豬瘟病毒疫苗株反向遺傳學(xué)改造
發(fā)布時間：2019-03-25 07:59
【摘要】：豬瘟(Classical swine fever,CSF),別名豬霍亂,俗稱“爛腸瘟”,是豬的一種高感染性的疾病,致死率高達(dá)90%以上,是威脅養(yǎng)豬業(yè)的主要傳染病之一。豬瘟的爆發(fā)會導(dǎo)致嚴(yán)重的經(jīng)濟(jì)損失,被世界動物衛(wèi)生組織列為A類傳染病,受到了各個國家的高度重視,成為了各個國家研究的熱點。豬瘟的病原豬瘟病毒是一種包膜病毒,大小約40-60nm,它的基因組大小約12.3kb,是單股正鏈RNA,包含了一個大的開放閱讀框(ORF),一個5'非編碼區(qū)(5'UTR)和一個3'非編碼區(qū)(3'UTR)。ORF能夠編碼大約3900個氨基酸的多聚蛋白,然后在信號肽酶等作用下產(chǎn)生12個成熟蛋白,順序依次為Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B。其中,C、Erns、E1、E2為4個結(jié)構(gòu)蛋白,Npro、P7、NS2、NS3、NS4A、NA4B、NS5A、NS5B為8個非結(jié)構(gòu)蛋白。本研究第一部分根據(jù)實驗室已構(gòu)建的豬瘟病毒感染性的c DNA,通過插入CMV啟動子和錘頭狀核酶構(gòu)建了重組質(zhì)粒p MC18-CMV-HM-CSFV,并用于豬瘟病毒的拯救。為了驗證錘頭狀核酶的功能,我們構(gòu)建了p GL2.0-CMV、p GL2.0-CMV-HM、p GL2.0-C MV-HM'和p GL2.0-CMV-HM-5'UTR四個質(zhì)粒。通過雙熒光素酶報告基因檢測系統(tǒng)和實時熒光定量核酸擴(kuò)增檢測系統(tǒng)(Real-time Quantitative PCR Detecting Syste m,QPCR)檢測發(fā)現(xiàn),重組質(zhì)粒轉(zhuǎn)染進(jìn)細(xì)胞后,錘頭狀核酶能夠發(fā)揮約62%的剪切作用。經(jīng)過QPCR檢測后發(fā)現(xiàn),改造后的質(zhì)粒p MC18-CMV-HM-CSFV通過轉(zhuǎn)染進(jìn)細(xì)胞成功拯救出了病毒。本研究為豬瘟疫苗的發(fā)展奠定了基礎(chǔ)。本研究第二部分探究了豬瘟病毒C蛋白與5'UTR的關(guān)系。在實驗室的前期研究中發(fā)現(xiàn),豬瘟病毒的C蛋白對3'UTR具有調(diào)節(jié)作用。為了確定C蛋白與5'UTR之間是否存在相互作用關(guān)系,我們分別構(gòu)建了雙熒光素酶表達(dá)質(zhì)粒p MIR-5'UTR-RLUC和p MIRRLUC。通過雙熒光素酶報告基因檢測系統(tǒng)檢測證明,p MIR-5'UTR-RLUC轉(zhuǎn)染進(jìn)細(xì)胞后,5'UTR能夠發(fā)揮相應(yīng)的作用。將豬瘟病毒C蛋白的真核表達(dá)載體p CMV-C與5’UTR的真核表達(dá)載體p MIR-5'UTR-RLUC共轉(zhuǎn)染至PK-15細(xì)胞,然后通過western blotting檢測、實時熒光定量核酸擴(kuò)增檢測系統(tǒng)(Real-time Quantitative PCR Detecting System)和雙熒光素酶報告基因檢測系統(tǒng)(Dual-Luciferase Reporter Assay System)證明,C蛋白與5'UTR之間不存在相互作用關(guān)系。本部分研究為探究豬瘟病毒蛋白對5'UTR功能的影響奠定了基礎(chǔ)。
[Abstract]:Swine cholera (Classical swine fever,CSF), also known as swine cholera, is a highly infectious disease of pigs with a mortality rate of more than 90%. It is one of the main infectious diseases that threaten the pig industry. The outbreak of classical swine fever will lead to serious economic losses. It has been classified as a class A infectious disease by the World Organization for Animal Health (OIE). It has been attached great importance to by various countries and has become the focus of research in various countries. The pathogen of classical swine fever (CSFV) is a envelope virus with a genome size of about 12.3kb and a large open reading frame (ORF) (ORF), contained in the single strand positive strand RNA, which is about 40 脳 60 nm in size and has a genome size of about 12.3 kb.Swine fever virus is the pathogen of classical swine fever. A 5 'non-coding region (5'UTR) and a 3' non-coding region (3'UTR). ORF encodes a Polymeric protein of about 3900 amino acids, and then produces 12 mature proteins under the action of signal peptidase, etc. Order is Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. Among them, C, Erns, E1, E2 and Npro,P7,NS2,NS3,NS4A,NA4B,NS5A,NS5B are four structural proteins and eight non-structural proteins. In the first part of this study, the recombinant plasmid p-DNA, was constructed by inserting CMV promoter and hammerhead ribozyme into the infectious c-MC18-CMV-HM-CSFV, of classical swine fever virus (CSFV) which had been constructed in the laboratory and used for the rescue of CSFV. To verify the function of hammerhead ribozyme, four plasmids, p GL2.0-CMV,p GL2.0-CMV-HM,p GL2.0-CMV-HM 'and p GL2.0-CMV-HM-5'UTR, were constructed. Double luciferase reporter gene detection system and real-time fluorescence quantitative nucleic acid amplification system (Real-time Quantitative PCR Detecting Syste-m, QPCR) showed that the hammerhead ribozyme could perform about 62% of the cleavage effect after the recombinant plasmid was transfected into the cells. After QPCR detection, it was found that the modified plasmid p-MC18-CMV-HM-CSFV successfully saved the virus by transfection into the cells. This study laid a foundation for the development of swine fever vaccine. In the second part of this study, the relationship between C protein and 5'UTR of classical swine fever virus (CSFV) was investigated. In previous laboratory studies, it was found that the C protein of Classical Swine Fever virus (CSFV) has a regulatory effect on 3'UTR. In order to determine the interaction between C protein and 5'UTR, we constructed double luciferase expression plasmids p MIR-5'UTR-RLUC and p MIRRLUC., respectively. The double luciferase reporter gene detection system showed that 5'UTR could play a role in the transfection of p-MIR-5'UTR-RLUC into cells. The eukaryotic expression vector p-CMV-C of CSFV C protein was co-transfected with the eukaryotic expression vector p-MIR-5'UTR-RLUC of 5'UTR into PK-15 cells, and then detected by western blotting. Real-time fluorescent quantitative nucleic acid amplification system (Real-time Quantitative PCR Detecting System) and double luciferase reporter gene detection system (Dual-Luciferase Reporter Assay System) showed that there was no interaction between C protein and 5'UTR. This part of the study laid a foundation for exploring the effect of classical swine fever virus protein on the function of 5'UTR.