| 378 | 7 | 109 |
| 下载次数 | 被引频次 | 阅读次数 |
痘苗病毒是一种在细胞质中复制的DNA病毒，曾作为天花疫苗在消除天花的过程中起着重要作用。近年来，随着分子生物学的发展，痘苗病毒作为基因表达载体，被广泛应用于疫苗研发、肿瘤治疗等不同方面。研究者们通过自然选择或基因工程技术减毒获得了复制缺陷型痘苗病毒，除了国际上广泛使用的MVA和NYVAC以外，国内还有非复制型痘苗病毒天坛株(NTV)。由于NTV缺失了多种宿主范围基因，导致其免疫原性低下，因此仍须对其进行基因改造以获得更好的疫苗载体。目前改造方法主要是同源重组，但是传统同源重组方法的重组率很低，限制了痘苗病毒载体疫苗载体的研发。近年来随着CRISPR/Cas9技术的发展，使得建立基于CRISPR/Cas9的高效重组体系成为可能。痘苗病毒目前的研究热点主要集中在抗凋亡基因上。有实验表明，抗凋亡基因F1L的缺失在不影响痘苗病毒的复制能力的同时，还降低了毒力，改善了CD8~+T细胞免疫反应水平，提高了作为疫苗载体的安全性。这些发现表明该基因的缺失是优化痘苗病毒的有效策略之一。本研究设计了痘苗病毒F1L区重组质粒，通过运用CRISPR/Cas9技术，对TTV的F1L基因进行缺失重组。经重组病毒的噬斑纯化与PCR、Western Blot验证后，证明了该重组系统的有效性与正确性。我们建立了痘苗病毒F1L区高效重组技术方法并筛选出最佳的gRNA。这为后续NTV的改造提供了更多基因靶点选择。
Abstract：The vaccinia virus is a DNA virus that replicates in cytoplasm. As a vaccine,it once played an important part in eliminating smallpox. In recent years,with the development of molecular biology,vaccinia virus as a gene expression vector has been widely used in the research and development of vaccine and tumor treatment. Researchers had undertaken replication of defective vaccinia virus through natural selection or genetic engineering technology. In addition to the internationally used MVA and NYVAC,the non-replicating vaccinia virus Tiantan(NTV)strain is present in China. NTV lacks various host-range genes,which results in low immunogenicity. Hence, NTV must be genetically modified to obtain a better vaccine vector. The transformation method is mainly homologous recombination. However,the recombination rate of the traditional method for homologous recombination is very low,which limits the research and development of the vaccine vector of the vaccinia virus. In recent years,with the development of clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9) technology,an efficient restructuring system based on CRISPR/Cas9 has been established. The current research of the vaccinia virus mainly focus on anti-apoptosis genes. Studies have shown that deletion of the anti-apoptotic gene F1L does not affect the replication ability,but reduces the virulence,improves the immune response of cluster of differentiation(CD)8~+T cells,and improves the safety as a vaccine vector. Those findings suggest that deletion of the F1L gene could be an effective strategy for optimizing a vaccine for the vaccinia virus. In the present study,the F1L region recombinant plasmid of the vaccinia virus was designed,and the F1L gene of TTV was deleted and recombined using CRISPR/Cas9. After plaque purification of the recombinant virus had been verified with polymerase chain reaction(PCR) and Western blotting. The effectiveness and correctness of the recombinant system were demonstrated. We established an efficient recombinant method for the F1L region of vaccinia virus,and screened for optimal guideRNA(gRNA). Our method provides greater selection of genetic targets for subsequent modifications of NTV.
[1] E. Gomez C,L. Najera J,Krupa M,Perdiguero B,Esteban M. MVA and NYVAC as Vaccines against Emergent Infectious Diseases and Cancer[J]. Current Gene Therapy,2011,11(3):189-217.
[2] Pastoret P P,Vanderplasschen A. Poxviruses as vaccine vectors[J]. Microbiol Infect Dis,2003,26(5):343-355.
[3]庞聪，田厚文.非复制型痘苗病毒的生物学性状研究进展[J].生物技术通讯，2014,25(3):415-420.
[4] Ober B T, BruH l P, Schmidt M, Wieser V,Gritschenberger W,Coulibaly S,Savidis-Dacho H,Gerencer M,Falkner F G. Immunogenicity and safety of defective vaccinia virus lister:comparison with modified vaccinia virus ankara[J]. J Virol,2002,76(15):7713-7723.
