O型口蹄疫病毒蛋白酶3C基因在哺乳细胞中的表达

doi:10.11924/j.issn.1000-6850.2013-3399

中国农学通报 ›› 2014, Vol. 30 ›› Issue (20): 17-20.doi: 10.11924/j.issn.1000-6850.2013-3399

所属专题：生物技术

O型口蹄疫病毒蛋白酶3C基因在哺乳细胞中的表达

马琪陈豪泰薛慧文

收稿日期:2013-12-30 修回日期:2014-03-24 出版日期:2014-07-15 发布日期:2014-07-15
基金资助:
中央级公益性科研院所基本科研业务费专项 “动物病毒诊断新技术研究” (1610322013023)。

Expression of 3C Proteinase of Type O Foot and Mouth Disease Virus in Mammalian Cells

Received:2013-12-30 Revised:2014-03-24 Online:2014-07-15 Published:2014-07-15

摘要/Abstract

摘要： 为构建O型口蹄疫病毒(FMDV)蛋白酶3C基因的真核表达质粒，并在BHK-21细胞中进行表达，以RT-PCR扩增O型口蹄疫病毒蛋白酶3C基因，利用XhoⅠ和HindⅢ限制性内切酶将3C基因克隆至真核表达载体pcDNA3.1(-)，通过脂质体法转染重组质粒pcDNA3.1(-)-3C。PCR，双酶切鉴定及测序结果表明：重组质粒pcDNA3.1(-)-3C构建成功；Western-blot分析结果表明：重组质粒pcDNA3.1(-)-3C在BHK-21细胞中能够表达O型FMDV蛋白酶3C基因。该真核表达质粒的构建，为进一步研究O型口蹄疫空衣壳的体外组装以及O型口蹄疫空衣壳疫苗的开发建立了实验基础。

关键词: 副热带高压, 副热带高压

Abstract:

To construct the eukaryotic expression plasmid of tpye O foot-and-mouth disaese virus(FMDV) 3C proteinase, and to express in BHK-21 cells, 3C gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from type O FMDV strain and then cloned into the eukaryotic expression vector pcDNA3.1(-) using the restriction enzyme XhoⅠand Hind Ⅲ, the recombinant plasmid pcDNA3.1(-)-3C was transfected into the BHK-21 cells by Lipofectamine ^TM 2000 Reagent. After the confirmation by PCR, enzyme digestion and sequence analyses, the resulting plasmid pcDNA3.1(-)-3C was constructed correctly; Western-blot analyses showed that the plasmid pcDNA3.1(-)-3C was successfully expressed type O FMDV 3C proteinase in BHK-21 cells. The construction of the eukaryotic expression plasmid would help to study the assembly of FMDV empty capsid in vitro and establish a basic experiment to develop the type O FMDV empty capsid vaccine in future.

马琪陈豪泰薛慧文. O型口蹄疫病毒蛋白酶3C基因在哺乳细胞中的表达[J]. 中国农学通报, 2014, 30(20): 17-20.

参考文献

[1] Ana C M, Vanesa R. Transient Gene Expression in Serum- Free Suspension-Growing Mammalian Cells for the Production of Footand-Mouth Disease Virus Empty Capsids[J]. PLoS ONE,2013,8(8): 72800.
[2] Thompson D, Muriel P, Russell D, et al. Economic costs of the foot and mouth disease outbreak in the United Kingdom in 2001[J]. Rev Sci Tech,2002(21):675-687.
[3] Grubman M J, Baxt B. Foot-and-mouth disease[J]. Clin Microbiol Rev,2004,17(2):465-493.
[4] Parida S. Vaccination against foot- and- mouth disease virus: strategies and effectiveness[J].Expert Rev Vaccines,2009,8:347-365.
[5] Rodriguez L L, Grubman M J. Foot and mouth disease virus vaccines[J].Vaccines,2009(27):90-94.
[6] Grubman M J. Development of novel strategies to control foot and mouth disease: marker vaccines and antivirals[J].Biologicals,2005 (33):227-234.
[7] Rowlands D J, Sangar D V, Brown F. A comparative chemical and serological study of the full and empty particles of foot-and mouth disease virus[J]. J Gen Virol,1975(26):227-238.
[8] Rweyemamu M M, Terry G, Pay T W F. Stability and Immnnogenicity of Empty Particles of Foot- and- Mouth Disease Virus[J].Arch Virol,1979,59(1-2):69-79.
[9] Abrams C C, King A M Q, Belsham G J. Assembly of foot- and mouth disease virus empty capsids synthesized by a vaccinia virus expression system[J]. J Gen Virol,1995,76:3089-3098.
[10] Zhou J H, Cong G Z, Gao S D, et al. Advances in Leader Protein of Foot- and- mouth Disease Virus[J].Biotechnology Bulletin,2007(3): 63-66.
[11] Sarkany Z, Polgar L. The unusual catalytic triad of poli virus protease 3C[J]. Biochemistry,2003,42:516-522.
[12] Belsham G J, Abrams C C, King A M., et al. Myristoylation of footand-mouth disease virus capsid protein precursor is independent of other viral proteins and occurs in both mammalian and insect cells [J]. J Gen Virol,1991,72:747-751.
[13] Cedillo-Barron L, Foster-Cuevas M, Belsham G.J, et al. Induction of a protective response in swine vaccinated with DNA encoding foot- and- mouth disease virus empty capsid proteins and the 3D RNA polymerase[J].J Gen Virol, 2001,82:1713-1724.
[14] 李志勇.AsiaⅠ型口蹄疫病毒空衣壳抗原的表达与免疫研究[D].北京:中国农业科学院,2007.
[15] 曹轶梅. Asia 1型口蹄疫病毒空衣壳在昆虫细胞中的组装及其免疫原性研究[D].北京:中国农业科学院,2009.
[16] Doel T. FMD vaccines[J]. Virus Res,2003,91(1):81-99.
[17] Mayr G A, Chinsangaram J. Development of replication- defective adenovirus serotype 5 containing the capsid and 3C protease coding regions of foot-and-mouth disease virus as a vaccine candidate[J]. Virology,1999,263(2):496-506.
[18] Belsham G J. Distinctive features of foot-and-mouth disease virus, a member of the picornavirus family; aspects of virus protein synthesis, protein processing and structure[J]. Prog Biophy Mol Biol,1993,60:241-60.
[19] Mayr G A, Donnell V O, Chinsangaram J, et al. Immune responses and protection against foot- and- mouth disease virus (FMDV) challenge in swine vaccinated with adenovirus- FMDV constructs [J]. Vaccine,2001,19:2152-2162.
[20] 袁立科.口蹄疫病毒 Asial型 P12A3C基因自杀式真核表达载体的构建[D].兰州:甘肃农业大学,2006.

O型口蹄疫病毒蛋白酶3C基因在哺乳细胞中的表达

Expression of 3C Proteinase of Type O Foot and Mouth Disease Virus in Mammalian Cells

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