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

摘要:

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-γ cNDA 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。

Abstract:

Abstract:［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-γrecombinant 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．