体外连接法构建重组人γ干扰素腺病毒并检测其在真核细胞中的表达

Construction of Recombinant Adenovirus Carrying Human IFNγ Gene by In Vitro Ligation and Its Expression

背景与目的:γ干扰素(interferon-γ,IFNγ)具有直接促进某些肿瘤细胞凋亡和调节细胞免疫的功能,因此在肿瘤的基因治疗中IFNγ将发挥重要的作用。本研究构建携带人IFNγcDNA的重组腺病毒,并检测它在真核细胞中的表达。方法:用逆转录聚合酶链反应(reversetranscription-polymerasechainreaction,RT-PCR)从人外周血单个核细胞中扩增出IFNγ的cDNA全长序列,插入腺病毒穿梭质粒pShuttle,经酶切和DNA测序鉴定,然后用体外连接法构建重组腺病毒Ad-IFNγ。用所构建的重组Ad-IFNγ感染HepG2细胞,通过RT-PCR和免疫组织化学法检测IFNγ的表达。结果:经测序,克隆的人IFNγcDNA序列与GenBank上公布的序列完全一致;Ad-IFNγ感染的HepG2细胞在转录和翻译水平均有IFNγ的表达。结论:体外连接法是一种简便易行的重组腺病毒载体构建方法;所构建的重组腺病毒Ad-IFNγ能表达IFNγ蛋白,可用于抗肿瘤和抗病毒等细胞及动物实验。

BACKGROUND ＆ OBJECTIVE： Interferon-γ （IFNγ） can promote directly the apoptosis of some kinds of tumor ceils and regulate cellular immunity, therefore, it may play an important role in gene therapy for tumors. This study was to construct recombinant adenovirus carrying human IFNγ cDNA, and detect its expression profile in eukaryotic cells. METHODS： IFNγ cDNA was amplified by reverse transcription-polymerase chain reaction （RT-PCR） from human peripheral blood mononuclear cells, and cloned into adenoviral shuttle plasmid pShuttle to construct recombinant adenovirus carrying human IFNγ cDNA （Ad-IFNγ） by in vitro ligation. Hepatocellular carcinoma cell line HepG2 was infected with Ad-IFNγ, and the expression of IFNγ was detected by RT-PCR and immunohistochemistry. RESULTS： The sequence of the cloned IFNγ cDNA was completely consistent with that reported in GenBank. PCR analysis confirmed the existence of IFNγ gene in Ad-IFNγ without wild adenovirus contamination, and the genome of Ad-IFNγ showed the predicted map of restricted endonuclease enzymes analysis. IFNγ mRNA and protein were detected in HepG2 cells infected with Ad-IFNγ. CONCLUSIONS： In vitro ligation is a simple and convenient method for construction of recombination adenovirus vector. We have constructed a functional recombinant adenovirus expressing human IFNγ, which could be a potential antivirus and antitumor agent in vitro or in vivo.