α 干扰素/白细胞介素 18 基因的融合表达及抗病毒活性研究
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广东省战略性新兴产业核心技术攻关(2012A020800006)


Expressions and antiviral bioactivities of chicken interferon α/ chicken interleukin-18 fusion protein
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    摘要:

    【目的】构建原核表达载体pET28a-rChIFN-α-ChIL-18、大肠埃希菌 Escherichia coli 表达及产物纯化与活性检测,研究高效广谱鸡基因工程重组复合抗病毒制剂,以对鸡的病毒性疾病进行防治.【方法】采用融合PCR(Fusion PCR )方法,利用具有互补末端的引物,形成具有重叠链的PCR产物,通过PCR产物重叠链的延伸将鸡α 干扰素(Chicken interferon alpha,ChIFN-α)与鸡白细胞介素18(Chicken interleukin-18,ChIL-18)基因构建 ChIFN-α-ChIL-18 融合基因并克隆入pET-28a原核表达载体中进行原核表达.通过镍柱亲和层析法纯化可溶性蛋白,并进行 SDS-PAGE分析、Western-blot鉴定.采用细胞病变抑制法检测rChIFN-α-ChIL-18蛋白在细胞上抑制水泡性口炎病毒(VSV)及新城疫病毒(NDV)增殖活性.【结果和结论】成功构建并克隆了 pET28a-rChIFN-α-ChIL-18 融合基因.融合基因在大肠埃希菌中表达的rChIFN-α-ChIL-18蛋白相对分子质量约为38 000,蛋白经纯化后纯度在90%以上.rChIFN-α-ChIL-18蛋白在鸡胚成纤维(CEF)细胞上具有明显抗病毒活性,其抗VSV和NDV的活性明显高于单一rChIFN-α、rChIL-18蛋白的抗病毒活性.

    Abstract:

    【Objective】This study was conducted to explore a highly efficient chicken gene engineering antiviral agent to prevent and control the chicken viral disease. A recombinant plasmid pET28a-rChIFNα-IL18 was constructed,which was expressed in Escherichia coli, purified by Ni-NTA and tested for antiviral bioactivitiy. 【Method】 This study introduced a novel method for constructing vectors by fusion PCR, which generated PCR products with overlapping chain using Primers complementary to the end. Through the extension of PCR products, ChIFN-α and ChIL-18 constituted the ChIFN-α-ChIL-18 fusion gene when cloned into the vector pET-28a, induced the expression in E. coli strain BL21(DE3). This protein was purified by Ni-NTA and detected by SDS-P AGE and Western-blot. Cytopathic effects(CPE)were applied to examine the potency of recombination rChIFN-α-ChIL-18 against vesicular stomatitis virus(VSV)and newcastle disease virus(NDV)proliferation.【Result and conclusion】 The fusion gene of ChIFN-α-ChIL-18 was successfully constructed and cloned into pET-28a vector. The rChIFN-α-ChIL-18 protein was expressed in E. coli and successfully purified with a molecular mass of about 38 000 and more than 90% on SDS-PAGE,which indicated that the correct rChIFN-α-ChIL-18 fusion protein had been obtained.The antiviral activity units of rChIFN-α-ChIL-18 protein inhibiting the reproduction of VSV and NDV on chicken embryo fibroblast(CEF) cells were much higher than those of the recombinant rChIFN-α,rChIL-18 protein.

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何静,张盼盼,孙敏华,康银峰,谢鹏,向斌,韩翡,谭阳通,任涛.鸡 α 干扰素/白细胞介素 18 基因的融合表达及抗病毒活性研究[J].华南农业大学学报,2015,36(1):18-22

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  • 收稿日期:2014-02-24
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  • 在线发布日期: 2015-01-06