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研究生:柯楚予
研究生(外文):Chu-Yu Ke
論文名稱:利用桿狀病毒系統共同表達非洲豬瘟病毒p72及pB602L之重組蛋白
論文名稱(外文):Co-expression of African swine fever virus p72 and pB602L recombinant proteins by baculovirus system
指導教授:賈敏原賈敏原引用關係
指導教授(外文):Min-Yuan Chia
口試委員:郭致榮劉正哲
口試委員(外文):Chih-Jung KuoCheng-Che Liu
口試日期:2022-01-17
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2022
畢業學年度:110
語文別:中文
論文頁數:52
中文關鍵詞:非洲豬瘟病毒p72蛋白pB602L蛋白桿狀病毒表現系統
外文關鍵詞:ASFVp72pB602Lbaculovirus expression system
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非洲豬瘟 (African swine fever;ASF) 是由非洲豬瘟病毒 (African swine fever virus;ASFV) 引起的惡性動物傳染病。自1921年首次在非洲肯亞報導以來,已傳播至三大洲,近年歐洲及亞洲各國陸續爆發疫情,造成全球養豬產業重大損失。至今仍無有效的疫苗及治療方法。ASFV為雙股DNA病毒,基因長度約為170至193 kbp,包含151至167個開放閱讀框,編碼超過150種蛋白,多數蛋白的功能未知。p72蛋白的編碼基因為B646L,是組成ASFV衣殼的主要結構蛋白,在病毒感染的後期表達,也是受感染豬隻被檢測到的主要抗原之一。p72蛋白所誘發的單株抗體已被證實有中和病毒的能力,並阻止ASFV與巨噬細胞結合。因此被認為可用來作為疫苗開發及血清學診斷之抗原。pB602L蛋白的編碼基因為B602L,為ASFV晚期表現之非結構蛋白,目前所知的功能為幫助p72蛋白完整折疊,形成正確的三聚體結構,是謂p72蛋白的伴護蛋白 (chaperone protein)。本論文研究目標為利用昆蟲桿狀病毒表現系統進行ASFV 結構蛋白p72與非結構蛋白pB602L之表達,以提供基礎科學研究與診斷試劑研發之需求。本實驗將B646L基因選殖至pENTR™/D-TOPO載體中,製備帶有His tag基因的Topo-p72-His質體,藉由Gateway Technology進行桿狀病毒基因重組。同時將帶有His tag基因的B646L基因和帶有Flag tag基因的B602L基因選殖至pOET5載體中,製備pOET5-co-express質體,並透過同源重組方式將目標基因轉移到桿狀病毒基因組中。利用西方墨點和免疫螢光染色法來分析重組蛋白。結果顯示p72和pB602L重組蛋白在桿狀病毒系統中成功共同表達。這些結果將有助於進一步研究與開發診斷試劑。
African swine fever (ASF) is a malignant animal infectious disease caused by African swine fever virus (ASFV). In recent years, there have been outbreaks of epidemics in various countries, causing significant losses to the global pig industry. Currently, there is still no effective vaccine and treatment for ASF. ASFV is a large, double stranded DNA virus that causes a highly lethal disease in domestic pigs, and its genome ranges in length from about 170 to 193 kbp that encodes 151 to 167 ORFs. p72 is the major structure protein encoded by the gene B646L of ASFV and is very important for forming the viral capsid in the late stage of viral infection. p72 also is one of the major antigens detected in infected pigs. Monoclonal antibodies recognizing p72 were shown to neutralize virulent ASFV isolates and to prevent ASFV binding to macrophages. pB602L is a non-structural protein expressed in the late stage of ASFV infecton. It serves as a chaperone for the folding of p72 to form the trimmer structure and prevents the aggregation of p72. The aim of this study is to co-express p72 and pB602L proteins by baculovirus system and to develop diagnostic techniques as ASFV control strategies in Taiwan. Two baculovirus expression vectors, Topo-p72-His and pOET5-co-express, were constructed in the present study. The gene B646L was cloned into pENTR™/D-TOPO vector which contained a hexahistidine tag sequence to generate Topo-p72-His plasmid and transferred into the baculovirus genome by the Gateway Technology. The gene B646L with a hexahistidine tag sequence and the gene B602L with flag tag sequence were cloned into pOET5 vector to generate pOET5-co-express plasmid and transferred into the baculovirus genome by homologous recombination. Western blotting and immunofluorescence assay were applied to analyze recombinant proteins. The results showed that p72 and pB602L were successfully co-expressed by the baculovirus system. These results will aid in further research for development diagnostic techniques.
