从江香猪源干扰素γ植物表达载体构建及在生菜中瞬时表达
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摘要
为构建从江香猪源干扰素γ(IFNγ)植物表达载体,通过农杆菌真空渗透法在新鲜生菜中实现瞬时表达。参考GenBank登录的猪IFNγ基因序列设计1对特异性IFNγ引物,采用RT-PCR方法从贵州从江香猪源肝脏组织扩增IFNγ基因序列,将其克隆至植物表达载体PBI121,转化根癌农杆菌LBA4404,筛选阳性菌株提取质粒,对重组质粒进行PCR扩增、双酶切及测序鉴定;携带重组质粒根癌农杆菌真空渗透法感染生菜,GUS染色检测感染的生菜叶片,ELISA检测感染生菜叶片中从江香猪源IFNγ蛋白,分析重组质粒在生菜中表达效果;采用细胞病变抑制实验检测生菜中从江香猪源IFNγ蛋白抗病毒活性。结果表明:本研究成功地从贵州从江香猪源肝脏组织扩增获得IFNγ基因序列并构建了从江香猪源IFNγ植物表达载体PBI121-IFNγ;GUS染色感染的生菜叶片可见蓝色物质;ELISA检测感染生菜叶片中从江香猪源IFNγ蛋白含量为169.397、170.657、170.37pg/mL;生菜中表达从江香猪源IFNγ经4-6稀释在Marc145细胞上仍能够抑制100 TCID50的PRRSV的致细胞病变作用。本实验成功构建从江香猪源IFNγ植物表达载体PBI121-IFNγ,生菜中瞬时表达蛋白具有免疫反应性和抗病毒活性,为从江香猪源IFNγ药用植物蛋白的研究开发提供参考依据。
In order to construct an IFNγ plant expression vector from Congjiangxiang pig, the transient expression was achieved in fresh lettuce by Agrobacterium vacuum infiltration method.A pair of specific IFNγ primers were designed with reference to the porcine IFNγ gene sequence registered in GeneBank. The IFNγ gene sequence was amplified from the liver tissue of Congjiangxiang pigs in Guizhou by RT-PCR and cloned into the plant expression vector PBI121 to transform Agrobacterium tumefaciens. LBA4404, screening positive strains to extract plasmids, PCR amplification, double enzyme digestion and sequencing identification of recombinant plasmids;the recombinant plasmid was infected with Agrobacterium tumefaciens by vacuum infiltration method, and the infected lettuce leaves were detected by GUS staining; ELISA was used to detect IFNγ protein from Congjiangxiang pigs in the leaves of infected lettuce, and the expression effect of recombinant plasmid in lettuce was analyzed; Detection of antiviral activity of IFNγ protein from Congjiangxiang pig by cytopathic inhibition assay.The results show:In this study, the IFNγ gene sequence was amplified from the liver tissue of Congjiangxiang pigs in Guizhou and the IFNγ plant expression vector PBI121-IFNγ was constructed from Congjiangxiang pig;GUS stained lettuce leaves are visible in blue;the content of IFNγ protein in the leaves of Congjiangxiang pigs was 169.397 pg/mL,170.657 pg/mL and 170.37 pg/mL in the leaves of infected lettuce;the cytopathic effect of PRRSV of 100 TCID 50 was still inhibited from lettuce-derived IFN-γ by 4-6 dilution on Marc145 cells.This experiment successfully constructed the IFNγ plant expression vector PBI121-IFNγ from Congjiangxiang pig. The transient expression protein in lettuce has immunoreactivity and antiviral activity, which provides a reference for the research and development of IFNγ medicinal plant protein from Congjiangxiang pig.
In order to construct an IFNγ plant expression vector from Congjiangxiang pig, the transient expression was achieved in fresh lettuce by Agrobacterium vacuum infiltration method.A pair of specific IFNγ primers were designed with reference to the porcine IFNγ gene sequence registered in GeneBank. The IFNγ gene sequence was amplified from the liver tissue of Congjiangxiang pigs in Guizhou by RT-PCR and cloned into the plant expression vector PBI121 to transform Agrobacterium tumefaciens. LBA4404, screening positive strains to extract plasmids, PCR amplification, double enzyme digestion and sequencing identification of recombinant plasmids;the recombinant plasmid was infected with Agrobacterium tumefaciens by vacuum infiltration method, and the infected lettuce leaves were detected by GUS staining; ELISA was used to detect IFNγ protein from Congjiangxiang pigs in the leaves of infected lettuce, and the expression effect of recombinant plasmid in lettuce was analyzed; Detection of antiviral activity of IFNγ protein from Congjiangxiang pig by cytopathic inhibition assay.The results show:In this study, the IFNγ gene sequence was amplified from the liver tissue of Congjiangxiang pigs in Guizhou and the IFNγ plant expression vector PBI121-IFNγ was constructed from Congjiangxiang pig;GUS stained lettuce leaves are visible in blue;the content of IFNγ protein in the leaves of Congjiangxiang pigs was 169.397 pg/mL,170.657 pg/mL and 170.37 pg/mL in the leaves of infected lettuce;the cytopathic effect of PRRSV of 100 TCID 50 was still inhibited from lettuce-derived IFN-γ by 4-6 dilution on Marc145 cells.This experiment successfully constructed the IFNγ plant expression vector PBI121-IFNγ from Congjiangxiang pig. The transient expression protein in lettuce has immunoreactivity and antiviral activity, which provides a reference for the research and development of IFNγ medicinal plant protein from Congjiangxiang pig.
