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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2015, Vol. 35 Issue (8): 23-29    DOI: 10.13523/j.cb.20150804
研究报告     
稳定表达鼠源整联蛋白αvβ6的CHO-677细胞系的构建
朱志坚1,2, 连凯琪2, 杨帆2, 张伟2, 郑海学2, 杨孝朴1
1. 甘肃农业大学 动物医学院 兰州 730070;
2. 中国农业科学院 兰州兽医研究所 家畜疫病病原生物学国家重点实验室 兰州 730046
Establishment of a Stable CHO-677 Cell Line Expressing Murine αvβ6 Integrin
ZHU Zhi-jian1,2, LIAN Kai-qi2, YANG Fan2, ZHANG Wei2, ZHENG Hai-xue2, YANG Xiao-pu1
1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2. State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
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摘要:

口蹄疫是由口蹄疫病毒(FMDV)引起的一种高度接触性传染病,主要侵害偶蹄动物。乳鼠常作为一种重要的实验动物模型用于FMDV的研究;整联蛋白αvβ6是FMDV的重要受体之一。为深入研究整联蛋白αvβ6在FMDV感染乳鼠中所发挥的作用,克隆了乳鼠整联蛋白αvβ6的两个亚基,并将其导入中国仓鼠卵巢细胞(Chinese hamster ovary, CHO-677)基因组中,构建了稳定表达乳鼠整联蛋白αv 和 β6亚基的细胞系CHO-677-mαvβ6,并分别选用两种不同血清型的野生型FMDV毒株Asia1/HN/CHA/06和O/BY/CHA/2010感染细胞系来分析细胞系对FMDV的易感性。首先通过PCR和间接免疫荧光试验证明了细胞系中整联蛋白αvβ6在基因水平成功导入,在蛋白水平成功表达。然后,通过实时荧光定量RT-PCR检测病毒RNA拷贝数,并结合TCID50试验测定了代表毒株在两个细胞上的生长曲线。结果表明,与亲本细胞CHO-677相比,细胞系CHO-677-mαvβ6对FMDV更易感,从αvβ6的功能性上进一步验证了细胞系被成功构建。

关键词: FMDV整联蛋白受体&alphav&beta6细胞系    
Abstract:

Foot-and-mouth disease (FMD) is a highly contagious disease of domestic and wild cloven-hoofed animals, and its causative agent is FMD virus (FMDV). The suckling mouse is an important experimental animal model for FMDV and integrin αvβ6 is an important receptor of FMDV. In order to further study the role of integrin αvβ6 in FMDV infection for suckling mice, two subunit genes of integrin αvβ6 from suckling mice were cloned and transduced the Chinese hamster ovary 677 (CHO-677) cell line to stably express both suckling mouse integrin subunits αv and β6 (CHO-677-mαvβ6). The presence of αvβ6 in CHO-677-mαvβ6 cell line was detected by PCR and indirect immunofluorescent assay (IFA) at the gene and protein levels, respectively. In order to analyze the susceptibility of FMDV to the cell line, the wild-type strain Asia1/HN/CHA/06 and O/BY/CHA/2010 were used to infect the cell line. Viral RNAs were detected by real-time quantitative RT-PCR. Growth curves of the representative strains in the cell line and its parental cell line were determined by TCID50 assay. The data showed that CHO-677-mαvβ6 cell line was more susceptible to FMDV than the CHO-677 cell line, indicating that CHO-677-mαvβ6 cell line was constructed successfully.

Key words: FMDV    Integrin receptor    αvβ6    Stable cell line
收稿日期: 2015-04-30 出版日期: 2015-08-25
ZTFLH:  Q819  
基金资助:

国家自然基金资助项目(31302118)

通讯作者: 郑海学,杨孝朴     E-mail: yangxpu@gsau.edu.cn;haixuezheng@163.com
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引用本文:

朱志坚, 连凯琪, 杨帆, 张伟, 郑海学, 杨孝朴. 稳定表达鼠源整联蛋白αvβ6的CHO-677细胞系的构建[J]. 中国生物工程杂志, 2015, 35(8): 23-29.

ZHU Zhi-jian, LIAN Kai-qi, YANG Fan, ZHANG Wei, ZHENG Hai-xue, YANG Xiao-pu . Establishment of a Stable CHO-677 Cell Line Expressing Murine αvβ6 Integrin. China Biotechnology, 2015, 35(8): 23-29.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150804        https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I8/23


[1] Mason P W, Grubman M J, Baxt B. Molecular basis of pathogenesis of FMDV. Virus Res, 2003, 91(1): 9-32.

[2] Grubman M J, Baxt B. Foot-and-mouth disease. Clin Microbiol Rev, 2004, 17(2): 465-493.

