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【目的】构建表达人 CTLA4Ig 的非复制型重组腺病毒载体并检测其在真核细胞中的表达。【方法】分离人外周血单个核细胞,提取细胞总 RNA,经 RT-PCR 分别扩增含抑瘤素 M 信号肽的 CTLA4胞外段及 IgG 恒定区(含绞链-CH2-CH3)基因片段。将 PCR 产物克隆人 pCDNA3,阳性重组子以双酶切和测序鉴定。将目的基因 CTLA4-Ig 亚克隆人腺病毒穿梭质粒 pAdTrack-CMV,与5型腺病毒骨架质粒 pAdeasy-1共转染 BJ5183,经细菌内同源重组构建重组腺病毒载体。采用脂质体法转染293细胞包装产生重组腺病毒,进一步感染 HepG2细胞,采用 RT-PCR 及 ELISA 测定 CTLA4Ig 表达情况。【结果】构建了表达人 CTLA4Ig 基因的重组腺病毒,病毒滴度可达6×10~9pfu/mL,并能在 HepG2和骨髓间充质干细胞中表达 CTLA4Ig。【结论】成功构建表达 CTLA4Ig 的重组腺病毒载体,为进一步研究肝细胞移植排异及自身免疫性疾病的免疫调节治疗提供有力的手段与工具。
【Objective】 To construct a non-replicating recombinant adenovirus vector expressing human CTLA4Ig and test its expression in eukaryotic cells. 【Methods】 Peripheral blood mononuclear cells (PBMCs) were isolated and total cellular RNA was extracted. The CTLA4 extracellular domain and constant region of IgG (including hinge-CH2-CH3) . The PCR product was cloned into human pCDNA3, and the positive recombinant was identified by double enzyme digestion and sequencing. The target gene CTLA4-Ig was subcloned into the adenoviral shuttle plasmid pAdTrack-CMV and co-transfected with adenovirus backbone plasmid pAdeasy-1 to construct BJ5183. The recombinant adenoviral vector was constructed by homologous recombination in bacteria. The recombinant adenovirus was transfected into 293 cells by lipofectamine 2000 and further infected into HepG2 cells. The expression of CTLA4Ig was detected by RT-PCR and ELISA. 【Results】 The recombinant adenovirus expressing human CTLA4Ig gene was constructed. The titer of virus was 6 × 10 ~ 9 pfu / mL, and CTLA4Ig was expressed in HepG2 and BMSCs. 【Conclusion】 The successful construction of recombinant adenovirus vector expressing CTLA4Ig will provide a powerful tool and tool for the further study on rejection of hepatocyte transplantation and immunomodulatory therapy of autoimmune diseases.