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.651
本文編號：2446793
[Abstract]:Swine cholera (Classical swine fever,CSF), also known as swine cholera, is a highly infectious disease of pigs with a mortality rate of more than 90%. It is one of the main infectious diseases that threaten the pig industry. The outbreak of classical swine fever will lead to serious economic losses. It has been classified as a class A infectious disease by the World Organization for Animal Health (OIE). It has been attached great importance to by various countries and has become the focus of research in various countries. The pathogen of classical swine fever (CSFV) is a envelope virus with a genome size of about 12.3kb and a large open reading frame (ORF) (ORF), contained in the single strand positive strand RNA, which is about 40 脳 60 nm in size and has a genome size of about 12.3 kb.Swine fever virus is the pathogen of classical swine fever. A 5 'non-coding region (5'UTR) and a 3' non-coding region (3'UTR). ORF encodes a Polymeric protein of about 3900 amino acids, and then produces 12 mature proteins under the action of signal peptidase, etc. Order is Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. Among them, C, Erns, E1, E2 and Npro,P7,NS2,NS3,NS4A,NA4B,NS5A,NS5B are four structural proteins and eight non-structural proteins. In the first part of this study, the recombinant plasmid p-DNA, was constructed by inserting CMV promoter and hammerhead ribozyme into the infectious c-MC18-CMV-HM-CSFV, of classical swine fever virus (CSFV) which had been constructed in the laboratory and used for the rescue of CSFV. To verify the function of hammerhead ribozyme, four plasmids, p GL2.0-CMV,p GL2.0-CMV-HM,p GL2.0-CMV-HM 'and p GL2.0-CMV-HM-5'UTR, were constructed. Double luciferase reporter gene detection system and real-time fluorescence quantitative nucleic acid amplification system (Real-time Quantitative PCR Detecting Syste-m, QPCR) showed that the hammerhead ribozyme could perform about 62% of the cleavage effect after the recombinant plasmid was transfected into the cells. After QPCR detection, it was found that the modified plasmid p-MC18-CMV-HM-CSFV successfully saved the virus by transfection into the cells. This study laid a foundation for the development of swine fever vaccine. In the second part of this study, the relationship between C protein and 5'UTR of classical swine fever virus (CSFV) was investigated. In previous laboratory studies, it was found that the C protein of Classical Swine Fever virus (CSFV) has a regulatory effect on 3'UTR. In order to determine the interaction between C protein and 5'UTR, we constructed double luciferase expression plasmids p MIR-5'UTR-RLUC and p MIRRLUC., respectively. The double luciferase reporter gene detection system showed that 5'UTR could play a role in the transfection of p-MIR-5'UTR-RLUC into cells. The eukaryotic expression vector p-CMV-C of CSFV C protein was co-transfected with the eukaryotic expression vector p-MIR-5'UTR-RLUC of 5'UTR into PK-15 cells, and then detected by western blotting. Real-time fluorescent quantitative nucleic acid amplification system (Real-time Quantitative PCR Detecting System) and double luciferase reporter gene detection system (Dual-Luciferase Reporter Assay System) showed that there was no interaction between C protein and 5'UTR. This part of the study laid a foundation for exploring the effect of classical swine fever virus protein on the function of 5'UTR.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.651
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 侯玉臻;趙丹彤;劉國英;賀番;劉斌;付紹印;郝永清;張文廣;;豬瘟病毒核心蛋白研究進(jìn)展[J];病毒學(xué)報;2015年05期
，本文編號：2446793
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