本文選題：豬流行性腹瀉病毒 + 豬輪狀病毒�。� 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：豬病毒性腹瀉是我國(guó)豬場(chǎng)常見的多發(fā)病,其主要病原有豬流行性腹瀉病毒(PEDV)、豬輪狀病毒(PRV)和豬傳染性腸胃炎病毒(TGEV)。該類疾病的傳染率和致死率都很高,同時(shí)各年齡階段的豬均有感染的危險(xiǎn),嚴(yán)重威脅了養(yǎng)豬業(yè)的發(fā)展,因此研究有效的相關(guān)疫苗具有重要意義。本試驗(yàn)在前期關(guān)于PRV病毒VP7蛋白在煙草中表達(dá)的基礎(chǔ)上進(jìn)行更深入的探索,以煙草瞬時(shí)表達(dá)系統(tǒng)、煙草穩(wěn)定表達(dá)系統(tǒng)和玉米胚乳特異性表達(dá)系統(tǒng)作為生物反應(yīng)器,分別構(gòu)建這幾種病毒相關(guān)抗原蛋白的重組表達(dá)載體,采用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)入受體植物材料,并進(jìn)行蛋白表達(dá)檢測(cè),為進(jìn)一步開發(fā)植物源病毒疫苗奠定基礎(chǔ)。試驗(yàn)主要取得以下結(jié)果:(1)選取PEDV病毒纖突蛋白S上的主要抗原位點(diǎn)所在區(qū)域248-789位氨基酸,用煙草核偏愛密碼子進(jìn)行優(yōu)化并由公司合成基因。根據(jù)受體植物材料不同,分別選取植物35S啟動(dòng)子和玉米14kD Zein啟動(dòng)子,成功構(gòu)建了pBI121-S植物表達(dá)載體和pBI121-pZein::S玉米胚乳特異性表達(dá)載體。用電擊法將載體轉(zhuǎn)入農(nóng)桿菌GV3101中,再用農(nóng)桿菌侵染法構(gòu)建植物轉(zhuǎn)基因再生體系,分別將S基因轉(zhuǎn)入普通煙草葉盤和玉米胚乳愈傷組織中。經(jīng)篩選和誘導(dǎo)培養(yǎng),獲得再生抗性植株。(2)以普通煙草為材料,用優(yōu)化的瞬時(shí)表達(dá)系統(tǒng)對(duì)PRV病毒中和抗原和血凝素抗原的融合蛋白VP4-7進(jìn)行轉(zhuǎn)化,提取侵染后的煙草總蛋白并用鎳柱純化,純化前后的蛋白我們分別進(jìn)行了SDS-PAGE和Western Blot分析。用His-tag抗體進(jìn)行檢測(cè),總蛋白Western的結(jié)果顯示目的蛋白獲得了表達(dá),純化后的結(jié)果顯示蛋白的大小發(fā)生了變化。(3)選取TGEV病毒纖突蛋白S上的抗原位點(diǎn)所在區(qū)域1-543位氨基酸,用煙草核偏愛密碼子進(jìn)行優(yōu)化并由公司合成基因。成功構(gòu)建了pBI121-TS植物表達(dá)載體,為后續(xù)植物疫苗的研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.651

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