【摘要】 目的 :构建带有 6个组氨酸纯化标签的hCGβ和hCGβ C3d3融合蛋白的分泌型真核表达质粒 ,在CHO细胞中获得具有生物学活性的重组蛋白的高效表达。方法 :利用高效真核表达载体phCMV1,构建phCMV1 6His hCGβ和phCMV1 6His hCGβ C3d3,脂质体法转染CHO细胞 ,G4 18(80 0 μg ml)筛选抗性克隆。放射免疫法检测抗性克隆培液中hCGβ表达量 ,Westernblotting和Raji细胞免疫化学法鉴定hCGβ C3d3融合蛋白。用镍柱和凝胶过滤层析纯化表达产物。 结果 :酶切及测序结果显示 ,phCMV1 6His hCGβ及phCMV1 6His hCGβ C3d3构建正确。在CHO细胞中成功地获得了hCGβ和hCGβ C3d3融合蛋白的高效表达 ,phCMV1载体的表达效率是pcDNA3的 1 6倍。表达产物经纯化后得到了所需的hCGβ和hCGβ C3d3融合蛋白。 结论 :hCGβ和hCGβ C3d3融合蛋白在CHO细胞的高效表达为下一步比较hCGβ抗原及hCGβ C3d3融合蛋白免疫动物的体液免疫效应和抗生育效果奠定了基础更多还原

【Abstract】 Objective:To construct the eukaryotic expression vectors of the secreted hCGβ and hCGβ- C3d3 fusion cDNA with a 6His tag and to express high-effectively the both recom binant proteins in CHO cells. Methods:phCMV1-6His-hCGβ and phCMV1-6His-hCGβ-C3d3 expression vectors, based on t he phCMV1, were constructed by way of molecular cloning. Then the recombinant ex pression vectors were transfected into CHO cells with Lipofectamine reagent. The resistant clones secreting the recombinant proteins were established under the selection of G418 (800 μg/ml). The secreted hCGβ and hCGβ-C3d3 fusion protei n concentration in the CHO cell supernatant was assayed by the radioimmunoassay. Both Western blotting and Raji cell immunocytochemistry were used to identify th e hCGβ-C3d3 fusion protein. Results:The authors have successfully constructed the plasmids of phCMV1-6His-hCGβ an d phCMV1 - 6His-hCGβ-C3d3 and gained a certain amounts of the correspondent recombina nt proteins from the transfected CHO cell culture supernatant,phCMV1 achieved 1 .6-fold higher expression levels than that of pcDNA3. Conclusion:The high expression of the hCGβ and hCGβ-C3d3 fusion protein in CHO cells are benefit for testifying immunogenicity of hCGβ and hCGβ-C3d3 fusion protein i n animal immunization in future. [更多还原