猪瘟病毒石门重组标记毒株的构建与拯救

doi:10.3864/j.issn.0578-1752.2013.01.022

摘要/Abstract

摘要： 【目的】中国控制猪瘟主要采用疫苗免疫，现有的猪瘟兔化弱毒疫苗有较好的免疫保护效果，但是不能从血清学上区分疫苗免疫猪与自然感染猪，猪瘟标记疫苗的研制与配套检测技术的开发可有效解决这一问题。【方法】利用猪瘟病毒低温诱变弱毒T株感染性克隆，将其全长结构蛋白基因替换为猪瘟病毒石门毒株全长结构蛋白基因，得到猪瘟石门重组病毒的全长感染性克隆pSMT；随后在pSMT E1蛋白的C端插入Flag抗原基因作为鉴别标记，构建猪瘟石门重组标记病毒的感染性克隆pSMT-Flag。经电转染法拯救出两株重组病毒vSMT和vSMT-Flag，通过SK6细胞进行病毒传代以获得重组病毒。【结果】多组酶切鉴定表明两株重组质粒成功构建，PCR扩增及测序、免疫过氧化酶单层细胞试验（IPMA）表明重组病毒已成功拯救且能成功传代。【结论】该标记病毒能够与Flag单抗发生特异性反应，且不影响E2蛋白中和表位与猪瘟单抗的结合，因此该毒株为研制CSFV新型标记疫苗提供了思路。

关键词: 猪瘟 , 重组病毒 , Flag , 标记疫苗

Abstract: 【Objective】Vaccination is still the main strategy for CSFV control in China. CSFV HCLV(Hog Cholera Lapinized Virus, HCLV) strain could protect effectively, but it couldn’t be serologically differentiated between infected pigs and vaccinated pigs (DIVA principle), which could been effectively solved by marker vaccine and marker diagnostics.【Method】Based on infectious cDNA clone of CSFV T strain, which is a temperature-sensitive mutation strain, the complete structural protein-coding region of T strain had been replaced by that of ShiMen strain , and the recombinant plasmid SMT was constructed. Additionally, the Flag gene was inserted into C end of E1 protein of the recombinant virus as a positive marker, and the pSMT-Flag was constructed successfully. After electroporation, the recombinant viruses vSMT and vSMT-Flag were passaged by SK6 cell. 【Result】Two recombinant plasmids were identified by specific restriction enzymes，the rescued viruses could be passaged successfully and confirmed by RT-PCR, sequencing and immunoperoxidase monolayer assay(IPMA). 【Conclusion】 This marker virus could react with the Flag specific monoclonal antibody while could not influence the combination between E2 protein and its specific monoclonal antibody, which could be a potential CSF novel marker vaccine.

Key words: classical swine fever , recombinant virus , Flag , marker vaccine

朱元源, 韩焘, 邹兴启, 范学政, 徐璐, 王琴, 赵启祖. 猪瘟病毒石门重组标记毒株的构建与拯救[J]. 中国农业科学, 2013, 46(1): 187-194.

ZHU Yuan-Yuan, HAN Tao, ZOU Xing-Qi, FAN Xue-Zheng, XU Lu, WANG Qin, ZHAO Qi-Zu. Construction and Rescue of Recombinant Classical Swine Fever Virus with Shimen Structure Protein and Flag Marker[J]. Scientia Agricultura Sinica, 2013, 46(1): 187-194.

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参考文献

[1]Fauquet C M, Fauquet C M, Mayo M A, Maniloff J, Desselberger U, Ball L A. Virus Taxonomy: Classification and Nomenclature of Viruses. London: Elsevier/Academic Press, 2005: 819-841.

[2]Dong X N, Chen Y H. Marker vaccine strategies and candidate CSFV marker vaccines. Vaccine, 2007, 25: 205-230.

[3]Rice C M. Flaviviridae: the viruses and their replication//Fields B N, Howley P M. Fields Virology. 3rd ed. Philadelphia: Lippincott Raven Publishers, 1996: 931-959.

[4]Thiel H J, Stark R, Weiland E, Rümenapf T, Meyers G. Hogcholera virus: molecular composition of virions from a pestivirus. Journal of Virology, 1991,65: 4705-4712.

[5]Lindenbach B D, Thiel H J, Rice C M. Flaviviridae: The Viruses and Their Replication, Field Virology. 5th ed. Philadelphia: Lippincott- Raven Publishers. 2007: 1101-1152.

[6]Risatti G R, Holinka L G, Lu Z, Kutish G F, Tulman E R, French R A, Rock D L, Borca M V. Mutation of E1 glycoprotein of classical swine fever virus affects viral virulence in swine. Virology, 2005, 343(1): 116-127.

[7]Fernandez-Sainz I, Risatti G R, Borca M V. Alteration of the N-linked glycosylation condition in E1 glycoprotein of Classical Swine Fever Virus strain Bresic alters virulence in swine. Virology, 2009,386: 210-216.

