非洲马瘟病毒VP7基因的克隆与表达

摘要/Abstract

摘要： 为获得非洲马瘟病毒VP7蛋白，以用于制备AHS血清学诊断试剂并为AHS新型疫苗的构建奠定基础，笔者采用原核表达系统表达VP7蛋白。在GeneBank中查找非洲马瘟病毒VP7基因序列，人工合成VP7基因，将VP7基因片段克隆插入pBAD/Thio-TOPO表达载体，将重组质粒转入大肠杆菌TOP10。经测序鉴定，筛选获得VP7基因正向插入、有正确读码框的阳性克隆，经L-Arabinose诱导表达VP7融合蛋白抗原。SDS-PAGE分析表明以终浓度为0.2%的L-阿拉伯糖进行诱导，4h后表达可达到高峰，融合蛋白质量约54.72 kDa。Western blotting检测和间接ELISA表明诱导表达的融合蛋白能与非洲马瘟病毒VP7单克隆抗体发生特异性反应，说明蛋白有特异的免疫学活性。

关键词: 小麦, 小麦, 行套, 陕西商洛

Abstract: To obtain the African horse sickness virus (AHSV) VP7 protein, which is used to preparing serological diagnostic reagent and constructing AHS new generation vaccine, the prokaryotic expression is used to express VP7 protein. The VP7 gene was synthesized from the database of GeneBank, then the VP7gene was cloned into pBAD/Thio-TOPO vector, and the recombinant plasmid was transferred into Escherichia coli TOP10. The recombinant plasmid was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted VP7 gene. The results of SDS-PAGE revealed that the VP7 recombinant fusion protein was expressed in TOP10 in a high level and the optimal amount of the expressed fusion protein is being induced with L-arabinose at 0.2% concentration for 4 hours. It had a molecular mass of approximately 54.72 kDa. The results of Western blotting and indirect ELISA revealed that the fusion protein is able to react with ASHV VP7 monoclonal antibody and they suggest that the fusion protein has immunologically reactive activity.

中图分类号:

S852.65+2

曾昭文,花群义,段纲,周晓黎,董俊,杨云庆,尹尚莲,项勋,常华. 非洲马瘟病毒VP7基因的克隆与表达[J]. 中国农学通报, 2006, 22(10): 49-49.

Zeng Zhaowen, Hua Qunyi, Duan Gang, Zhou Xiaoli, Dong Jun, Yang Yunqing, Yin Shanglian，Xiang Xun, Chang Hua. Cloning and expression of VP7 gene of African horse sickness virus[J]. Chinese Agricultural Science Bulletin, 2006, 22(10): 49-49.

参考文献

1 Du Plessis M, Cloete M, Aitchison H, et al. Protein aggregation complicates the development of baculovirus-expressed African horsesickness virus serotype 5 VP2 subunit vaccines.Onderstepoort J Vet Res,1998,65(4):321~329
2 Coetzer J A, Erasmus B J. African horsesickness. In: Coetzer J A, Thomson G R, Tustin R C. Infectious diseases of livestock with special reference to southern Africa. Vol, 1. Cape Town: Oxford University Press, 1994.460~475
3 Roy P, Mertens P P, Casal I. African horse sickness virus structure. Comp Immunol Microbiol Infect Dis,1994,17(3~4): 243~273
4 Howell P G. The isolation and identification of further antigenic types of African horse sickness virus. Onderstepoort J Vet Res, 1962,(29): 139~149
5 Iwata H, Yamagawa M, Roy P. Evolutionary relationships among the gnat-transmitted orbiviruses that cause African horse sickness, bluetongue, and epizootic hemorrhagic disease as evidenced by their capsid protein sequences. Virology, 1992,191(1): 251~261
6 Bremer C W, Huismans H, Van Dijk A A. Characterization and cloning of the African horsesickness virus genome.J Gen Virol,1990,(71): 793~799
7 Vreede F T, Huismans H. Cloning, characterization and expression of the gene that encodes the major neutralization-specific antigen of African horsesickness virus serotype 3.J Gen Virol, 1994,(75): 3629~3633
8 Martinez-Torrecuadrada J L, Langeveld J P, Venteo A, et al. Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus. Virology, 1999,257(2):449~459
9 Oellermann R A, Els H J, Erasmus B J. Characterization of African horsesickness virus.Arch Gesamte Virusforsch, 1970,29(2):163~174
10 Maree S, Paweska J T. Preparation of recombinant African horse sickness virus VP7 antigen via a simple method and validation of a VP7-based indirect ELISA for the detection of group-specific IgG antibodies in horse sera.J Virol Methods, 2005,125(1): 55~65
11 Chuma T, Le Blois H, Sanchez-Vizcaino J M, et al. Expression of the major core antigen VP7 of African horsesickness virus by a recombinant baculovirus and its use as a group-specific diagnostic reagent.J Gen Virol,1992,(73): 925~931
12 Basak A K, Gouet P, Grimes J, et al. Crystal structure of the top domain of African horse sickness virus VP7: comparisons with bluetongue virus VP7.J Virol, 1996,70(6): 3797~3806
13 Maree S, Durbach S, Huismans H. Intracellular production of African horsesickness virus core-like particles by expression of the two major core proteins, VP3 and VP7, in insect cells.J Gen Virol,1998,(79):333~337
14 Wade-Evans A M, Pullen L, Hamblin C, et al. African horsesickness virus VP7 sub-unit vaccine protects mice against a lethal, heterologous serotype challenge.J Gen Virol,1997,(78):1611~1616

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