本文關(guān)鍵詞：利用遺傳密碼子擴(kuò)充技術(shù)構(gòu)建可控性柯薩奇B3病毒顆粒的研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 遺傳密碼子擴(kuò)充 非天然氨基酸 柯薩奇B3病毒

【摘要】：遺傳密碼子擴(kuò)充技術(shù)是利用正交性氨酰tRNA合成酶/tRNA(aaRS/tRNA)識(shí)別mRNA上的無義密碼子(終止密碼子或移碼突變),實(shí)現(xiàn)在特定位點(diǎn)處插入非天然氨基酸。這些非天然氨基酸通常都具有特殊的物理、化學(xué)或生物特性,因此實(shí)現(xiàn)非天然氨基酸插入的遺傳密碼子擴(kuò)充技術(shù)已在多個(gè)研究領(lǐng)域中展現(xiàn)出豐富多樣的應(yīng)用潛質(zhì)�？滤_奇B3病毒(coxsackievirus B3,CVB3)屬于小RNA病毒科的一員,在人群中分布廣泛,被認(rèn)為是病毒性心肌炎最為重要的誘發(fā)因子之一,除此之外還與無菌性腦膜炎、胰腺炎以及I型糖尿病等疾病相關(guān)。盡管CVB3對(duì)人類健康具有一定的威脅,但是至今仍無可以臨床使用的疫苗或藥物。對(duì)于CVB3的防治困境,結(jié)合遺傳密碼子擴(kuò)充技術(shù),本課題設(shè)想為CVB3的翻譯包裝設(shè)置一道遺傳屏障。即在CVB3基因組內(nèi)引入琥珀密碼子UAG,在自然環(huán)境下,CVB3的翻譯會(huì)因?yàn)橐氲溺晖蛔兌崆敖K止,不能包裝出完整病毒;在人工環(huán)境下,遺傳密碼子擴(kuò)充能夠正確識(shí)別引入的琥珀突變,并在相應(yīng)位點(diǎn)處插入氨基酸,使CVB3的翻譯可以繼續(xù)下去,包裝出完整病毒。本課題中所構(gòu)建的CVB3突變體具有可控性,在人工環(huán)境中包裝出的CVB3具有免疫原性,但不能在自然環(huán)境中進(jìn)行病毒擴(kuò)增,從而為CVB3的疫苗研制提供一個(gè)新的思路。本課題選用甲烷八疊球菌來源的PylRS/tRNA_(CUA)。PylRS在真核細(xì)胞中不存在,所選用的Boc-lysine是人工化學(xué)合成的不存在于天然環(huán)境中,只能通過人工添加,從而進(jìn)一步提升了CVB3的可控性以及安全性。主要研究內(nèi)容及結(jié)果如下:1.根據(jù)文獻(xiàn)調(diào)研,選取構(gòu)建Methanosarcina mazei來源的MmBocRS和U6-PyltRNA_(CUA)。經(jīng)文獻(xiàn)查閱以及Gene Bank比對(duì)確定最終片段序列進(jìn)行全基因合成。將MmBocRS基因片段連入載體pcDNA3.1,再反向依次連入4個(gè)串聯(lián)的PyltRNA_(CUA)片段。經(jīng)酶切驗(yàn)證,遺傳密碼子擴(kuò)充系統(tǒng)質(zhì)粒構(gòu)建成功,依次命名為MmBocRS-pcDNA3.1、MmBocRS-tRNA、MmBocRS-2tRNA、MmBocRS-3tRNA和MmBocRS-4tRNA。2.將上述構(gòu)建質(zhì)粒瞬時(shí)轉(zhuǎn)染入HEK 293T細(xì)胞內(nèi),利用Western Blot驗(yàn)證MmBocRS能在細(xì)胞中正常表達(dá)。通過實(shí)時(shí)定量PCR驗(yàn)證PyltRNA_(CUA)也能在細(xì)胞中正常表達(dá),并且PyltRNA_(CUA)的表達(dá)量確是隨拷貝數(shù)的增加而增加。結(jié)果表明構(gòu)建的遺傳密碼子擴(kuò)充系統(tǒng)質(zhì)粒能在細(xì)胞中正常表達(dá)。3.在pEGFP-N1上EGFP開放閱讀框的39位進(jìn)行琥珀突變,構(gòu)建EGFP琥珀突變體質(zhì)粒pEGFP-N1-UAG。pEGFP-N1-UAG與遺傳密碼子擴(kuò)充系統(tǒng)質(zhì)粒共轉(zhuǎn)染細(xì)胞,顯微鏡下觀察熒光及Western Blot結(jié)果均表明遺傳密碼子擴(kuò)充系統(tǒng)質(zhì)粒在HEK 293T細(xì)胞內(nèi)能正常發(fā)揮功能,提示通過非天然氨基酸Boc-lysine能夠?qū)崿F(xiàn)控制蛋白表達(dá)。又進(jìn)一步利用流式細(xì)胞術(shù)分析非天然氨基酸的插入效率,pEGFP-N1-UAG的綠色熒光蛋白并沒有如預(yù)期隨著PyltRNA_(CUA)拷貝數(shù)的增加而增加,最高大概為43.10%左右,與野生型EGFP的表達(dá)率66.10%相比,本課題中所構(gòu)建的遺傳密碼子擴(kuò)充系統(tǒng)可以使Boc-lysine的相對(duì)插入效率達(dá)到65.20%(相對(duì)值=43.10%/66.10%)。4.利用反向遺傳學(xué)技術(shù)在CVB3基因組的不同位置分別進(jìn)行了琥珀突變,共構(gòu)建了16個(gè)CVB3琥珀突變體,經(jīng)酶切以及測(cè)序表明CVB3琥珀突變體構(gòu)建成功。CVB3琥珀突變體質(zhì)粒與MmBocRS-4tRNA質(zhì)粒共轉(zhuǎn)染細(xì)胞,對(duì)轉(zhuǎn)染產(chǎn)物進(jìn)行反復(fù)凍融,收獲病毒液,感染下一代細(xì)胞,分別對(duì)兩代所表達(dá)出來的CVB3進(jìn)行檢測(cè)。針對(duì)CVB3的5’-UTR設(shè)計(jì)引物,利用實(shí)時(shí)定量PCR分析表明Boc-lysine的添加與否確實(shí)可以使CVB3琥珀突變體的表達(dá)有明顯差異,提示實(shí)現(xiàn)了通過非天然氨基酸控制病毒擴(kuò)增。通過病毒RNA提取、逆轉(zhuǎn)錄、PCR以及擴(kuò)增產(chǎn)物送測(cè)序發(fā)現(xiàn)突變體存在回復(fù)突變的現(xiàn)象。5.為了保證每次試驗(yàn)的時(shí)空一致性,并且避免每次轉(zhuǎn)染受細(xì)胞狀態(tài)、細(xì)胞密度以及實(shí)驗(yàn)操作者經(jīng)驗(yàn)影響等因素,利用慢病毒包裝技術(shù)成功構(gòu)建可以表達(dá)遺傳密碼子擴(kuò)充系統(tǒng)的穩(wěn)定細(xì)胞株BocRS-tRNA_(CUA)。對(duì)穩(wěn)定細(xì)胞株進(jìn)行了Western Blot以及實(shí)時(shí)定量PCR檢測(cè),還進(jìn)一步轉(zhuǎn)染pEGFP-N1-UAG驗(yàn)證其功能。結(jié)果表明穩(wěn)定細(xì)胞株BocRS-tRNA_(CUA)中MmBocRS和PyltRNA_(CUA)可以穩(wěn)定表達(dá),轉(zhuǎn)染結(jié)果可以觀察到綠色熒光表明穩(wěn)定細(xì)胞株確具有遺傳密碼子擴(kuò)充功能。綜上所述,本課題針對(duì)CVB3尚無臨床可用預(yù)防性疫苗的現(xiàn)狀,構(gòu)建了CVB3琥珀感染性克隆,并通過遺傳密碼子擴(kuò)充及非天然氨基酸Boc-lysine實(shí)現(xiàn)了為CVB3設(shè)置一道遺傳屏障,隔離人工環(huán)境與自然環(huán)境,使改造的病毒基因組在人工條件下能正常翻譯,在自然條件下不能自我復(fù)制,即利用遺傳密碼子擴(kuò)充技術(shù)獲得了具有可控性的CVB3突變體。而所構(gòu)建的突變體雖然在自然條件下不能自我復(fù)制但并不會(huì)失去病毒原有的免疫原性,為CVB3及其他小RNA病毒疫苗的研制提供了一個(gè)新的思路。
