迹试验发现CHO细胞培养上清液中含有三组蛋白成分 ,其相对分子质量分别为12 0 0 0 0、90 0 0 0和 6 5 0 0 0。利用新型分子佐剂C3d与hCGβ的连接 ,构建了pcDNA3 hCGβ C3d3真核细胞表达质粒 ,为提高hCGβDNA免疫避孕疫苗的免疫原性及抗生育作用奠定了基础
In order to improve the immunogenicity of hCGβ, hCGβ eukaryotic expression plasmid pcDNA3 hCGβ C3d3 containing novel molecular adjuvant C3d was constructed and transfected into Chinese hamster ovary (CHO) cells. The secretion of fusion protein was detected. After the CHO cells were transfected with the pcDNA3 hCGβ C3d3 plasmid, 48 h after culture, 10 6 × 10 6 CHO cells secreted 6 6 ng recombinant fusion hCGβ C3d3 protein. Immunocytochemistry analysis showed that the expression product contained both the components of hCGβ and C3d. The results of SDS PAGE and Western blotting showed that there were three protein components in the culture supernatant of CHO cells. The relative molecular mass of CHO cells were 120,000, 90,000 and 65,000 respectively. The construction of pcDNA3 hCGβ C3d3 eukaryotic expression plasmid by using the new molecular adjuvant C3d and hCGβ laid the foundation for enhancing immunogenicity and anti-fertility of hCGβ DNA immunocontraceptive vaccine