摘要 i
Abstractii
目次 iii
表目次 vi
圖目次 vii
第一章 前言 1
第二章 文獻回顧 2
第一節 非洲豬瘟病毒 2
2.1.1 歷史簡介 2
2.1.2 病毒構造 3
2.1.2.1 p72蛋白 3
2.1.2.2 pB602L蛋白 4
2.1.3 病毒複製 5
2.1.4 傳播方式 5
2.1.5 實驗室診斷方法 7
2.1.5.1 抗原檢測 7
2.1.5.2 抗體檢測 8
2.1.5.3 側向流動試驗法 (lateral flow assay) 9
第二節 昆蟲桿狀病毒表現系統 9
2.2.1 桿狀病毒簡介 9
2.2.2 桿狀病毒生活史 10
2.2.3 桿狀病毒基因表現 10
2.2.4 桿狀病毒表現系統發展與應用 11
2.2.5 flashBACTM Baculovirus Expression System 11
2.2.6 BaculoDirect™ Baculovirus Expression System 12
2.2.7 Bac-to-Bac™ Baculovirus Expression System 12
2.2.8 研究動機與目的 13
第三章 材料與方法 14
第一節 實驗目的 14
第二節 表現p72重組蛋白 14
3.2.1 核酸引子設計 14
3.2.2 聚合酶鏈鎖反應 15
3.2.3 瓊脂糖膠體電泳 15
3.2.4 膠體純化 15
3.2.5 質體構築 16
3.2.6 轉型作用 16
3.2.7 重組Topo-p72-His質體之篩選 16
3.2.8 序列分析 16
3.2.9 菌種保存 16
3.2.10 質體萃取 17
3.2.11 桿狀病毒基因重組 17
3.2.12 昆蟲細胞之轉染作用 17
第三節 共同表現p72與pB602L重組蛋白 18
3.3.1 核酸引子設計 18
3.3.2 聚合酶鏈鎖反應 18
3.3.3 限制酶切割反應 19
3.3.4 接合反應 19
3.3.5 轉型作用 19
3.3.6 重組pOET5-co-express質體之篩選 19
3.3.7 昆蟲細胞之轉染作用20
第四節 昆蟲細胞培養 20
3.4.1 細胞培養 20
3.4.2 細胞冷凍 21
3.4.3 細胞解凍 21
第五節 確認重組病毒帶有目標基因 21
第六節 重組病毒純化及力價測定 22
第七節 蛋白質電泳 22
第八節 蛋白質電泳膠片染色 22
第九節 西方墨點法 23
第十節 免疫螢光染色 23
第四章 結果 24
第一節 重組蛋白p72及pB602L之基因選殖 24
第二節 重組質體之定序結果 24
第三節 重組桿狀病毒DNA轉染Sf21細胞 24
第四節 重組桿狀病毒帶有目標基因之檢測 24
第五節 p72及pB602L重組蛋白西方墨點法之分析 25
第六節 p72及pB602L重組蛋白免疫螢光染色法之分析 25
第五章 討論 26
第六章 參考文獻 30
附錄一、Gateway 重組反應 46
附錄二、pENTR™/D-TOPO®圖譜 47
附錄三、BaculoDirectTM C-Term Linear DNA圖譜 48
附錄四、第一型拓樸異構酶在pENTR™/D-TOPO®的作用機制 49
附錄五、pOET5圖譜 50
附錄六、Topo-p72-His與pOET5-co-express質體之p72胺基酸序列比對結果 51
附錄七、pOET5-co-express 質體之pB602L胺基酸序列比對結果 52
1.Sánchez-Vizcaíno JM, Mur L, Gomez-Villamandos JC, et al. An update on the epidemiology and pathology of African swine fever. J. Comp. Pathol. 2015;152:9-21.