[Abstract]:Genetic codon expansion technology is the use of orthogonal aminoacyl tRNA synthetase /tRNA (aaRS/tRNA) nonsense codon recognition on the mRNA (stop codon or frameshift mutations), achieve site-specific insertion of non natural amino acids. These non natural amino acids usually have special physical, chemical or biological characteristics, therefore non natural amino acid insertion codon expansion technology has been in many research fields to show diversity. The potential application of Coxsackie virus B3 (coxsackievirus B3 CVB3) is a member of small RNA virus, in the crowd are widely distributed, is considered one of the most important factors to induce viral in addition, myocarditis, and aseptic meningitis, pancreatitis related disease and type I diabetes. Although CVB3 is a threat to human health, but still can be clinically used vaccines or drugs. The prevention and control of difficulties in CVB3, combining the genetic code expansion technology, this topic ideas for the translation of the CVB3 package set up a genetic barrier. The introduction of the amber codon UAG in CVB3 genome, in the natural environment, the translation will be CVB3 because of the introduction of the amber mutation and premature termination, not a complete package in the artificial virus; under the environment, genetic code expansion can correctly identify the amber mutation and insertion of amino acids in the corresponding site, the CVB3 translation can continue, packaging intact virus. CVB3 mutants constructed in this study is controllable, packaging in the artificial environment of CVB3 immunogenic, but not in the natural environment for virus amplification, which provides a new method for CVB3 vaccine development. This study selected eight fold methane derived PylRS/tRNA_ aureus (CUA).PylRS does not exist in eukaryotic cells, selected The Boc-lysine is using artificial chemical synthesis does not exist in the natural environment, only by artificial additives, so as to further enhance the CVB3 controllability and safety. The main research contents and results are as follows: 1. according to the literature research, construction of Methanosarcina mazei selected sources of MmBocRS and U6-PyltRNA_ (CUA). Through literature review and comparison of Gene Bank to determine the final total gene synthesis sequence. MmBocRS gene fragment into vector pcDNA3.1, then reverse connected into the 4 series of PyltRNA_ (CUA) fragment. After enzyme digestion verification, genetic code expansion system was successfully constructed and were named MmBocRS-pcDNA3.1, MmBocRS-tRNA, MmBocRS-2tRNA, MmBocRS-3tRNA and MmBocRS-4tRNA.2. of the constructed plasmids were transiently HEK was transfected into 293T cells, using Western Blot MmBocRS to verify the normal expression in cells. By real-time quantitative PCR verification PyltR NA_ (CUA) can be expressed in normal cells, and the expression of PyltRNA_ (CUA) is increased with the increase of the copy number. The results show that the constructed genetic codon expansion system plasmid in cells in the normal expression of.3. in pEGFP-N1 EGFP open reading frame of 39 EGFP amber amber mutation, construction the mutation plasmid