密碼子優(yōu)化的輪狀病毒VP6蛋白在煙草中的高效表達
發(fā)布時間：2018-10-24 09:20
【摘要】： 輪狀病毒(Rotavirus,RV)是引起全世界嬰幼兒和幼齡動物嚴重腹瀉的最重要、最常見的病原。在發(fā)展中國家,每年因RV感染發(fā)生中度和嚴重腹瀉以及死亡的兒童高達1887萬人;在發(fā)達國家,因RV感染造成的經(jīng)濟損失是一個不容忽視的問題。據(jù)估計美國每年就此一項的醫(yī)藥損失高達2億6千萬美元。鑒于RV危害嚴重且無有效的治療手段,世界衛(wèi)生組織(WHO)將Rv疫苗列為最優(yōu)先發(fā)展的疫苗項目之一。我們對于新型RV疫苗的研究仍在探索之中。 植物生物反應器是一種安全和廉價的生物系統(tǒng)。隨著分子生物學的發(fā)展,特別是植物遺傳工程的快速發(fā)展,人類可以通過自身的意愿設計和改造植物,高效的生產(chǎn)出廉價的人類生產(chǎn)和生活所需要的各種產(chǎn)品。這種植物反應器的概念,尤其是利用植物生產(chǎn)藥用蛋白和工業(yè)化酶類的概念,以及植物直接作為口服疫苗的概念,漸漸受到人們的重視和慢慢被人們廣泛的接受。 本實驗嘗試利用煙草作為生物反應器生產(chǎn)VP6蛋白。將密碼子優(yōu)化的VP6基因克隆到質(zhì)粒中,構(gòu)建植物表達載體pBI121-V6(U)。利用農(nóng)桿菌介導法轉(zhuǎn)化煙草,經(jīng)卡那霉素(Kan)多重篩選,獲得抗性植株,對抗性植株提取DNA進行PCR分析,表明VP6基因已經(jīng)整合到煙草的基因組中。轉(zhuǎn)基因植株的western blot顯示pBI121-V6(U)的轉(zhuǎn)基因煙草可以有效的表達VP6蛋白。實驗結(jié)果表明,我們所構(gòu)建的重組輪狀病毒多表位具有一定的免疫原性。本研究為研制重組輪狀病毒抗原表位亞單位疫苗、轉(zhuǎn)基因植物口服疫苗提供一條新的途徑和方法,具有一定的理論意義和應用前景。
[Abstract]:Rotavirus (Rotavirus,RV) is the most important and common cause of severe diarrhea in infants and young animals around the world. In developing countries, as many as 18.87 million children die each year from moderate and severe diarrhoea from RV infection, and economic losses from RV infection are a problem that cannot be ignored in developed countries. The United States is estimated to lose $260 million a year on this item. In view of the serious harm of RV and the lack of effective treatment, (WHO) of the World Health Organization (WHO) lists Rv vaccine as one of the most priority vaccine projects. We are still exploring new RV vaccines. Plant bioreactor is a safe and cheap biological system. With the development of molecular biology, especially the rapid development of plant genetic engineering, human beings can design and transform plants through their own will, and efficiently produce all kinds of products needed by cheap human production and life. The concept of this kind of plant reactor, especially the concept of using plants to produce medicinal proteins and industrializing enzymes, and the concept of plants as oral vaccines, have gradually been paid more and more attention to and accepted widely by people. In this experiment, tobacco was used as a bioreactor to produce VP6 protein. The codon optimized VP6 gene was cloned into the plasmid and the plant expression vector pBI121-V6 (U). Was constructed. Transgenic tobacco was transformed by Agrobacterium tumefaciens and kanamycin (Kan) was used to obtain resistant plants. DNA was extracted from resistant plants for PCR analysis. The results showed that VP6 gene had been integrated into the genome of tobacco. Western blot of transgenic plants showed that pBI121-V6 (U) transgenic tobacco could express VP6 protein effectively. The results showed that the recombinant rotavirus polyepitopes were immunogenicity. This study provides a new approach and method for the preparation of recombinant rotavirus epitope subunit vaccine and oral vaccine for transgenic plants, which has certain theoretical significance and application prospect.
【學位授予單位】：天津師范大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392;Q943.2
本文編號：2290974
[Abstract]:Rotavirus (Rotavirus,RV) is the most important and common cause of severe diarrhea in infants and young animals around the world. In developing countries, as many as 18.87 million children die each year from moderate and severe diarrhoea from RV infection, and economic losses from RV infection are a problem that cannot be ignored in developed countries. The United States is estimated to lose $260 million a year on this item. In view of the serious harm of RV and the lack of effective treatment, (WHO) of the World Health Organization (WHO) lists Rv vaccine as one of the most priority vaccine projects. We are still exploring new RV vaccines. Plant bioreactor is a safe and cheap biological system. With the development of molecular biology, especially the rapid development of plant genetic engineering, human beings can design and transform plants through their own will, and efficiently produce all kinds of products needed by cheap human production and life. The concept of this kind of plant reactor, especially the concept of using plants to produce medicinal proteins and industrializing enzymes, and the concept of plants as oral vaccines, have gradually been paid more and more attention to and accepted widely by people. In this experiment, tobacco was used as a bioreactor to produce VP6 protein. The codon optimized VP6 gene was cloned into the plasmid and the plant expression vector pBI121-V6 (U). Was constructed. Transgenic tobacco was transformed by Agrobacterium tumefaciens and kanamycin (Kan) was used to obtain resistant plants. DNA was extracted from resistant plants for PCR analysis. The results showed that VP6 gene had been integrated into the genome of tobacco. Western blot of transgenic plants showed that pBI121-V6 (U) transgenic tobacco could express VP6 protein effectively. The results showed that the recombinant rotavirus polyepitopes were immunogenicity. This study provides a new approach and method for the preparation of recombinant rotavirus epitope subunit vaccine and oral vaccine for transgenic plants, which has certain theoretical significance and application prospect.
【學位授予單位】：天津師范大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392;Q943.2
【引證文獻】
相關(guān)碩士學位論文 前1條
1 邸萌亮;人脂聯(lián)素基因（ADI）轉(zhuǎn)化水稻的研究[D];揚州大學;2011年
，本文編號：2290974
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