摘要
为了研究开发植物来源的轮状病毒疫苗,试验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通过农杆菌介导法转化野生型拟南芥,获得了含有VP7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基因,构建植物表达载体p PHAP1301-His-HRVVP7;然后通过农杆菌介导的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、Western、ELISA法等进行分析。结果表明:VP7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0.31%,即每克转基因拟南芥种子中所含目的蛋白为43.3μg。以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,可使小鼠获得免疫原性。说明通过植物作为生物反应器生产轮状病毒疫苗方法可行。
To explore the development of rotavirus vaccine in the plant,the VP7 was genetically cloned to the up stream of the His and codon-optimized in Arabidopsis thaliana. p PHAP1301-His-HRVVP7 expression vecter was constructed and transformed into Arabidpsis thaliana.The VP7 gene was analyzed by RT-PCR,Western and ELISA. Furthermore,the protective response of plant derived VP7 was evaluated in mice by oral immunizations. The results revealed that expression vector of the p PHAP1301-His-HRVVP7 was successfully constructed,and transformed into Arabidpsis thaliana. Molecular analysis revealed that VP7 was efficiently expressed in plant seeds where the VP7 protein accounted for 0. 31% in the sample of TSP in Arabidopsis thaliana carrying VP7 gene. A high level VP7( 43. 3 μg) protein was expressed in each gram of VP7 transgenic seed. The immunized mice with the TSP of transformed seeds could elicit serum Ig G and mucosal Ig A aganinst VP7 protein. These data indicate that the VP7 protein produced in Arabidopsis thaliana is a potential candidate for subunit vaccine against rotavirus.
出处
《黑龙江畜牧兽医》
CAS
北大核心
2017年第5期24-29,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
"863"国家高技术研究发展计划项目"油体生物反应器研制及产品开发"(2011AA100606)
关键词
轮状病毒
VP7
拟南芥
免疫原性
疫苗
Rotavirs
VP7
Arabidopsis thaliana
immunogenicity
vaccine
