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
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摘要
【目的】构建原核表达载体p ET28a-r Ch IFN-α-Ch IL-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,Ch IFN-α)与鸡白细胞介素18(Chicken interleukin-18,Ch IL-18)基因构建Ch IFN-α-Ch IL-18融合基因并克隆入p ET-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SDSPAGE分析、Western-blot鉴定.采用细胞病变抑制法检测r Ch IFN-α-Ch IL-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p ET28a-r Ch IFN-α-Ch IL-18融合基因.融合基因在大肠埃希菌中表达的r Ch IFN-α-Ch IL-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.r Ch IFN-α-Ch IL-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r Ch IFN-α、r Ch IL-18蛋白的抗病毒活性.
【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 p ET28a-r Ch IFNα-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,Ch IFN-α and Ch IL-18 constituted the Ch IFN-α-Ch IL-18 fusion gene when cloned into the vector p ET-28 a,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 r Ch IFN-α-Ch IL-18 against vesicular stomatitis virus( VSV) and newcastle disease virus( NDV) proliferation. 【Result and conclusion】The fusion gene of Ch IFN-α-Ch IL-18 was successfully constructed and cloned into p ET-28 a vector. The r Ch IFN-α-Ch IL-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 r Ch IFN-α-Ch IL-18 fusion protein had been obtained. The antiviral activity units of r Ch IFN-α-Ch IL-18 protein inhibiting the reproduction of VSV and NDV on chicken embryo fibroblast( CEF) cells were much higher than those of the recombinant r Ch IFN-α,r Ch IL-18 protein.
【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 p ET28a-r Ch IFNα-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,Ch IFN-α and Ch IL-18 constituted the Ch IFN-α-Ch IL-18 fusion gene when cloned into the vector p ET-28 a,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 r Ch IFN-α-Ch IL-18 against vesicular stomatitis virus( VSV) and newcastle disease virus( NDV) proliferation. 【Result and conclusion】The fusion gene of Ch IFN-α-Ch IL-18 was successfully constructed and cloned into p ET-28 a vector. The r Ch IFN-α-Ch IL-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 r Ch IFN-α-Ch IL-18 fusion protein had been obtained. The antiviral activity units of r Ch IFN-α-Ch IL-18 protein inhibiting the reproduction of VSV and NDV on chicken embryo fibroblast( CEF) cells were much higher than those of the recombinant r Ch IFN-α,r Ch IL-18 protein.
引文
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[3]王宪文,刘兴友,王岩.鸡白细胞介素18研究进展[J].安徽农业科学,2008,36(16):6776-6777.
[4]李敏,杨谦.一种高效构建同源重组DNA片段的方法—融合PCR[J].中国生物工程杂志,2007,27(8):53-58.
[5]韦琴,彭贵青,金梅林,等.鸡α干扰素基因的克隆、原核表达及抗病毒效果研究[J].生物工程学报,2006,22(5):737-743.
[6]SAGANUWAN S A.A modified arithmetical method of reed and muench for determination of a relatively ideal median dose(LD50)[J].Afr J Pharm Pharmacol,2011,5(12):1543-1546.
[7]闫若潜,谢彩华,吴志明.鸡α干扰素/白细胞介素2基因的融合表达及活性研究[J].中国农业科学,2010,43(3):594-604.
[8]YEH H Y,WINSLOW B J,JUNKER D E,et al.In vitro effects of recombinant chicken interferon-gamma on immune cells[J].J Interf Cytok Res,1999,19(6):687-691.
[9]MEAGER A.Biological assays for interferons[J].J Immunol Methods,2002,261(1/2):21-36.
[10]VOLPINI L M,CALNEK B W.Interferon modulation of marek's disease virus genome expression chicken cell lines[J].Avian Dis,1996,40(1):78-87.
[11]XING Z,SCHAT K A.Inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of Marek's disease virus[J].J Virol,2000,74(8):3605-3612.
[2]JOHNSON H M,BAZE F W,SZENTE B E.How interferons fight disease[J].Sci Am,1994,270(5):68-75.
[3]王宪文,刘兴友,王岩.鸡白细胞介素18研究进展[J].安徽农业科学,2008,36(16):6776-6777.
[4]李敏,杨谦.一种高效构建同源重组DNA片段的方法—融合PCR[J].中国生物工程杂志,2007,27(8):53-58.
[5]韦琴,彭贵青,金梅林,等.鸡α干扰素基因的克隆、原核表达及抗病毒效果研究[J].生物工程学报,2006,22(5):737-743.
[6]SAGANUWAN S A.A modified arithmetical method of reed and muench for determination of a relatively ideal median dose(LD50)[J].Afr J Pharm Pharmacol,2011,5(12):1543-1546.
[7]闫若潜,谢彩华,吴志明.鸡α干扰素/白细胞介素2基因的融合表达及活性研究[J].中国农业科学,2010,43(3):594-604.
[8]YEH H Y,WINSLOW B J,JUNKER D E,et al.In vitro effects of recombinant chicken interferon-gamma on immune cells[J].J Interf Cytok Res,1999,19(6):687-691.
[9]MEAGER A.Biological assays for interferons[J].J Immunol Methods,2002,261(1/2):21-36.
[10]VOLPINI L M,CALNEK B W.Interferon modulation of marek's disease virus genome expression chicken cell lines[J].Avian Dis,1996,40(1):78-87.
[11]XING Z,SCHAT K A.Inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of Marek's disease virus[J].J Virol,2000,74(8):3605-3612.