VP7-CTB融合蛋白在拟南芥种子中的表达及免疫原性分析
发布时间：2019-05-23 12:14
【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。
[Abstract]:In order to develop a plant-derived rotavirus vaccine, Arabidopsis thaliana, a model plant, was used as a bioreactor to fuse the main neutralizing antigen gene VP7 of rotavirus into wild type Arabidopsis thaliana by Agrobacterium tumefaciens mediated method. A transgenic Arabidopsis thaliana stable strain containing VP7-CTB was obtained and the immunogenicity of its seed expression protein was analyzed. Firstly, according to the preference of Arabidopsis codon, the optimized VP7 and CTB genes were designed and synthesized, and the plant expression vector pPHAP1301-his-VP7-linker-CTB; was constructed. Then Arabidopsis thaliana was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated Floral Dip method, then seeded after harvesting T0 generation seeds, then T1 generation seeds were obtained by spraying ammonium phosphate to screen transgenic Arabidopsis thaliana seeds, and T3 generation Arabidopsis thaliana seeds were obtained by repeating the above process. Through RT-PCR,W estern-bolt, ELISA and so on carry on the analysis. The results showed that his-VP7-linker-CTB gene had been successfully integrated into the genome of Arabidopsis thaliana and transcribed and expressed, and VP7 protein accounted for 0.39% of the total soluble protein. That is to say, the target protein contained in transgenic Arabidopsis thaliana seeds was 52.65 g 路kg ~ (- 1). Finally, the mice were immunized with soluble proteins from the seeds of transgenic Arabidopsis thaliana stable strain to obtain immunogenicity. The challenge test of neonatal mice showed that the antibodies produced by immunization of female mice could be effectively transmitted to their offspring and had a good protective effect.
【作者单位】： 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;吉林农业科技学院中药学院;吉林农业大学中药材学院;温州大学;
【基金】：国家高技术研究发展计划(863)项目(2011AA100606)
【分类号】：Q943.2;S859.797
，
本文编号：2483888
[Abstract]:In order to develop a plant-derived rotavirus vaccine, Arabidopsis thaliana, a model plant, was used as a bioreactor to fuse the main neutralizing antigen gene VP7 of rotavirus into wild type Arabidopsis thaliana by Agrobacterium tumefaciens mediated method. A transgenic Arabidopsis thaliana stable strain containing VP7-CTB was obtained and the immunogenicity of its seed expression protein was analyzed. Firstly, according to the preference of Arabidopsis codon, the optimized VP7 and CTB genes were designed and synthesized, and the plant expression vector pPHAP1301-his-VP7-linker-CTB; was constructed. Then Arabidopsis thaliana was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated Floral Dip method, then seeded after harvesting T0 generation seeds, then T1 generation seeds were obtained by spraying ammonium phosphate to screen transgenic Arabidopsis thaliana seeds, and T3 generation Arabidopsis thaliana seeds were obtained by repeating the above process. Through RT-PCR,W estern-bolt, ELISA and so on carry on the analysis. The results showed that his-VP7-linker-CTB gene had been successfully integrated into the genome of Arabidopsis thaliana and transcribed and expressed, and VP7 protein accounted for 0.39% of the total soluble protein. That is to say, the target protein contained in transgenic Arabidopsis thaliana seeds was 52.65 g 路kg ~ (- 1). Finally, the mice were immunized with soluble proteins from the seeds of transgenic Arabidopsis thaliana stable strain to obtain immunogenicity. The challenge test of neonatal mice showed that the antibodies produced by immunization of female mice could be effectively transmitted to their offspring and had a good protective effect.
【作者单位】： 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;吉林农业科技学院中药学院;吉林农业大学中药材学院;温州大学;
【基金】：国家高技术研究发展计划(863)项目(2011AA100606)
【分类号】：Q943.2;S859.797
，
本文编号：2483888
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