腺病毒介导犬干扰素-γ基因的表达及其体外抗犬细小病毒的作用  

Adenovirus-mediated canine interferon-γ expression and its antiviral activity against canine parvovirus

作　　者：张考[1] 靳慧君[1] 仲飞[1] 李秀锦[2] 能昌爱[2] 陈慧慧[1] 李文艳[1] 温洁霞[1] 

机构地区：[1]河北农业大学动物医学院基础兽医部,农业部动物疫病病原生物学华北科学观测实验站,河北省兽用生物制品工程技术研究中心,保定071001 [2]燕山大学环境与化学工程学院生物工程系,秦皇岛066004

出　　处：《微生物学报》2012年第11期1400-1407,共8页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30771586);河北省自然科学基金(C2008000244)~~

摘　　要：【目的】构建含犬干扰素-γ(cIFN-γ)基因的重组腺病毒,并在培养的犬肾细胞MDCK中分析其抗犬细小病毒的活性。【方法】首先将cIFN-γcNDA基因克隆到腺病毒穿梭质粒中,构建成含cIFN-γ基因的腺病毒穿梭质粒pShuttle3-cIFN-γ。利用特异的酶切位点,通过直接连接法将cIFN-γ表达盒插入到腺病毒基因组质粒pAdeno-X中,构建成含cIFN-γ基因的腺病毒基因组质粒pAd-cIFN-γ。pAd-cIFN-γ质粒经酶切线性化后转染人胚胎肾细胞HEK293T,在细胞中拯救出含有cIFN-γ基因的复制缺陷型重组腺病毒。然后用该重组腺病毒处理(感染)培养的犬肾细胞MDCK,再用犬细小病毒感染重组腺病毒处理的细胞,分析重组腺病毒在体外抗犬细小病毒的活性。【结果】通过连接法构建了含cIFN-γ基因的重组腺病毒,构建的重组腺病毒能够介导cIFN-γ在MDCK细胞中进行分泌表达。用含cIFN-γ基因的重组腺病毒处理MDCK细胞,可明显地抑制犬细小病毒在细胞中的增殖,表明构建的重组腺病毒具有明显的抗犬细小病毒的活性。【结论】构建了含cIFN-γ基因的重组腺病毒,并证明该重组病毒在体外具有明显的抗犬细小病毒的活性。[Objective] To construct recombinant adenovirus containing canine interferon-γ（cIFN-γ） gene and to investigate its antiviral activity against canine parvovirus in Madin-Darby canine kidney cells（MDCK）.[Methods] The cIFN-γ gene was inserted into adenovirus shuttle plasmid to construct pShuttle3-cIFN-γ expression vector,from which the cIFN-γ expression cassette was transferred into the adenovirus genomic plasmid pAdeno-X by specific restriction sites to generate recombinant adenovirus genomic plasmid pAd-cIFN-γ.The pAd-cIFN-γ plasmid was linearized by digestion and transfected into human embryonic kidney（HEK） 293T cells to generate the replication-defective cIFN-γ recombinant adenovirus（Ad-cIFN-γ）.To analyze its anti-canine parvovirus activity,the MDCK cells were pre-infected by Ad-cIFN-γ recombinant adenovirus,and then infected by canine parvovirus.The antiviral activity of the Ad-cIFN-γ recombinant adenovirus against parvovirus was analyzed.[Results] The recombinant adenovirus containing cIFN-γ gene was constructed by the ligation method.The recombinant adenovirus could mediates recombinant cIFN-γ secretory expression in MDCK cells.The Ad-cIFN-γ recombinant adenovirus could significantly inhibit canine parvovirus replication in MDCK cells pre-infected with the recombinant adenovirus.These results indicate that the Ad-cIFN-γ recombinant adenovirus has the potent antiviral activity against canine parvovirus.[Conclusion] The Ad-cIFN-γ recombinant adenovirus was successfully constructed by the ligation method and possessed a powerful antiviral activity against canine parvovirus.

关 键 词：干扰素-Γ 腺病毒载体 抗病毒活性 犬细小病毒 

分 类 号：S858.292[农业科学—临床兽医学]