重组腺病毒Ad-sTNFRI-IgGFc转染人气道上皮细胞及其表达的实验研究  

Construction of adenoviral vector encoding soluble human TNFRI-IgGFc cDNA and its expression in human bronchial epithelial cells

作　　者：苏瑾[1] 尤长宣[1] 蔡绍曦[1] 马骊[2] 温茜[2] 罗微[2] 黄泳塔[2] 

机构地区：[1]南方医科大学南方医院呼吸科,广东广州510515 [2]南方医科大学生物技术学院分子免疫学研究所,广东广州510515

出　　处：《南方医科大学学报》2008年第4期517-521,共5页Journal of Southern Medical University

基　　金：国家863计划(2007AA02Z458);广东省科技攻关计划(2005B34001004);广州市科技攻关计划(2005Z3-E0151);广东省医学科学技术研究基金(B2005083)~~

摘　　要：目的构建携带人可溶性肿瘤坏死因子受体I胞外区和IgGFc融合基因的重组腺病毒载体(Ad-sTNFRI-IgGFc),体外转染正常人气道上皮细胞株(HBE135-E6E7),探讨重组腺病毒Ad-sTNFRI-IgGFc转染气道上皮治疗支气管哮喘的可行性。方法将sTNFRI-IgGFc基因插入腺病毒穿梭质粒pDC316,与辅助质粒pBHGloxΔE1,3Cre共转染HEK293细胞,重组产生Ad-sTNFRI-IgGFc,PCR鉴定毒种正确后,进行扩增、纯化和滴度测定,转染培养HBE135-E6E7,流式细胞术检测转染效率,RT-PCR、免疫组化检测转染后细胞中sTNFRI-IgGFc的表达和转录。结果成功构建了Ad-sTNFRI-IgGFc,感染性滴度达3×1010TCID50/ml,转染后HBE135-E6E7在mRNA和蛋白水平均有sTNFRI-IgGFc表达。结论构建的Ad-sTNFRI-IgGFc可有效转染HBE135-E6E7,并在其中高效表达,并能拮抗TNF活性,为将表达sTNFRI-IgGFc腺病毒基因治疗哮喘的实验研究提供了基础。Objective To construct a recombinant adenovirus vector carrying soluble extracellular region of tumor necrosis factor alpha receptor I-IgGFc （sTNFRI-IgGFc） and express the fusion protein in human bronchial epithelial HBE135-E6E7 cells. Methods sTNFRI-IgGFc fusion gene was subcloned into the adenovirus shuttle plasmid pDC316, which was co-transfected with helper plasmid pBHGlox△E1,3Cre into HEK293 cells. The recombinant adenovirus （Ad-sTNFRI-IgGFc） was generated by homologous recombination of the 2 plasmids in HEK293 cells. After identification with PCR, Ad-sTNFRI-IgGFc was amplified and purified, and its titer measured using TCID50 assay. The transcription and expression of sTNFRI-IgGFc gene in the ttansfected HBE135-E6E7 were detected by RT-PCR and immunohistochemistry. Results Ad-sTNFRI-IgGFc was successfully constructed with a viral titer of 3 ×10^10 TCID50/ml. The expression of sTNFRI-IgGFc mRNA and protein was confirmed in the txansfected HBE135-E6E7 cells. Conclusion The constructed Ad-sTNFRI-IgGFc can effectively infect HBE 135-E6E7 cells for efficient expression of sTNFRI-IgGFc protein, which antagonizes the cytolytic effect of TNFα in L929 cells, suggesting the potential of adenovirus expressing sTNFRI-IgGFc for local treatment of asthma.

关 键 词：可溶性肿瘤坏死因子受体 腺病毒载体 气道上皮细胞 支气管哮喘 

分 类 号：R562.25[医药卫生—呼吸系统]