【摘要】 本研究利用基因重组技术构建人IL35-IgG4(Fc)融合基因真核表达载体,稳定转染CHO/DG44细胞并检测重组蛋白的表达。主要采用聚合酶链式反应(PCR)从脂多糖(Lipopolysaccharides,LPS)诱导的人髓性白血病细胞株KG-IcDNA文库中克隆EBI3和IL-12p35cDNA,重叠PCR法连接2个片段,并克隆到IgG4(Fc)-pOptiVECTM-TOPO载体上,对新构建的IL-35-IgG4(Fc)pOptiVECTM-TOPO真核表达载体并进行酶切、测序、PCR鉴定;脂质体法转染CHO/DG44细胞;RT-PCR检测转染结果,采用a-MEM-培养基筛选实验组细胞,对筛选的阳性克隆细胞再进行氨甲喋呤(Methotrexate,MTX)的加压筛选,ProteinG-Agarose纯化阳性克隆培养上清,免疫印迹检测目的蛋白表达。结果显示IL-35-IgG4(Fc)pOptiVECTM-TOPO表达载体稳定转染CHO/DG44细胞并获得阳性克隆;SDS-PAGE电泳得到一条与预期相对分子质量大小相符的蛋白条带;该蛋白能与羊抗人IgG4抗体特异结合。本实验获得了能够稳定表达具有稳定结构的IL35-IgG4(Fc)融合蛋白的CHO/DG44细胞株。更多还原

【Abstract】 We constructed the eukaryotic expression vector of human IL-35-IgG4(Fc)-pOptiVECTM-TOPO by gene recombination technique and expressed the fusion protein human IL-35-IgG4(Fc) in CHO/DG44 cells.The two components of the newly discovered cytokine human IL-35,EBI3 and IL-12p35,were amplified by PCR from the cDNA library derived from the KG-I cells after LPS induction.The two PCR-amplified cDNA fragments of human IL-35 were linked by over-lapping PCR and then cloned into the IgG4(Fc)-pOptiVECTM-TOPO vector.The constructed plasmid with the recombinant cDNA IL-35-IgG4(Fc) was verified by restriction enzyme digestion analysis,PCR and DNA sequencing.The verified plasmid with the recombinant cDNA was transfected into CHO/DG44 cells using LipofectamineTM 2000.The success of the transfection was examined and confirmed by RT-PCR.After selection in α-MEM(-) medium,the IL-35-Ig G4(Fc) positive CHO/DG44 clones were chosen and the media from these positive clones were collected to be used to purify the fusion protein.The positive CHO/DG44 clones were further cultured in increasing concentrations of MTX and the expression levels of the fusion protein IL-35-Ig G4(Fc) were repetitively induced by MTX-induced gene amplification.The IL-35-Ig G4(Fc) fusion protein was purified from the media collected from the positive CHO/DG44 clones by protein G affinity chromatography and then identified by SDS-PAGE and Western blotting.The results showed that one protein band was found to match well with the predicted relative molecular mass of human IL-35-IgG4(Fc) and this protein could specifically bind to anti-human IgG4(Fc) monoclonal antibody.In conclusion,our study successfully established an IL-35-IgG4(Fc) positive DG44 cell line which could stably express IL-35-IgG4(Fc) fusion protein.更多还原