利用遗传密码子扩充技术构建可控性柯萨奇B3病毒颗粒的研究

发布时间：2017-12-31 04:09

本文关键词：利用遗传密码子扩充技术构建可控性柯萨奇B3病毒颗粒的研究　出处：《中国人民解放军军事医学科学院》2017年硕士论文　论文类型：学位论文

【摘要】：遗传密码子扩充技术是利用正交性氨酰tRNA合成酶/tRNA(aaRS/tRNA)识别mRNA上的无义密码子(终止密码子或移码突变),实现在特定位点处插入非天然氨基酸。这些非天然氨基酸通常都具有特殊的物理、化学或生物特性,因此实现非天然氨基酸插入的遗传密码子扩充技术已在多个研究领域中展现出丰富多样的应用潜质。柯萨奇B3病毒(coxsackievirus B3,CVB3)属于小RNA病毒科的一员,在人群中分布广泛,被认为是病毒性心肌炎最为重要的诱发因子之一,除此之外还与无菌性脑膜炎、胰腺炎以及I型糖尿病等疾病相关。尽管CVB3对人类健康具有一定的威胁,但是至今仍无可以临床使用的疫苗或药物。对于CVB3的防治困境,结合遗传密码子扩充技术,本课题设想为CVB3的翻译包装设置一道遗传屏障。即在CVB3基因组内引入琥珀密码子UAG,在自然环境下,CVB3的翻译会因为引入的琥珀突变而提前终止,不能包装出完整病毒;在人工环境下,遗传密码子扩充能够正确识别引入的琥珀突变,并在相应位点处插入氨基酸,使CVB3的翻译可以继续下去,包装出完整病毒。本课题中所构建的CVB3突变体具有可控性,在人工环境中包装出的CVB3具有免疫原性,但不能在自然环境中进行病毒扩增,从而为CVB3的疫苗研制提供一个新的思路。本课题选用甲烷八叠球菌来源的PylRS/tRNA_(CUA)。PylRS在真核细胞中不存在,所选用的Boc-lysine是人工化学合成的不存在于天然环境中,只能通过人工添加,从而进一步提升了CVB3的可控性以及安全性。主要研究内容及结果如下:1.根据文献调研,选取构建Methanosarcina mazei来源的MmBocRS和U6-PyltRNA_(CUA)。经文献查阅以及Gene Bank比对确定最终片段序列进行全基因合成。将MmBocRS基因片段连入载体pcDNA3.1,再反向依次连入4个串联的PyltRNA_(CUA)片段。经酶切验证,遗传密码子扩充系统质粒构建成功,依次命名为MmBocRS-pcDNA3.1、MmBocRS-tRNA、MmBocRS-2tRNA、MmBocRS-3tRNA和MmBocRS-4tRNA。2.将上述构建质粒瞬时转染入HEK 293T细胞内,利用Western Blot验证MmBocRS能在细胞中正常表达。通过实时定量PCR验证PyltRNA_(CUA)也能在细胞中正常表达,并且PyltRNA_(CUA)的表达量确是随拷贝数的增加而增加。结果表明构建的遗传密码子扩充系统质粒能在细胞中正常表达。3.在pEGFP-N1上EGFP开放阅读框的39位进行琥珀突变,构建EGFP琥珀突变体质粒pEGFP-N1-UAG。pEGFP-N1-UAG与遗传密码子扩充系统质粒共转染细胞,显微镜下观察荧光及Western Blot结果均表明遗传密码子扩充系统质粒在HEK 293T细胞内能正常发挥功能,提示通过非天然氨基酸Boc-lysine能够实现控制蛋白表达。又进一步利用流式细胞术分析非天然氨基酸的插入效率,pEGFP-N1-UAG的绿色荧光蛋白并没有如预期随着PyltRNA_(CUA)拷贝数的增加而增加,最高大概为43.10%左右,与野生型EGFP的表达率66.10%相比,本课题中所构建的遗传密码子扩充系统可以使Boc-lysine的相对插入效率达到65.20%(相对值=43.10%/66.10%)。4.利用反向遗传学技术在CVB3基因组的不同位置分别进行了琥珀突变,共构建了16个CVB3琥珀突变体,经酶切以及测序表明CVB3琥珀突变体构建成功。CVB3琥珀突变体质粒与MmBocRS-4tRNA质粒共转染细胞,对转染产物进行反复冻融,收获病毒液,感染下一代细胞,分别对两代所表达出来的CVB3进行检测。针对CVB3的5’-UTR设计引物,利用实时定量PCR分析表明Boc-lysine的添加与否确实可以使CVB3琥珀突变体的表达有明显差异,提示实现了通过非天然氨基酸控制病毒扩增。通过病毒RNA提取、逆转录、PCR以及扩增产物送测序发现突变体存在回复突变的现象。5.为了保证每次试验的时空一致性,并且避免每次转染受细胞状态、细胞密度以及实验操作者经验影响等因素,利用慢病毒包装技术成功构建可以表达遗传密码子扩充系统的稳定细胞株BocRS-tRNA_(CUA)。对稳定细胞株进行了Western Blot以及实时定量PCR检测,还进一步转染pEGFP-N1-UAG验证其功能。结果表明稳定细胞株BocRS-tRNA_(CUA)中MmBocRS和PyltRNA_(CUA)可以稳定表达,转染结果可以观察到绿色荧光表明稳定细胞株确具有遗传密码子扩充功能。综上所述,本课题针对CVB3尚无临床可用预防性疫苗的现状,构建了CVB3琥珀感染性克隆,并通过遗传密码子扩充及非天然氨基酸Boc-lysine实现了为CVB3设置一道遗传屏障,隔离人工环境与自然环境,使改造的病毒基因组在人工条件下能正常翻译,在自然条件下不能自我复制,即利用遗传密码子扩充技术获得了具有可控性的CVB3突变体。而所构建的突变体虽然在自然条件下不能自我复制但并不会失去病毒原有的免疫原性,为CVB3及其他小RNA病毒疫苗的研制提供了一个新的思路。
[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.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373

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