VP7-CTB融合蛋白在拟南芥种子中的表达及免疫原性分析  

Expression and immunogenicity analysis of VP7-CTB fusion protein in transgenic Arabidopsis thaliana seeds

作　　者：李余先[1,2,3] 官丽莉[1] 周永刚[1] 刘秀明[1] 杨晶[1] 郭咏昕[1] 杨赫[1] 王红雨[1] 李海燕[1] 李校堃[4] 

机构地区：[1]吉林农业大学教育部生物反应器与药物开发工程研究中心,吉林长春130118 [2]吉林农业科技学院中药学院,吉林吉林132101 [3]吉林农业大学中药材学院,吉林长春130118 [4]温州大学,浙江温州325035

出　　处：《中国兽医科学》2017年第4期403-410,共8页Chinese Veterinary Science

基　　金：国家高技术研究发展计划(863)项目(2011AA100606)

摘　　要：为了研发植物来源的轮状病毒疫苗,采用模式植物拟南芥作为生物反应器,将轮状病毒主要中和抗原基因VP7融合CTB,通过农杆菌介导法转化野生型拟南芥,获得了含有VP7-CTB的转基因拟南芥稳定株系,并分析其种子表达蛋白的免疫原性。首先,根据拟南芥密码子的偏好性,设计合成优化的VP7和CTB基因,构建植物表达载体pPHAP1301-his-VP7-linker-CTB;再通过农杆菌介导的Floral Dip法转化拟南芥,收获T0代种子后播种,喷用草铵膦筛选转基因拟南芥获得T1代种子,重复上述过程最终获得T3代拟南芥种子,通过RT-PCR、W estern-bolt、ELISA等进行分析。结果表明,his-VP7-linker-CTB基因已成功整合到拟南芥的基因组并进行了转录和蛋白表达,并且VP7蛋白占总可溶性蛋白的0.39%,即转基因拟南芥种子中所含目的蛋白为52.65 g/g。最后,用转基因拟南芥稳定株系种子中的可溶性蛋白口服免疫小鼠,使小鼠获得免疫原性;乳鼠攻毒试验证明,母鼠免疫后产生的抗体能够有效传递给子代,具有很好的保护作用。To research and develop rotavirus vaccine production in plant,the neutralization antigen gene VP7 was fused with CTB gene,and p PHAP1301-his-VP7-liner-CTB expression vecter was constructed and transformed into Arabidpsis thaliana.The his-VP7-liner-CTB was analyzed by RT-PCR,Westernblot and ELISA.Further,the protective response of plant derived his-VP7-liner-CTB in mice was investigated by oral immunizations.The results showed that p PHAP1301-his-VP7-liner-CTB expression vecter was successfully constructed and transformed into Arabidopsis thaliana.Molecular analysis revealed that his-VP7-liner-CTB was efficiently expressed in plant seeds.The account of VP7 was 0.39% in the sample of TSP that was expressed in Arabidopsis thaliana containing his-VP7-liner-CTB.A high level VP7（56.25 g） protein was expressed in each gram of transgenic seeds.Mice was immunized and got immunogenicity with the TSP from trangenic seeds.The test of neonatal rats challenged with rotavirs his-VP7-liner-CTB proved that it had good protection for rotavirs and immune antibodies was effectively passed on to the offspring from mother rats.The results indicated that the VP7 protein produced by Arabidopsis thaliana was a candidate for a subunit vaccine specifically for the rotavirus strain.

关 键 词：轮状病毒 VP7融合蛋白 拟南芥 免疫原性 疫苗 

分 类 号：S852.659.4[农业科学—基础兽医学]