Tamdy病毒糖蛋白基因的重组表达及抗体制备Recombinant Expression of Glycoprotein Gene of Tamdy Virus and Antibody Preparation
刘利平;沈姝;刘希佳;张敏;王岚;何明宇;孙素荣;张渝疆;丁军涛;
摘要(Abstract)：
原核表达Tamdy病毒（Tamdy virus,TAMV）糖蛋白Gn和Gc，分别制备兔抗融合蛋白Gn和Gc多克隆抗体。利用RT-PCR扩增Gn和Gc基因片段，分别构建原核表达质粒pET-28a-Gn和pET-32a-Gc，并在E.coli BL21(DE3)中诱导表达，镍柱亲和层析纯化融合蛋白Gn和Gc之后，以皮下注射方式分别免疫新西兰兔，制备兔抗融合蛋白Gn和Gc多克隆抗体。经Western Blotting和间接免疫荧光（IFA）鉴定多克隆抗体抗原识别能力，ELISA法测定抗体效价。结果显示，pET-28a-Gn、pET-32a-Gc原核表达质粒构建正确，融合蛋白Gn和Gc大小分别约为45 kD、74 kD，制备的兔抗融合蛋白Gn和Gc多克隆抗体能够分别识别原核表达的融合蛋白Gn和Gc和真核表达产物，效价分别为1∶409 600、1∶204 800。制备的多克隆抗体效价高，能够特异性识别原核系统和真核系统表达的TAMV糖蛋白，为TAMV的流行学分析提供基础。
关键词(KeyWords)： Tamdy病毒;糖蛋白;原核表达;真核表达;蛋白纯化;多克隆抗体
基金项目(Foundation): 国家自然科学基金项目（项目号：81960369,81760365）,题目：新型布尼亚样病毒（SFTSV-Like）结构蛋白（SP/GP）线性抗原表位及其自然疫源性研究,准噶尔盆地鼠类和媒介蜱携带重要虫媒病毒病原及其流行病学调查;; 新疆高校科研计划自然科学重点项目（项目号：XJEDU2019I002）;题目：准噶尔盆地新疆出血热自然疫源地调查研究~~
作者(Authors): 刘利平;沈姝;刘希佳;张敏;王岚;何明宇;孙素荣;张渝疆;丁军涛;
DOI: 10.13242/j.cnki.bingduxuebao.004259
参考文献(References)：
- [1] Fuente J, Estrada-Pena A, Venzal J M, Kocan K M,Sonenshine D E. Overview:Ticks as vectors of pathogens that cause disease in humans and animals[J].Front Biosci, 2008, 13:6938-6946.
- [2] Shi J, Hu Z, Deng F, Shen S. Tick-Borne Viruses[J].Virol Sin, 2018, 33(1):21-43.
- [3] Abudurexiti A, Adkins S, Alioto D, Alkhovsky S V,Avsic-Zupanc T, Ballinger M J. Taxonomy of the order Bunyavirales:update 2019[J]. Arch Virol, 2019, 164(7):1949-1965.
- [4] Lvov D K, Sidorova G A, Gromashevsky V L,Kurbanov M, Skvoztsova L M, Gofman Y P, Berezina L K, Klimenko S M, Zakharyan V A, Aristova V A,Neronov V M. Virus"Tamdy"--a new arbovirus,isolated in the Uzbee S.S.R. and Turkmen S.S.R. from ticks Hyalomma asiaticum asiaticum Schulee et Schlottke, 1929, and Hyalomma plumbeum plumbeum Panzer, 1796[J]. Arch Virol, 1976, 51(1-2):15-21.
- [5] Ma J, Lv X L, Zhang X, Han S Z, Wang Z D, Li L,Sun H T, Ma L X, Cheng Z L, Shao J W, Chen C,Zhao Y H, Sui L, Liu L N, Qian J, Wang W, Liu Q.Identification of a new orthonairovirus associated with human febrile illness in China[J]. Nat Med, 2021, 27(3):434-439.
- [6] Zhou H, Ma Z, Hu T, Bi Y, Mamuti A, Yu R, Carr M J, Shi M, Li J, Sharshov K, Gao G F, Shi W.Tamdy virus in ixodid ticks infesting bactrian camels,Xinjiang, China, 2018[J]. Emerg Infect Dis, 2019, 25(11):2136-2138.
