【摘要】 目的　比较真核表达载体 phCMV1和pcDNA3在中国仓鼠卵巢 (CHO)细胞中表达人绒毛膜促性腺激素 β(hCGβ) C3d3融合蛋白的表达效率 ,以获得hCGβ C3d3融合蛋白的高效表达。　 方法　利用高效真核表达载体 phCMV1,构建带有 6个组氨酸纯化标签的融合蛋白表达质粒phCMV1 6His hCGβ C3d3;脂质体法介导phCMV1 6His hCGβ C3d3和pcDNA3 hCGβ C3d3转染CHO细胞 ,G4 18(80 0 μg/ml)筛选抗性克隆 ,G4 18(30 0 μg/ml)维持培养、扩增 ,待阳性克隆 95 % 汇片时换用无血清培养基培养。放射免疫法检测hCGβ表达量 ,Westernblot鉴定表达产物 ,Raji细胞免疫化学染色法鉴定hCGβ与C3d分子的成功融合。反复冻存和复苏高效表达hCGβ C3d3融合蛋白的阳性克隆 ,比较表达效率的变化。用镍柱和凝胶过滤层析纯化表达产物。　结果　酶切鉴定及测序证实 phCMV1 6His hCGβ C3d3构建正确 ,并成功地在CHO细胞中获得了 6His hCGβ C3d3融合蛋白的表达。无血清培液中 2 4、4 8、72h融合蛋白的表达量分别为 4 96、5 2 7、6 33mIU/ml/ 1× 10 6细胞 (以hCGβ含量计算 ) ,phCMV1比 pcDNA3载体的表达效率高 1.4倍。 3次冻存和复苏高效表达hCGβ C3d3融合蛋白的阳性克隆 ,表达效率无明显变化。表达产物经纯化后获得了所需的hCGβ C3d3?更多还原

【Abstract】 Objective: To compare the expression efficiency of hCGβ-C3d3 fusion protein in CHO cells transfected by phCMV1 and pcDNA3 vector,aiming to produce a great deal of the recombinant fusion protein expressed in CHO cells. Methods: phCMV1-6His-hCGβ-C3d3 expression vectors,based on the phCMV1,were constructed to allow fusion of hCGβ and C3d3 and a 6His-tag at the N-terminal end. Then the recombinant eukaryotic expression vectors,phCMV1-6His-hCGβ-C3d3 and pcDNA3-hCGβ-C3d3,were transfected into CHO cells with the aid of lipofectamine reagent. The resistant clones that can secrete the recombinant fusion proteins were established under the selection of G418 (800 μg/ml). The concentration of secreted hCGβ protein in culture media was assayed by the radioimmunoassay. Both Western blot and Raji cell immunocytochemistry were used to identify the hCGβ-C3d3 fusion protein. The recombinant proteins were purified using the immobilized metal-ion affinity chromatography (IMAC) under native conditions and SephadexG-150 column gel filtration chromatography.Results: We have successfully constructed the plasmid of phCMV1-6His-hCGβ-C3d3 and gained a comparable amount of recombinant fusion protein with the biological activity in CHO cells. The expression levels of hCGβ in the supernatants from phCMV1-6his-hCGβ stably transfected CHO cells at 24,48 and 72 h under serum-free condition are 496,527 and 633 mIU/ml/1×10 6cells respectively. The phCMV1 vector can produce 1.4 times higher level of hCGβ-C3d3 fusion protein than pcDNA3 vector in CHO cells.Conclusion: The phCMV1 vector can achieve higher expression efficiency of hCGβ-C3d3 fusion protein in CHO cells than pcDNA3 vector. With its 6His-tag,hCGβ-C3d3 fusion protein can be successfully purified.更多还原