猪瘟病毒表面抗原E2-Erns蛋白在拟南芥种子中的表达及抗原性分析  

Expression in Arabidopsis thaliana seeds and actigenic analysis of E2-Erns protein of classical swine fever virus

作　　者：杨贺 李晓微[1] 刘伟灿 陈欢 李余先 张敏 周永刚 李海燕 YANG He;LI Xiao-wei;LIU Wei-can;CHEN Huan;LI Yu-xian;ZHANG Min;ZHOU Yong-gang;LI Hai-yan(College of Life Sciences, Jilin Agricultural University/Engineering Research Center of Bioreactor and Pharmaceutical Develop ment, Ministry of Education, Changchun 130118, China;Changchun Medical College, Changchun 130031, China)

机构地区：[1]吉林农业大学生命科学学院生物反应器与药物开发教育部工程研究中心,吉林长春130118 [2]长春医学高等专科学校,吉林长春130031

出　　处：《中国兽医科学》2018年第7期830-837,共8页Chinese Veterinary Science

基　　金：国家高技术研究发展计划(863)项目(2011AA100606);吉林省科技厅重点科技攻关项目(20150204027NY);吉林省发改委项目(JF2012C002-4)

摘　　要：根据拟南芥密码子偏好性对猪瘟病毒表面抗原基因E2和E^rns的序列进行优化,并将优化的E2-E^rns基因克隆入植物表达载体中;利用农杆菌介导的花序浸染法转化拟南芥,通过1%草铵膦喷洒筛选阳性植株,利用PCR鉴定阳性植株;提取阳性种子总蛋白进行ELISA检测评价重组E2-E^rns蛋白的免疫反应原性;用E2-E^rns融合蛋白灌胃免疫小鼠并检测其血清中的特异抗体效价。结果,成功构建了由菜豆蛋白启动子驱动的种子特异性表达目的基因的植物表达载体p PHAP1301-E2-E^rns,并利用拟南芥成功表达了猪瘟病毒表面抗原E2-E^rns融合蛋白。免疫试验结果表明,通过拟南芥表达的E2-E^rns融合蛋白具有良好的免疫反应原性。结果表明,本研究获得了表达E2-E^rns蛋白的转基因拟南芥,小鼠口服植物源E2-E^rns融合蛋白具有良好的抗原性,这为深入研究利用植物表达猪瘟病毒表面抗原奠定了基础。The sequences of E2 and E^rnsgenes of classical swine fever virus were optimized according to the codon bias of Arabidopsis thaliana and then the optimized E2-E^rnssequence was cloned into plant expression vectors to construct p PHAP1301-E2-E^rns,in which E2-E^rnswas driven by the promoter of Pha P of Phaseolus vulgaris.The exogenous gene was transferred into A.thaliana bioreactor by Floral-dip method,and transgenic plant was obtained by Basta resistibility.The selected lines were confirmed to contain the E2-E^rnssequence by PCR,The immunogenicity of E2-E^rnsfusion protein was confirmed by ELISA.The E2-E^rnsfusion protein was expressed by A.thaliana after the test of target protein and confirmed by SDS-PAGE and Western-blot.The expressed E2-E^rnsprotein was used to immunize mice orally,which induced greater titer serum Ig G.The results showed that the expressed E2-E^rnsprotein had better immunogenicity, which provide supports for production of glycoprotein of CSFV via plant bioreactor.

关 键 词：猪瘟病毒 E2-E^rns蛋白 拟南芥 转化 抗原性 

分 类 号：S852.651[农业科学—基础兽医学]