2.Gallardo C, Nieto R, Soler A, et al. Assessment of African swine fever diagnostic techniques as a response to the epidemic outbreaks in eastern european union countries: How to improve surveillance and control programs. J. Clin. Microbiol. 2015;53:2555-2565.
3.Kollnberger SD, Gutierrez-Castañeda B, Foster-Cuevas M, et al. Identification of the principal serological immunodeterminants of African swine fever virus by screening a virus cDNA library with antibody. J. Gen. Virol. 2002;83:1331-1342.
4.Lopez-Otin C, Freije J, Parra F, et al. Mapping and sequence of the gene coding for protein p72, the major capsid protein of African swine fever virus. Virology. 1990;175.2:477-484.
5.Bastos A, Penrith M, Cruciere C, et al. Genotyping field strains of African swine fever virus by partial p72 gene characterisation. Arch. Virol. 2003;148:693-706.
6.Sanz A, García-Barreno B, Nogal ML, et al. Monoclonal antibodies specific for African swine fever virus proteins. J. Virol. 1985;54:199-206.
7.Yu M, Morrissy C, Westbury, H, et al. Strong sequence conservation of African swine fever virus p72 protein provides the molecular basis for its antigenic stability. Arch. Virol. 1996;141.9:1795-1802.
8.Lokhandwala S, Waghela SD, Bray J, et al. Adenovirus-vectored novel African swine fever virus antigens elicit robust immune responses in swine. PLoS One. 2017;12:e0177007.
9.Busch F, Haumont C, Penrith ML, et al. Evidence-based African swine fever policies: Do we address virus and host adequately? Front. Vet. Sci. 2021;8:224.
10.Matthews, F. The classification and nomenclature of viruses: summary of results of meetings of the International Committee on Taxonomy of Viruses in Strasbourg, August 1981. Intervirology. 1981;16.2: 53-60.
11.Martelli AW, Mayo GP. Virus Taxonomy. Sixth report of the International Committee on Taxonomy of Viruses. Arch. Virol. 1995;10:293-299.
12.Pringle CR. Virology division news virus taxonomy-San Diego 1998. Arch Virol. 1998;143.
13.Eustace Montgomery R. On a form of swine fever occurring in British East Africa (Kenya Colony). J. Comp. Pathol. and Therapeut. 1921;34:159-191.
14.Danzetta ML, Marenzoni ML, Iannetti S, et al. African swine fever: Lessons to learn from past eradication experiences. A systematic review. Front. Vet. Sci. 2020;7.
15.Sánchez-Vizcaíno JM, Mur L, Martínez-López B. African swine fever: An epidemiological update. Transbound Emerg Dis. 2012;59:27-35.
16.Vepkhvadze NG, Menteshashvili I, Kokhreidze M, et al. Active surveillance of African swine fever in domestic swine herds in Georgia, 2014. Rev. Off. Int. Epizoot. 2017;36:879-887.
17.Turlewicz-Podbielska H, Kuriga A, Niemyjski R, et al. African swine fever virus as a difficult opponent in the fight for a vaccine–Current data. Viruses. 2021;13:1212.
18.Gaudreault NN, Madden DW, Wilson WC, et al. African swine fever virus: An emerging DNA arbovirus. Front. Vet. Sci. 2020;7.
19.Lubisi BA, Bastos ADS, Dwarka RM, et al. Intra-genotypic resolution of African swine fever viruses from an East African domestic pig cycle: A combined p72-CVR approach. Virus Res. 2007;35:729-735.
20.Guo F, Shi Y, Yang M, et al. The structural basis of African swine fever virus core shell protein p15 binding to DNA. FASEB J. 2021;35.
21.Dixon LK, Chapman DAG, Netherton CL, et al. African swine fever virus replication and genomics. Virus Res. 2013;173:3-14.
