轮状病毒外壳蛋白VP7基因在拟南芥种子中表达及免疫原性分析

发布时间：2018-05-14 10:26

本文选题：轮状病毒 + VP　；参考：《黑龙江畜牧兽医》2017年09期

【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。
[Abstract]:In order to develop a rotavirus vaccine from plants, the model plant Arabidopsis thaliana was used as a bioreactor. The VP7 gene of rotavirus neutralizing antigen was transformed into wild type Arabidopsis thaliana by Agrobacterium tumefaciens. The transgenic Arabidopsis thaliana stable strain containing VP7 was obtained and the immunogenicity of the expressed protein was analyzed. According to the preference of codon in Arabidopsis thaliana, the optimized VP7 gene was designed and synthesized, and the plant expression vector pPHAP1301-His-HRVVP7 was constructed, then transformed Arabidopsis thaliana into Arabidopsis thaliana by Floral Dip mediated by Agrobacterium tumefaciens, the seeds of T0 generation were harvested and seeded. T1 generation seeds of transgenic Arabidopsis thaliana were screened by spraying ammonium phosphate, and T3 generation seeds were obtained by repeating the above process. The results were analyzed by RT-PCR- Western Elisa. The results showed that the 1: VP7 gene had been successfully integrated into the genome of Arabidopsis thaliana for transcription and protein expression, and the VP7 protein accounted for 0.31% of the total soluble protein, that is, 43.3 渭 g of the target protein per gram of transgenic Arabidopsis thaliana seeds. Mice were immunized orally with soluble protein in stable strain seeds of transgenic Arabidopsis thaliana. The results showed that it was feasible to produce rotavirus vaccine by using plant as bioreactor.
【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】：“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)
【分类号】：S859.797

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