抗HBc-ScFv基因复制缺陷型腺病毒载体的构建及其体外表达  

Construction and expression of replication-deficient recombinant adenoviral vector carrying anti-HBc ScFv gene

作　　者：汤正好[1] 马会慧[2] 臧国庆[1] 余永胜[1] 李刚[2] 姚集鲁[2] 

机构地区：[1]上海交通大学附属第六人民医院感染科,上海市200233 [2]中山大学附属第三医院传染科实验室,广东省广州市510630

出　　处：《世界华人消化杂志》2005年第6期734-738,共5页World Chinese Journal of Digestology

摘　　要：目的:构建表达抗乙型肝炎病毒核心蛋白单链抗体(抗HBc ScFv)的复制缺陷型腺病毒载体,并检测其能否在真核细胞中有效表达目的基因. 方法:采用DNA重组技术将特异性人源性抗卜HBc单链抗体基因克隆于腺病毒穿梭质粒pAdTrack-CMV,与5 型腺病毒骨架质粒pAdeasy-1共转染B15183细菌,经细菌内同源重组产生携带抗HBc ScFv基因的重组腺病毒载体pAd—ScFV,经脂质体法转染293细胞,包装产生携带抗HBc scFv基因的重组腺病毒Ad—scFv,体外转染HepG2细胞,PCR和ELISA法检测目的基因及其表达. 结果:构建了表达抗HBc ScFv基因的复制缺陷型腺病毒,病毒滴度达4×1015 PFU/L,并能在真核细胞中有效表达目的基因. 结论:成功构建表达抗HBc ScFv的复制缺陷型腺病毒载体,为进一步开展抗HBc ScFv在抗HBV基因治疗中的作用研究提供实验基础.AIM: To construct recombinant adenoviral vector carrying anti-HBc ScFv gene and to express the gene efficiently in eukaryotic cells. METHODS: The gene of human variable region of single chain antibody against hepatitis B virus core antigen (anti-HBc ScFv) was amplified by polymerase chain reaction(PCR) and was cloned into adenoviral shuttle vector pAdTrack-CMV. Then, the recombinant vector pAdTrack-CMV-ScFv was linearized by digesting with restriction endonuclease Pme I, and co-transformed into E. coli BJ5183 cells with adenoviral backbone plasmid pAdeasy-1. The recombinant adenoviral plasmid pAd-ScFv was obtained by homologous recombination in bacteria and then transfected into 293 cells for packaging of recombinant adenovirus Ad-ScFv. Finally, HepG2 cells were infected with the adenoviruses and anti-HBc ScFv was detected by PCR and ELISA in vitro. RESULTS: The titer of recombinant adenovirus Ad-ScFv was up to 4×1015 PFU/L in infected 293 cells. Anti-HBc ScFv was expressed efficiently in HepG2 cells after infection. CONCLUSION: The recombinant adenovirus Ad-ScFv expressing anti-HBc ScFv has been constructed successfully, which can be used in study of gene therapy for HBV.

关 键 词：缺陷型腺病毒载体 体外表达 基因复制 人源性抗HBc单链抗体 ScFv基因 复制缺陷型腺病毒 ELISA法检测 DNA重组技术 重组腺病毒载体 HepG2细胞 病毒核心蛋白 目的基因 构建表达 真核细胞 293细胞 乙型肝炎 穿梭质粒 基因克隆 

分 类 号：R346[医药卫生—基础医学]