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  

Construction of Interferon-γ Plant Expression Vector from Congjiangxiang Pig and Transient Expression of Lettuce

作　　者：田浪 温贵兰[1,2] 张升波 杨佰启 李昌红 徐丽[1,2] 陈广 张喜懿[1,2] TIAN Lang;WEN Gui-lan;ZHANG Sheng-bo;YANG Bai-qi;LI Chang-hong;XU Li;CHEN Guang;ZHANG Xi-yi(Laboratory of Preventive Veterinary Medicine,Institute of Animal Science,Guizhou University,Guizhou Guiyang 550025,China;Animal Biological Engineering Technology Research Center in Guizhou Province,Guizhou Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院预防兽医实验室,贵州贵阳550025 [2]贵州省动物生物制品工程技术研究中心,贵州贵阳550016

出　　处：《中国畜牧杂志》2019年第7期96-100,共5页Chinese Journal of Animal Science

基　　金：国家自然科学基金(31460668);贵州省科学技术基金(黔科合J字[2015]2046号);贵州省高层次创新型人才培养(黔财教[2017]号);贵州大学博士基金(贵大人基合字(2013)12号);贵州省研究生工作站计划项目([GZZ2017002]);贵州省科技平台及人才团队计划(黔科合平台人才[2018]5253)

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、170.37 pg/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 IFNγplant expression vector from Congjiangxiang pig,the transient expression was achieved in fresh lettuce by Agrobacterium vacuum in filtration method.A pair of speci fic IFNγprimers were designed with reference to the porcine IFNγgene sequence registered in GeneBank.The IFNγgene 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 identi fication of recombinant plasmids;the recombinant plasmid was infected with Agrobacterium tumefaciens by vacuum in filtration method,and the infected lettuce leaves were detected by GUS staining;ELISA was used to detect IFNγprotein 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 IFNγprotein from Congjiangxiang pig by cytopathic inhibition assay.The results show:In this study,the IFNγgene sequence was ampli fied from the liver tissue of Congjiangxiang pigs in Guizhou and the IFNγplant expression vector PBI121-IFNγwas constructed from Congjiangxiang pig;GUS stained lettuce leaves are visible in blue;the content of IFNγprotein 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-γby 4^-6 dilution on Marc145 cells.This experiment successfully constructed the IFNγplant expression vector PBI121-IFNγfrom Congjiangxiang pig.The transient expression protein in lettuce has immunoreactivity and antiviral activity,which provides a reference for the research and development of IFNγmedicinal plant protein from Congjiangxiang pig.

关 键 词：从江香猪源干扰素γ 植物表达载体 生菜 瞬时表达 抗病毒活性 

分 类 号：Q78[生物学—分子生物学]