[8]Fan Y F, Zhao Q Z, Zhao Y, Wang Q, Ning Y B, Zhang Z Q. Complete genome sequence of attenuated low-temperature Thiverval strain of classical swine fever virus. Virus Genes, 2008, 36:531-538.

[9]范运峰, 赵启祖, 赵耘, 邹兴启, 张仲秋, 王琴, 宁宜宝. 猪瘟病毒低温诱变疫苗Thiverval株感染性克隆的构建及病毒拯救. 畜牧兽医学报, 2009, 40(9): 1420-1425.

Fan Y F , Zhao Q Z , Zhao Y , Zou X Q, Xu L, Zhang Z Q, Wang Q , Ning Y B. Recovery of the infectious Classical Swine Fever Virus from the cloned cDNA of low temperature attenuated thiverval strain. Chinese Journal of Animal and Veterinary Sciences, 2009, 40(9): 1420-1425. (in Chinese)

[10]Holinka L G, Fernandez-Sainz I, O'donnell V K, Prarat M V, Gladue D P, Lu Z, Risatti G R, Borca M V. Development of a live attenuated antigenic marker classical swine fever vaccine. Virology, 2009, 384:106-113.

[11]Nie Y C, Chen J G, Ding M X. Sequencing and rescuing a highly virulent classical swine fever virus: Chinese strain cF114 from a full-length. Chinese Science Bulletin , 2003, 48(11):1124-1128.

[12]Van Gennip H G, Van Rijn P A,Widjojoatmodjo M N, De Smit A J, Moormann R J. Chimeric classical swine fever viruses containing envelopeprotein E(RNS) or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response. Vaccine, 2000, 19(4/5):447-459.

[13]Moormann R J, van Gennip H G, Miedema G K, Hulst M M , van Rijn P A. Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus. Journal of Virology, 1996, 70( 2): 763-770.

[14]Wang Y, Wu H Y, Zhang C Y. Construction and pathogenicity of infectious cDNA clone of classical swine fever virus (CSFV). Chinese Jouanal of Virology, 2005, 21(1):43-46.

[15]Risatti G R, Borca M V, kutish G F, Lu Z, Holinka G, French R A, Tulman E R, Rock D L.The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine. Journal of Virology,2005, 79:3787-3796.

[16]Kortekaas J, Vloet R P M, Weerdmeester K, Ketelaar J, van Eijk M, Leoffen W L. Rational design of a classical swine fever C-strain vaccine virus that enables the differentiation between infected and vaccinated animals. Journal of Virological Methods, 2010, 163: 175-185.

[17]Flores E F, Kreutz L C, Donis R O. Swine and ruminant pestiviruses require the same cellular factor to enter bovine cells. Journal of General Virology, 1996, 77: 1295-1303.

[18]Meyers G, Thiel H J, Rümenapf T.Classical swine fever virus: recovery of infectious viruses from cDNA constructs and generation of recombinant cytopathogenic defective interfering particles. Journal of Virology, 1996, 70(3):1588-1595.

[19]Aberle J H, Aberle S W, Kofler R M, Mandl C W. Humoral and cellular immune response to RNA immunization with flavivirus replicons derived from tick-borne encephalitis virus. Virology, 2005, 79:15107-15113.

[20]邹兴启, 赵启祖, 范运峰, 朱元源, 王琴, 徐璐, 范学政, 宁宜宝. 猪瘟病毒C株全长cDNA感染性克隆的构建及病毒拯救. 中国农业科学, 2011, 44(2): 409-416.

Zou X Q, Zhao Q Z, Fan Y F, Zhu Y Y, Wang Q, Xu L, Fan X Z, Ning Y B. Construction of the full length infectious CDNA clone of CSFV C strain and virus rescue. Scientia Agricultura Sinica, 2011, 44(2): 409-416.(in Chinese)

[21]Traschin J D, Moser C, Ruggli N, Hofmann M A.Classical swine fever virus leader proteinase Npro is not required for viral replication in vell culture. Journal of Virology, 1998, 72(9): 7681-7684.

[22]殷震, 刘景华. 动物病毒学. 2版. 北京: 科学出版社, 1997: 645-664.

Yin Z, Liu J H. Animal Virology. 2nd ed. Beijing: Science Press, 1997:645-664. (in Chinese)

[23]Hulst M M, Van Gennip H G P, Vlot A C, Schooten E, De Smit A J, Moormann R J M. Interaction of Classical Swine Fever Virus with membrane-associated heparan sulfate: Role for virus replication in vivo and virulence. Journal of Virology, 2001, 75(20):9585-9595.

[24]Hulst M M, Van Gennip H G P, Moormann R J M. Passage of Classical Swine Fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein Erns. Journal of Virology, 2000, 74(20): 9553-9561.