- [7] Liu X, Zhang X, Wang Z, Dong Z, Xie S, Jiang M,Song R, Ma J, Chen S, Chen K, Zhang H, Si X, Li C, Jin N, Wang Y, Liu Q. A tentative Tamdy orthonairovirus related to febrile illness in Northwestern China[J]. Clin Infect Dis, 2020, 70(10):2155-2160.
- [8] Albornoz A, Hoffmann A B, Lozach P Y, Tischler N D. Early bunyavirus-host cell interactions[J]. Viruses,2016, 8(5):143.
- [9] Hulswit R J G, Paesen G C, Bowden T A, Shi X.Recent advances in bunyavirus glycoprotein research:Precursor processing, receptor binding and structure[J].Viruses, 2021, 13(2):353.
- [10]Estrada D F, De Guzman R N. Structural characterization of the Crimean-Congo hemorrhagic fever virus Gn tail provides insight into virus assembly[J]. J Biol Chem, 2011, 286(24):21678-21686.
- [11]Moming A, Shen S, Fang Y, Zhang J, Zhang Y, Tang S, Li T, Hu Z, Wang H, Zhang Y, Sun S, Wang L F, Deng F. Evidence of human exposure to Tamdy virus, Northwest China[J]. Emerg Infect Dis, 2021, 27(12):3166-3170.
- [12]Arai K Y, Tsuchida K, Li C, Watanabe G, Sugino H,Taya K, Nishiyama T. Purification of recombinant activin A using the second follistatin domain of follistatinrelated gene(FLRG)[J]. Protein Expr and Purif,2006, 49(1):78-82.
- [13]Xu T, Li M, Wang C, Yuan M, Chang X, Qian Z, Li B, Sun M, Wang H. Codon optimization, soluble expression and purification of PE_PGRS45 gene from and preparation of its polyclonal antibody protein[J]. J Microbiol Biotechnol, 2021, 31(11):1583-1590.
- [14]Chen Z, Yin C, Liu J, Zeng M, Wang Z, Yu D, Bi L,Jin L, Yang S, Song B. Methodology for antibody preparation and detection of southern rice black-streaked dwarf virus[J]. Arch Virol, 2012, 157(12):2327-2333.
- [15]Hu X-y, Fang Q, Wang J-s, Xie J-q, Chai B-s, Li Fq, Cui X, Yang Y. Over-expression of aldehyde dehydrogenase-2 protects against H2O2-induced oxidative damage and apoptosis in peripheral blood mononuclear cells[J]. Acta Pharmacol Sin, 2011, 32(2):245-252.
- [16]Garrison A R, Alkhovsky S V, Avsic-Zupanc T, Bente D A, Bergeron E, Burt F. ICTV virus taxonomy profile:Nairoviridae[J]. J Gen Virol, 2020, 101(8):798-799.
- [17]Lasecka L, Baron M D. The molecular biology of nairoviruses, an emerging group of tick-borne arboviruses[J]. Arch Virol, 2014, 159(6):1249-1265.
- [18]Brinkmann A, Dincer E, Polat C, Hekimoglu O,Hacioglu S, Foldes K, Ozkul A, Oktem I M A,Nitsche A, Ergunay K. A metagenomic survey identifies Tamdy orthonairovirus as well as divergent phlebo-，rhabdo-，chu-and flavi-like viruses in Anatolia, Turkey[J]. Ticks Tick Borne Dis, 2018, 9(5):1173-1183.
- [19]寇春，段晓梅，张渝疆，孙素荣.新疆地区虫媒病毒调查研究状况[J].中国人兽共患病学报，2016, 32(3):301-311.
- [20]Spiegel M, Plegge T, Pohlmann S. The role of phlebovirus glycoproteins in viral entry, assembly and release[J]. Viruses, 2016, 8(7):202.
- [21]Tani H, Shimojima M, Fukushi S, Yoshikawa T,Fukuma A, Taniguchi S, Morikawa S, Saijo M.Characterization of glycoprotein-mediated entry of severe fever with thrombocytopenia syndrome virus[J]. J Virol, 2016, 90(11):5292-5301.