[3] Steinberger J, Grishkovskaya I, Cencic R, et al. Foot-and-mouth disease virus leader proteinase: structural insights into the mechanism of intermolecular cleavage. Virology, 2014, 468-470: 397-408.

[4] Santos J A, Gouvea I E, Judice W A, et al. Hydrolytic properties and substrate specificity of the foot-and-mouth disease leader protease. Biochemistry, 2009, 48(33): 7948-7958.

[5] Zunszain P A, Knox S R, Sweeney T R, et al. Insights into cleavage specificity from the crystal structure of foot-and-mouth disease virus 3C protease complexed with a peptide substrate. J Mol Biol, 2010, 395(2): 375-389.

[6] Belsham G J. Translation and replication of FMDV RNA. Curr Top Microbiol Immunol, 2005, 288: 43-70.

[7] O'Donnell V, Larocco M, Duque H, et al. Analysis of foot-and-mouth disease virus internalization events in cultured cells. J Virol, 2005, 79(13): 8506-8518.

[8] Ruiz-Saenz J, Goez Y, Tabares W, et al. Cellular receptors for foot and mouth disease virus. Intervirology, 2009, 52(4): 201-212.

[9] Gullberg M, Muszynski B, Organtini L J, et al. Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells. J Gen Virol, 2013, 94(8): 1769-1779.

[10] Bai X, Bao H, Li P, et al. Effects of two amino acid substitutions in the capsid proteins on the interaction of two cell-adapted PanAsia-1 strains of foot-and-mouth disease virus serotype O with heparan sulfate receptor. Virol J, 2014, 11: 132.

[11] Wang G, Wang Y, Shang Y, et al. How foot-and-mouth disease virus receptor mediates foot-and-mouth disease virus infection. Virol J, 2015, 12(1): 9.

[12] Berryman S, Clark S, Kakker N K, et al. Positively charged residues at the five-fold symmetry axis of cell culture-adapted foot-and-mouth disease virus permit novel receptor interactions. J Virol, 2013, 87(15): 8735-8744.

[13] Mohapatra J K, Pandey L K, Rai D K, et al. Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions. J Gen Virol, 2015, 96(3): 553-564.

[14] Rieder E, Henry T, Duque H, et al. Analysis of a foot-and-mouth disease virus type A24 isolate containing an SGD receptor recognition site in vitro and its pathogenesis in cattle. J Virol, 2005, 79(20): 12989-12998.

[15] Monaghan P, Gold S, Simpson J, et al. The alpha(v)beta6 integrin receptor for Foot-and-mouth disease virus is expressed constitutively on the epithelial cells targeted in cattle. J Gen Virol, 2005, 86(10): 2769-2780.

[16] Berryman S, Clark S, Monaghan P, et al. Early events in integrin alphavbeta6-mediated cell entry of foot-and-mouth disease virus. J Virol, 2005, 79(13): 8519-8534.

[17] Monaghan P, Simpson J, Murphy C, et al. Use of confocal immunofluorescence microscopy to localize viral nonstructural proteins and potential sites of replication in pigs experimentally infected with foot-and-mouth disease virus. J Virol, 2005, 79(10): 6410-6418.

[18] Zhao Q, Pacheco J M, Mason P W. Evaluation of genetically engineered derivatives of a Chinese strain of foot-and-mouth disease virus reveals a novel cell-binding site which functions in cell culture and in animals. J Virol, 2003, 77(5): 3269-3280.

[19] Zhang Y, Zheng H X, Zhang Z D. Establishment of a Murine alpha nu beta 1 transgenic CHO-K1 cell line and its susceptibility to foot-and-mouth disease virus type Asia 1/HN/2006 in China. J Ani Vet Adv, 2013,12 (1): 108.

[20] Ma X, Li P, Bai X, et al. Sequences outside that of residues 93-102 of 3A protein can contribute to the ability of foot-and-mouth disease virus (FMDV) to replicate in bovine-derived cells. Virus Res, 2014, 191: 161-171.

[21] Gu Y X, Gao Z L, Zhou J H, et al. Establishment and evaluation of stable cell lines inhibiting foot-and-mouth disease virus by RNA interference. Biomed Res Int, 2014, 2014: 109428.

[22] Zhang Z D, Hutching G, Kitching P, et al. The effects of gamma interferon on replication of foot-and-mouth disease virus in persistently infected bovine cells. Arch Virol, 2002, 147(11): 2157-2167.

[23] Knowles N J, Samuel A R. Molecular epidemiology of foot-and-mouth disease virus. Virus Res, 2003, 91(1): 65-80.

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