22.Wang G, Xie M, Wu W, et al. Structures and functional diversities of ASFV proteins. Viruses. 2021;13:2124.
23.Breese SS, DeBoer CJ. Electron microscope observations of African swine fever virus in tissue culture cells. Virology. 1966;28:420-428.
24.Wang N, Zhao D, Wang J, et al. Architecture of African swine fever virus and implications for viral assembly. Science. 2019;366:640-644.
25.Hawes PC, Netherton CL, Wileman TE, et al. The envelope of intracellular African swine fever virus is composed of a single lipid bilayer. J. Virol. 2008;82:7905-7912.
26.Jia N, Ou Y, Pejsak Z, et al. Roles of African swine fever virus structural proteins in viral infection. J. Vet. Res. 2017;61:135-143.
27.Lithgow P, Takamatsu H, Werling D, et al. Correlation of cell surface marker expression with african swine fever virus infection. Vet. Microbiol. 2014;168.
28.Revilla Y, Perez-Nunez D, Richt A, et al. African swine fever virus biology and vaccine approaches. Adv. Virus Res. 2018;100:41-74.
29.Neilan JG, Zsak L, Lu Z, et al. Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection. Virology. 2004;319:337-342.
30.Cobbold C, Wileman T. The major structural protein of African swine fever virus, p73, is packaged into large structures, indicative of viral capsid or matrix precursors, on the endoplasmic reticulum. J. Virol. 1998;72.
31.Borca M, Irusta P, Carrillo C, et al. African swine fever virus structural protein p72 contains a conformational neutralizing epitope. Virology. 1994;201:413-418.
32.Liu Q, Ma B, Qian N, et al. Structure of the African swine fever virus major capsid protein p72. Cell Res. 2019;29:953-955.
33.Heimerman ME, Murgia MV, Wu P, et al. Linear epitopes in African swine fever virus p72 recognized by monoclonal antibodies prepared against baculovirus-expressed antigen. J. Vet. Diagn. Invest. 2018;30:406-412.
34.Sastre P, Pérez T, Costa S, et al. Development of a duplex lateral flow assay for simultaneous detection of antibodies against African and classical swine fever viruses. J. Vet. Diagn. Invest. 2016;28:543-549.
35.Penrith ML, Duarte Bastos A, Etter EMC, et al. Epidemiology of African swine fever in Africa today: Sylvatic cycle versus socio‐economic imperatives. Transbound Emerg Dis. 2019;66:672-686.
36.Costard S, Mur L, Lubroth J, et al. Epidemiology of African swine fever virus. Virus Res. 2013;173.1:191-197.
37.Rowlands RJ, Michaud V, Heath L, et al. African swine fever virus isolate, Georgia, 2007. Emerging Infect. Dis. 2008;14:1870-1874.
38.Epifano C, Krijnse-Locker J, Salas ML, et al. The African swine fever virus nonstructural protein pB602L is required for formation of the icosahedral capsid of the virus particle. J. Virol. 2006;80:12260-12270.
39.Gallardo C, Reis AL, Kalema-Zikusoka G, et al. Recombinant antigen targets for serodiagnosis of African swine fever. Clin. Vaccine Immunol. 2009;16:1012-1020
40.Valdeira ML, Geraldes A. Morphological study on the entry of African swine fever virus into cells. Cell Biol. Int. 1985;55:35-40.
41.Alcamí A, Carrascosa AL, Viñuela E. Saturable binding sites mediate the entry of African swine fever virus into VERO cells. Virology. 1989;168:393-398.
42.Popescu L, Gaudreault N, Whitworth K, et al. Genetically edited pigs lacking CD163 show no resistance following infection with the African swine fever virus isolate, Georgia 2007/1. Virology. 2017;501:102-106.
43.Cuesta-Geijo MA, Galindo I, Hernáez B, et al. Endosomal maturation, Rab7 GTPase and phosphoinositides in African swine fever virus entry. PLoS One. 2012;7:e48853.
