猪病毒性腹泻病原相关蛋白在植物系统中的表达

发布时间：2018-06-15 21:57

本文选题：猪流行性腹泻病毒 + 猪轮状病毒　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：猪病毒性腹泻是我国猪场常见的多发病,其主要病原有猪流行性腹泻病毒(PEDV)、猪轮状病毒(PRV)和猪传染性肠胃炎病毒(TGEV)。该类疾病的传染率和致死率都很高,同时各年龄阶段的猪均有感染的危险,严重威胁了养猪业的发展,因此研究有效的相关疫苗具有重要意义。本试验在前期关于PRV病毒VP7蛋白在烟草中表达的基础上进行更深入的探索,以烟草瞬时表达系统、烟草稳定表达系统和玉米胚乳特异性表达系统作为生物反应器,分别构建这几种病毒相关抗原蛋白的重组表达载体,采用农杆菌介导法转入受体植物材料,并进行蛋白表达检测,为进一步开发植物源病毒疫苗奠定基础。试验主要取得以下结果:(1)选取PEDV病毒纤突蛋白S上的主要抗原位点所在区域248-789位氨基酸,用烟草核偏爱密码子进行优化并由公司合成基因。根据受体植物材料不同,分别选取植物35S启动子和玉米14kD Zein启动子,成功构建了pBI121-S植物表达载体和pBI121-pZein::S玉米胚乳特异性表达载体。用电击法将载体转入农杆菌GV3101中,再用农杆菌侵染法构建植物转基因再生体系,分别将S基因转入普通烟草叶盘和玉米胚乳愈伤组织中。经筛选和诱导培养,获得再生抗性植株。(2)以普通烟草为材料,用优化的瞬时表达系统对PRV病毒中和抗原和血凝素抗原的融合蛋白VP4-7进行转化,提取侵染后的烟草总蛋白并用镍柱纯化,纯化前后的蛋白我们分别进行了SDS-PAGE和Western Blot分析。用His-tag抗体进行检测,总蛋白Western的结果显示目的蛋白获得了表达,纯化后的结果显示蛋白的大小发生了变化。(3)选取TGEV病毒纤突蛋白S上的抗原位点所在区域1-543位氨基酸,用烟草核偏爱密码子进行优化并由公司合成基因。成功构建了pBI121-TS植物表达载体,为后续植物疫苗的研究奠定了基础。
[Abstract]:Porcine viral diarrhea is a common disease in pig farms in China. The main pathogens are Porcine epidemic diarrhea virus (PEDVV), Porcine Rotavirus (PRV) and Porcine Infectious gastroenteritis virus (TGEV). The infection rate and fatality rate of this kind of disease are very high, and at the same time, pigs of all ages are at risk of infection, which seriously threatens the development of pig industry. Therefore, it is of great significance to study effective related vaccines. In this study, the expression of PRV VP7 protein in tobacco was further explored. Tobacco transient expression system, tobacco stable expression system and maize endosperm specific expression system were used as bioreactor. The recombinant expression vectors of these virus-associated antigen proteins were constructed, and then transferred to the recipient plant material by Agrobacterium tumefaciens, and the protein expression was detected, which laid a foundation for the further development of plant-derived virus vaccine. The main results were as follows: (1) the amino acids at the 248-789 amino acid site of the main antigen site of PEDV fibrin S were selected and optimized by tobacco nucleopolymeric codon and synthesized by the company. The plant expression vector pBI121-S and the specific expression vector pBI121-pZein: s of maize endosperm were successfully constructed by selecting plant 35s promoter and maize 14kD Zein promoter according to the different plant materials of the receptor. The plant expression vector pBI121-S and pBI121-pZein: 1: s maize endosperm specific expression vector were successfully constructed. The vector was transferred into Agrobacterium tumefaciens GV3101 by electric shock method, and then the transgenic regeneration system was constructed by Agrobacterium tumefaciens. The S gene was transferred into the leaf disk of common tobacco and the callus of maize endosperm, respectively. The regenerated resistant plant was obtained by screening and inducing culture. The recombinant protein VP4-7 of PRV neutralizing antigen and hemagglutinin antigen was transformed by the optimized transient expression system, using common tobacco as the material, and the fusion protein VP4-7 of PRV neutralizing antigen and hemagglutinin antigen was obtained. The total protein of infected tobacco was extracted and purified by nickel column. The protein before and after purification was analyzed by SDS-PAGE and Western blot, respectively. His-tag antibody was used to detect the protein. Western results of the total protein showed that the target protein was expressed. The purified protein showed that the size of the protein had changed.) the amino acid at the antigenic site of TGEV fibrin protein S was selected, and the amino acid at position 1-543 was selected. Tobacco nucleus preference codon is used to optimize and synthesize genes from companies. The plant expression vector pBI121-TS was successfully constructed, which laid a foundation for the further study of plant vaccine.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651

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