[5] Zhao Y,Zhao L,Huang P,Ren J,Zhang P,Tian H,Tan W. Non-replicating vaccinia virus tiantan strain(ntv)translation arrest of viral late protein synthesis associated with anti-viral host factor samd9[J]. Front Cell Infect Microbiol,2020,10:116-131.
[6] Veyer D L,Carrara G,Maluquer de Motes C,Smith G L. Vaccinia virus evasion of regulated cell death[J].Immunol Lett,2017,186:68-80.
[7] Ludwig H,Suezer Y,Waibler Z,Kalinke U,Schnierle B S,Sutter G. Double-stranded RNA-binding protein E3 controls translation of viral intermediate RNA,marking an essential step in the life cycle of modified vaccinia virus Ankara[J]. J Gen Virol,2006,87(5):1145-1155.
[8] Stewart T L,Wasilenko S T,Barry M. Vaccinia virus F1L protein is a tail-anchored protein that functions at the mitochondria to inhibit apoptosis[J]. J Virol,2005,79(2):1084-1098.
[9] Pelin A,Foloppe J,Petryk J,Singaravelu R,Hussein M,Gossart F,Jennings V A,Stubbert L J,Foster M,Storbeck C,Postigo A,Scut E,Laight B,Way M,Erbs P,Le Boeuf F,Bell J C. Deletion of apoptosis inhibitor f1l in vaccinia virus increases safety and oncolysis for cancer therapy[J]. Molecular TherapyOncolytics,2019,14:246-252.
[10]Perdiguero B,Gomez C E,Najera J L,Sorzano C O,Delaloye J,Gonzalez-Sanz R,Jimenez V,Roger T,Calandra T,Pantaleo G,Esteban M. Deletion of the viral anti-apoptotic gene F1L in the HIV/AIDS vaccine candidate MVA-C enhances immune responses against HIV-1 antigens[J/OL]. PLoS One,2012,7(10):e48524.
[11]Yuan M,Zhang W,Wang J,Al Yaghchi C,Ahmed J,Chard L,Lemoine N R,Wang Y. Efficiently editing the vaccinia virus genome by using the CRISPR-Cas9system[J]. J Virol,2015,89(9):5176-5179.
[12]Di Gioia C,Yuan M,Wang Y. Vaccinia virus genome editing using crispr[J]. Methods Mol Biol,2019,2023:109-117.
[13]Yao X D,Evans D H. Effects of DNA structure and homology length on vaccinia virus recombination[J]. J Virol,2001,75(15):6923-6932.
[14]Coupar B E H,Oke P G,Andrew M E. Insertion sites for recombinant vaccinia virus construction:effects on expression of a foreign protein[J]. Microbiology,2000,81(2):431-439.
[15]吴雅彬，赵莉，任皎，袁航，张鹏，叶飞，田厚文，王文玲，谭文杰.基于CRISPR-Cas9的痘苗病毒高效重组体系的建立[J].中华实验和临床病毒学杂志，2021,35(02):199-204.
[16]Mehta N,Enwere E K,Santos T D,Saffran H A,Hazes B,Evans D,Barry M,Smiley J R,SandriGoldin R M. Expression of the vaccinia virus antiapoptotic f1 protein is blocked by protein kinase r in the absence of the viral e3 protein[J/OL]. J Virol,2018,92(19):e01167-01118.
[17]Gowripalan A,Smith S,Stefanovic T,Tscharke D C.Rapid poxvirus engineering using CRISPR/Cas9 as a selection tool[J]. Commun Biol,2020,3(1):643-655.
[18]Yuan M,Gao X,Chard L S,Ali Z,Ahmed J,Li Y,Liu P,Lemoine N R,Wang Y. A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9[J]. Mol Ther Methods Clin Dev,2015,2:15035-15043.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004173
中图分类号:R392-33
引用信息:
[1]袁航,任皎,赵莉,等.痘苗病毒天坛株F1L区高效重组方法的建立[J].病毒学报,2022,38(04):791-798.DOI:10.13242/j.cnki.bingduxuebao.004173.
基金信息:
国家自然科学基金专项项目（项目号：82041041），题目：自然感染和疫苗接种人群中的广谱保护性免疫应答规律~~
2021-12-03
2021
2022-01-29
2022-07-08
2022
1
2022-07-11
2022-07-11
2022-07-11