44.Hernáez B, Guerra M, Salas ML, et al. African swine fever virus undergoes outer envelope disruption, capsid disassembly and inner envelope fusion before core release from multivesicular endosomes. PLOS Pathog. 2016;12:e1005595.
45.Simões M, Freitas FB, Leitão A, et al. African swine fever virus replication events and cell nucleus: New insights and perspectives. Virus Res. 2019;270:197667.
46.Muñoz-Moreno R, Galindo I, Cuesta-Geijo MÁ, et al. Host cell targets for African swine fever virus. Virus Res. 2015;209:118-127.
47.Kleiboeker S, Reviews G. Pathogenesis of African swine fever virus in Ornithodoros ticks. Anim. Health Res. Rev. 2001; 2.2:121-128.
48.Kleiboeker S, Scoles G, Burrage T, et al. African swine fever virus replication in the midgut epithelium is required for infection of Ornithodoros ticks. J. Virol. 1999;73:8587-8598.
49.Frant M, Woźniakowski G, Pejsak Z. African swine fever (ASF) and ticks. No risk of tick-mediated ASF spread in Poland and Baltic states. J. Vet. Res. 2017;61:375-380.
50.Gogin A, Gerasimov V, Malogolovkin A, et al. African swine fever in the North Caucasus region and the Russian Federation in years 2007–2012. Virus Res. 2013;173:198-203.
51.Zhou X, Li N, Luo Y, et al. Emergence of African swine fever in China, 2018. Transbound Emerg Dis. 2018;65:1482-1484.
52.Boklund A, Cay B, Depner K, et al. Epidemiological analyses of African swine fever in the European Union (November 2017 until November 2018). EFSA Journal. 2018;16.
53.Fernández-Pinero J, Gallardo C, Elizalde M, et al. Molecular diagnosis of African swine fever by a new real-time PCR using universal probe library. Transbound Emerg Dis. 2013;60:48-58.
54.Agüero M, Fernández J, Romero L, et al. Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clinical samples. J. Clin. Microbiol. 2003;41:4431-4434.
55.World Organisation for Animal Health (OIE). African swine fever. Manual of diagnostic tests and vaccines for terrestrial animals 2019, chapter 3.8.1.
56.Enjuanes L, Carrascosa, A. L, Moreno, M. A, et al. Titration of African swine fever (ASF) virus. J. Gen. Virol. 1976;32.3:471-477.
57.Bustos MJ, de León P, Carrascosa AL, et al. Methods for growing and titrating African swine fever virus: field and laboratory samples. Curr Protoc Cell Biol. 2011;53.1:26.14.1-26.14.25.
58.Gallardo C, Fernández-Pinero J, Arias M. African swine fever (ASF) diagnosis, an essential tool in the epidemiological investigation. Virus Res. 2019;271:197676.
59.Gallardo C, Soler A, Rodze I, et al. Attenuated and non‐haemadsorbing (non‐HAD) genotype II African swine fever virus (ASFV) isolated in Europe, Latvia 2017. Transbound Emerg Dis. 2019;66:1399-1404.
60.Oura C, Edwards L, Batten C, et al. Virological diagnosis of African swine fever–comparative study of available tests. Virus Res. 2013;173.1:150-158.
61.Wardley R, Elzein E, Crowther J, et al. A solid-phase enzyme linked immunosorbent assay for the detection of African swine fever virus antigen and antibody. Epidemiol. Infect. 1979;83.2:363-370.
62.Vidal M, Stiene M, Henkel J, et al. A solid-phase enzyme linked immunosorbent assay using monoclonal antibodies, for the detection of African swine fever virus antigens and antibodies. J. Virol. Methods. 1997;66.2:211-218.
63.Arias M, Sánchez-Vizcaíno M, Yoon K, et al. African swine fever. Trends in emerging viral infections of swine. 2002;119-124.
64.Pastor M, Laviada D, Sanchez-Vizcaino M, et al. Detection of African swine fever virus antibodies by immunoblotting assay. Can. J. Vet. Res. 1989;53.1:105.
65.Sastre P, Gallardo C, Monedero A, et al. Development of a novel lateral flow assay for detection of African swine fever in blood. BMC Vet. Res. 2016;12:1-8.
