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目的:构建含有FRT序列的CHO/dhfr-工程细胞株,并在此细胞株中用定点整合系统表达抗基孔肯亚病毒人鼠嵌合抗体。方法:首先克隆FRT序列与HBsAg的融合基因FRT-HBsAg,然后将FRT-HBsAg亚克隆到pCI neo的MCS中,构建表达载体pCI FRT-HBsAg。以此载体转染CHO/dhfr-细胞,用1g/L的G418筛选抗性克隆。检测抗性克隆HBsAg的表达,筛选表达量高的克隆作为宿主细胞株,命名为CHO/dhfr-FRT+。将表达质粒pA FRTHFLF和pOG44以1∶9的质量比混合,取2μg转染CHO/dhfr-FRT+。转染48h后,换选择培养基(撤掉H、T)用96孔板进行克隆化,2wk后对长成的单克隆进行检测。筛选正确整合到CHO/dhfr-FRT+细胞中FRT位点的克隆,并不断提高MTX的浓度,进一步筛选表达量更高的克隆。在MTX的浓度为2×10-7mol/L时,获得稳定表达细胞株,其最后的表达量为5mg/L。对此细胞株扩大培养后,收集上清并纯化,以纯化的嵌合抗体进行SDS-PAGE及Westernblot和抗原结合活性的检测。结果:构建了高转录活性位点整合FRT序列的CHO/dhfr-细胞株,并在此细胞株中表达抗基孔肯亚病毒人鼠嵌合抗体,表达量为5mg/L。结论:构建了含有FRT序列的CHO/dhfr-工程细胞株,并在此细胞株中用定点整合系统表达抗基孔肯亚病毒人鼠嵌合抗体,为用此系统表达抗体分子和其他蛋白分子奠定了基础?
OBJECTIVE: To construct a CHO / dhfr-engineered cell line containing FRT sequence and express chimeric antibody against chikungunya virus in this cell line by using the site-directed integration system. Methods: The fusion gene FRT-HBsAg between FRT sequence and HBsAg was cloned and subcloned into pCI neo MCS to construct expression vector pCI FRT-HBsAg. CHO / dhfr- cells were transfected with this vector and resistant clones were screened with 1 g / L of G418. The expression of HBsAg was detected, and the clone with high expression level was selected as the host cell line and named as CHO / dhfr-FRT +. The expression plasmids pA FRTHFLF and pOG44 were mixed at a mass ratio of 1: 9 and 2 μg of transfected CHO / dhfr-FRT + was taken. 48h after transfection, the selection medium (removal of H, T) with 96-well plates for cloning, after 2wk grown monoclonal detected. The clones correctly integrated into the FRT site in CHO / dhfr-FRT + cells were screened and the MTX concentration was continuously increased to further screen clones with higher expression levels. When the concentration of MTX was 2 × 10-7mol / L, stable expression cell lines were obtained, and the final expression was 5mg / L. After the cell lines were expanded and cultured, the supernatant was collected and purified, and the purified chimeric antibodies were subjected to SDS-PAGE, Western blot and antigen-binding activity tests. RESULTS: The CHO / dhfr- cell line with high transcriptional activity site-integrated FRT sequence was constructed and chimeric antibody against chikungunya virus was expressed in this cell line with an expression level of 5 mg / L. CONCLUSION: The CHO / dhfr-engineered cell line containing the FRT sequence was constructed and the chimeric antibody against chikungunya virus was expressed in this cell line by using the site-directed integration system. In order to express the antibody molecule and other protein molecules by this system, Foundation?