【作者】 魏欣；

【导师】 徐虹；

【作者基本信息】 西北农林科技大学 ， 生物工程（专业学位）， 2016， 硕士

【摘要】 近年来,植物疫苗的开发利用已经成为研究热点,因其具有安全、有效、成本低廉的优势,逐步得到科学界的认同。猪轮状病毒(PRV)是引起猪病毒性腹泻的主要病原微生物之一,是目前危害我国养猪业的一种主要病毒。VP7蛋白是轮状病毒(RV)粒子表面的外壳蛋白,也是RV主要的中和抗原,可以引起机体产生中和抗体,起到免疫保护作用。VP4和VP6蛋白分别是RV的外壳蛋白和内壳蛋白,都是重要的保护性抗原,在预防RV感染时,可与VP7一同刺激机体产生免疫效应。本研究通过构建高效植物表达载体,建立在烟草中表达VP7、VP4和VP6蛋白的遗传转化体系,为开发植物源PRV疫苗奠定基础。试验主要取得如下结果:(1)根据PRV VP7的氨基酸序列,选取能产生中和反应的51-312位氨基酸,用烟草偏爱密码子进行优化人工合成基因序列。在目的基因上游添加Ω序列以增强翻译,下游添加M细胞靶向肽-Col序列增强免疫效应,并添加内质网滞留信号ER序列定向表达蛋白,融合6个His标签用于蛋白的分离纯化。(2)成功构建了pBI121-VP7植物表达载体,通过冻融法转入农杆菌EHA105菌株中。利用农杆菌介导的注射侵染法将VP7基因转入本氏烟草中,经RT-PCR检测、SDS-PAGE及用His-Tag抗体进行Western Blotting分析,初步检测到VP7蛋白的表达。(3)用农杆菌EHA105介导的叶盘法将VP7基因转入普通烟草,经共培养、筛选、分化及生根过程最终得到2株转基因幼苗,通过烟草叶片直接PCR的方法进行鉴定,但未检测到目标条带,还需进一步验证VP7基因是否稳定转化。(4)设计并优化合成了PRV的VP4与VP7融合基因VP4-7以及VP6基因,成功构建了pBI121-VP4-7、pBI121-VP6植物表达载体,为后续在烟草中表达VP4-VP7融合蛋白、VP6蛋白及研发二价或二联植物疫苗奠定基础。更多还原

【Abstract】 In recent years, development and utilization of plant vaccine has become a hot topic, it has gradually recognized by the scientific community because it is safe, effective and low-cost. Porcine rotavirus(PRV) is one of the main pathogenic microorganisms which cause porcine virus diarrhea, it currently is a major virus hazard of our pig industry. VP7 protein is the coat protein of rotavirus(RV) particle surface, it is also the major antigen of RV which can cause the body produce neutralizing antibodies to play a role in immune protection. VP4 and VP6 protein are PRV coat protein and the inner shell protein respectively, they are also important protective antigens. In the prevention of PRV infection, together with the VP7, both of them will stimulate the body’s immune response.in the prevention of RV infection, together with the VP7 will stimulate the body’s immune response. In this study, we have constructed the efficient plant expression vector, in order to establish the genetic transformation system of VP7, VP4 and VP6 protein’s expression in tobacco, to lay foundation for the development of PRV plant vaccine.The main experiment results are as follows:(1) According to the amino acid sequence of PRV VP7, we selected the amino acids51-312 which can lead to neutralizing reaction, used the tobacco preferred codons to optimize,synthesized the target gene sequence. We have added the Ω sequence to the upstream of target gene to improve translation, added M cell targeting peptide- Col sequences to the downstream to enhance immune response, added endoplasmic reticulum ER retention signal sequence to express protein directionally, fused 6 His tag for separation and purification of VP7 protein.(2) We have successfully constructed plant expression vector pBI121-VP7, Transformed it into A.tumefaciens EHA105 by freeze-thaw method, used Agrobacterium-mediated injection infection method to transfer VP7 gene into Nicotiana benthamiana, conducted RT-PCR, SDS-PAGE analysis and used His-Tag antibody to perform Western Blotting,Finally preliminary detected the expression of VP7 protein.(3) We have transformed VP7 gene into Nicotiana tabacum using A.tumefaciens EHA105-mediated leaf disc transformation, through the co-culture, selective differentiationand rooting process, finally obtained two transgenic seedlings, tobacco leaves direct PCR result showed no target gene, it need further examination to verify whether VP7 gene stably transformed.(4) We have synthesized optimized VP4 and VP7 fusion gene VP4-7, VP6 gene of PRV,successfully constructed pBI121-VP4-7, pBI121-VP6 plant expression vectors, and laid foundation for the further expression of VP4-VP7 fused protein、VP6 Protein in tobacco and study of divalent or bivalent plant vaccine.更多还原