pEGFP-N1-UAG.pEGFP-N1-UAG and genetic codon expansion system cotransfected cells under the microscope, fluorescence and Western Blot results showed that the genetic code expansion function of the system in the HEK plasmid play normal 293T cells can be provided, shown by non natural amino acid Boc-lysine to achieve control of protein expression. Further analysis of the use of non natural amino acid insertion efficiency flow cytometry, green fluorescent protein pEGFP-N1-UAG is not as expected with PyltRNA_ (CUA) copy number increases, the maximum is about 43.1 About 0%, compared with the wild-type EGFP expression rate of 66.10%, the construction of the issue of the genetic code expansion system can make Boc-lysine the relative insertion efficiency reached 65.20% (relative value =43.10%/66.10%).4. in different locations of CVB3 genome were amber mutation using reverse genetics technology, constructed 16 CVB3 amber mutants. By enzyme digestion and sequencing showed that the CVB3 mutant was successfully constructed.CVB3 amber amber mutation plasmid and MmBocRS-4tRNA plasmid were co transfected cells, the transfection products were repeated freezing and thawing, harvest virus liquid, the next generation of infected cells, were detected on the two generation expressed by CVB3. According to CVB3 5 -UTR primers were designed using real-time quantitative PCR analysis showed that the addition of Boc-lysine or CVB3 can make the expression of amber mutants were significantly different, suggesting that through the implementation of non natural amino acid to control disease Virus amplification. The virus through RNA extraction, reverse transcription, PCR and PCR product sequencing revealed that the mutant exist mutation phenomenon of.5. in order to ensure the time-space consistency of each test, and avoid each transfection by cell, cell density and experimental factors of operator experience influence, successfully constructed can express genetic codon extension system stable cell line BocRS-tRNA_ using lentiviral packaging technology (CUA). The stable cell lines by Western Blot and real-time PCR detection, further verify the function of pEGFP-N1-UAG transfection. The results showed that BocRS-tRNA_ stable cell line (CUA) and MmBocRS PyltRNA_ (CUA) can be expressed stably, the transfection results can be observed green fluorescence showed that the stable cell line it has a genetic code expansion function. To sum up, the topic for the CVB3 situation that there is no clinically available prophylactic vaccine, construction The amber CVB3 infectious clone, and the genetic code expansion and non natural amino acids Boc-lysine to achieve the set a genetic barrier for CVB3 isolation, artificial environment and the natural environment, the transformation of the viral genome to normal translation under artificial conditions, can not replicate in natural conditions, which uses genetic codon expansion technique the CVB3 mutant is controllable. And the mutant although under natural conditions can not be self replicating but does not lose the original virus immunogenicity, for the development of CVB3 and other small RNA virus vaccine provides a new way of thinking.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R373

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