引文
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[9]罗熹,王涛,卢彪.植物体瞬时表达ChIFN-γ蛋白研究[J].广东农业科学,2013,40(4):128-130.
[10]郑鸣,边传周,王老七,等.猪干扰素α、β和γ的融合表达及活性[J].中国农业科学,2012,45(9):1840-1847.
[11]梁望旺,周丹娜,杨克礼,等.猪γ干扰素的表达及抗病毒活性研究[J].湖北农业科学,2011,50(11):2283-2286.
[12]吴文学,夏春.猪干扰素γcDNA的分子克隆与在大肠杆菌中的表达[J].农业生物技术学报,2002,10(3):255-258.
[13]罗雯,艾佐佐,刘艳梅,等.EV71抗原双元植物表达载体构建及生菜瞬时表达[J].生物技术,2018(1):18-23,29.
[14]张瑞华,刘春凌,李春红.毕赤酵母表达猪γ干扰素基因及其抗病毒作用研究[J].黑龙江畜牧兽医,2011(5):113-115.
[15]钟鲁龙.猪干扰素α和γ在家蚕中的共表达及抗病毒活性测定[D].兰州:中国农业科学院,2013.
[2]温贵兰,陈绍品,张升波,等.从江香猪γ干扰素基因克隆与序列分析[J].中国畜牧兽医,2018,45(6):1437-1446.
[3]Rodríguezcarre?o M P,Lópezfuertes L,Revilla C,et al.Phenotypic characterization of porcine IFN-gamma-producing lymphocytes by flow cytometry[J].J Immunol Methods,2002,259(1):171-179.
[4]Dijkmans R,Vandenbroeck K,Beuken E,et al.Sequence of the porcine interferon-gamma(IFN-gamma)gene[J].Nucleic Acids Res,1990,18(14):4259.
[5]舒银辉,漆世华,曹瑞兵,等.重组基因猪γ干扰素抑制PRRSV、PPV、PRV三种病毒在传代细胞上增殖的研究[A].中国畜牧兽医学会生物制品学分会第十次学术研讨会论文集[C].广州:中国畜牧兽医学会,2005.
[6]涂浩,赵星灿,杨占娜,等.猪重组IL-2、IL-4和IFN-γ对口蹄疫合成肽疫苗的免疫增强作用研究[J].畜牧兽医学报,2015,46(8):1390-1399.
[7]赵伟鸽.猪α、γ干扰素的原核表达、纯化及抗病毒活性初步检测[D].上海:上海交通大学,2010.
[8]Randall R E,Goodbourn S.Interferons and viruses:an interplay between induction,signalling,antiviral responses and virus countermeasures[J].J Gen Virol,2008,89(1):1-47.
[9]罗熹,王涛,卢彪.植物体瞬时表达ChIFN-γ蛋白研究[J].广东农业科学,2013,40(4):128-130.
[10]郑鸣,边传周,王老七,等.猪干扰素α、β和γ的融合表达及活性[J].中国农业科学,2012,45(9):1840-1847.
[11]梁望旺,周丹娜,杨克礼,等.猪γ干扰素的表达及抗病毒活性研究[J].湖北农业科学,2011,50(11):2283-2286.
[12]吴文学,夏春.猪干扰素γcDNA的分子克隆与在大肠杆菌中的表达[J].农业生物技术学报,2002,10(3):255-258.
[13]罗雯,艾佐佐,刘艳梅,等.EV71抗原双元植物表达载体构建及生菜瞬时表达[J].生物技术,2018(1):18-23,29.
[14]张瑞华,刘春凌,李春红.毕赤酵母表达猪γ干扰素基因及其抗病毒作用研究[J].黑龙江畜牧兽医,2011(5):113-115.
[15]钟鲁龙.猪干扰素α和γ在家蚕中的共表达及抗病毒活性测定[D].兰州:中国农业科学院,2013.
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