66.Cappai S, Loi F, Coccollone A, et al. Evaluation of a commercial field test to detect African swine fever. J. Wildl. Dis. 2017;53:602-606.
67.Herniou EA, Olszewski JA, Cory JS, et al. The genome sequence and evolution of baculoviruses. Annu. Rev. Entomol. 2003;48:211-234.
68.Francki B. Classification and nomenclature of viruses. Fifth report of the international committee on viruses. Arch. Virol. 1991;2:450.
69.Chambers AC, Aksular M, Graves LP, et al. Overview of the baculovirus expression system. Curr Protoc Protein Sci. 2018;91:5.4.1-5.4.6.
70.Volkman LE, Summers MD, Hsieh CH. Occluded and nonoccluded nuclear polyhedrosis virus grown in Trichoplusia ni: comparative neutralization comparative infectivity, and in vitro growth studies. J. Virol. 1976;19:820-832.
71.Slack J, Arif M. The baculoviruses occlusion‐derived virus: virion structure and function. Adv. Virus Res. 2006, 69: 99-165.
72.Volkman L, Goldsmith A. Mechanism of neutralization of budded autographs californica nuclear polyhedrosis virus by a monoclonal antibody: Inhibition of entry by adsorptive endocytosis. Virology.1985;143.1:185-195.
73.Smith G, Summers M, Fraser M, et al. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol. 1983:2156–2165.
74.Ana S, Miele B, Garavaglia J, et al. Baculovirus: molecular insights on their diversity and conservation. J. Evol. Biol. 2011;2011:15.
75.Hofmann C, Sandig V, Jennings G, et al. Efficient gene transfer into human hepatocytes by baculovirus vectors. PNAS. 1995;92.22:10099-10103.
76.Meng K, Zhang Y, Zhu W, et al. Expression and purification of African swine fever virus p72 trimers as subunit vaccine candidate. bioRxiv. 2020.
77.Freije P, Muñoz M, Viñuela E, et al. High-level expression in Escherichia coli of the gene coding for the major structural protein (p72) of African swine fever virus. Gene. 1993;123:259-262.
78.Wang P, Liu C, Wang S, et al. Production and application of mouse monoclonal antibodies targeting linear epitopes in pB602L of African swine fever virus. Arch. Virol. 2022;1:1-10.
79.Wang YB, Wang ZY, Chen HY, et al. Secretory expression of porcine interferon-gamma in baculovirus using HBM signal peptide and its inhibition activity on the replication of porcine reproductive and respiratory syndrome virus. Vet. Immunol. Immunopathol. 2009;132:314-317.
80.Cobbold C, Windsor M, Wileman T. A virally encoded chaperone specialized for folding of the major capsid protein of African swine fever virus. J. Virol. 2001;75:7221-7229.
81.Pérez-Núñez D, Sunwoo SY, Sánchez EG, et al. Evaluation of a viral DNA-protein immunization strategy against African swine fever in domestic pigs. Vet. Immunol. Immunopathol. 2019;208:34-43.
82.Tabares E, Salvador-Temprano E, Carnero ME, et al. A reliable enzyme linked immunosorbent assay for African swine fever using the major struetural protein as antigenie reagent. Arch. Virol.1981;70.3: 297-300.
83.Gallardo C, Blanco E, Rodríguez JM, et al. Antigenic properties and diagnostic potential of African swine fever virus protein pp62 expressed in insect cells. J. Clin. Microbiol. 2006;44:950–956.
84.Pérez-Filgueira DM, González-Camacho F, Gallardo C, et al. Optimization and Validation of Recombinant Serological Tests for African Swine Fever Diagnosis Based on Detection of the p30 Protein Produced in Trichoplusia ni. J. Clin. Microbiol. 2006;44:3114–3121.
85.Reis AL, Parkhouse RME, Penedos AR, et al. Systematic analysis of longitudinal serological responses of pigs infected experimentally with African swine fever virus. J. Gen. Virol. 2007;88:2426–2434.
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