[1]原 霖,张 瑜,刘 洋,等.A群猪轮状病毒VP6结构蛋白在杆状病毒系统中的表达及鉴定[J].中国预防兽医学报,2018,(06):476-480.[doi:0.3969/j.issn.1008-0589.201707005]
 YUAN Lin,ZHANG Yu,LIU Yang,et al.Expression and identification of recombinant structural protein VP6 of porcine rotavirus group A in baculovirus expression system[J].Chinese journal of preventive veterinary medicine,2018,(06):476-480.[doi:0.3969/j.issn.1008-0589.201707005]
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A群猪轮状病毒VP6结构蛋白在杆状病毒系统中的表达及鉴定()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2018年06
页码:
476-480
栏目:
病原生物学
出版日期:
2018-06-20

文章信息/Info

Title:
Expression and identification of recombinant structural protein VP6 of porcine rotavirus group A in baculovirus expression system
文章编号:
1008-0589(2018)6-0476-05
作者:
 

原 霖14张 瑜12刘 洋1 王婧怡13寇占英1亢文华1杨 林1王传彬1倪建强1*

 (1. 中国动物疫病预防控制中心,北京 朝阳 102618;2. 哈尔滨医科大学(大庆),黑龙江 大庆 163000;
3. 内蒙古农业大学 兽医学院,内蒙古 呼和浩特 010018;4. 中国农业大学 动物医学院,北京100193)
Author(s):
 

YUAN Lin14 ZHANG Yu12 LIU Yang1 WANG Jing-yi13 KOU Zhan-ying1 KANG Wen-hua1YANG Lin1 WANG Chuan-bin1 NI Jian-qiang1*

 (1. China Animal Disease Control Centre, Beijing 102618, China; 2. Harbin Medical University (Daqing), Daqing 163000, Chian;
3. College of Veterinary Medicine, Inner Mongolina, Hohhot 010018, China;
4. China Agricultural University College of Veterinary Medicine, Beijing 100193, China)
关键词:
A群猪轮状病毒VP6蛋白杆状病毒表达系统重组蛋白表达
Keywords:
porcine group A rotavirus  protein VP6  baculovirus expression system  recombinant protein expression
分类号:
S852.65
DOI:
0.3969/j.issn.1008-0589.201707005
文献标志码:
A
摘要:
为在杆状病毒表达系统中表达和纯化A群猪轮状病毒(PoRVA) VP6结构蛋白,在PoRVA VP6基因5’端融合添加组氨酸标签(6×His)后,克隆至杆状病毒表达载体pFastBac HTB中,得到重组转移载体pFastBac-VP6,并将其转化至DH10Bac感受态细胞进行蓝白斑筛选。将鉴定正确的重组杆状病毒杆粒转染Sf9细胞后收获重组杆状病毒rBac-PoRVA-VP6,经SDS-PAGE和western blot鉴定结果显示,表达的PoRVA 重组VP6蛋白约45 ku,并且能够被PoRVA阳性血清识别。上述结果表明,经杆状病毒表达系统表达的重组VP6蛋白具有良好的反应原性,为建立PoRVA抗体检测技术奠定了基础。
  
Abstract:
In order to express the structural protein VP6 of porcine rotavirus-A (PoRVA) in the baculovirus expression system, the VP6 gene fused with 6 Hiscodons in 5’-terminal was cloned into the baculovirus vector pFastBacHTB and then the recombinant plasmid pFastBac-VP6 was constructed. Subsequently, pFastBac-VP6 was transformed into DH10Bac competent cells for blue-white selection, and then the identified recombinant baculovirus bacmid was transfected into Sf9 cells to generate the recombinant baculovirus rBac-PoRVA-VP6. SDS-PAGE and western blot analysis showed that the molecular weight of recombination VP6 (rVP6) was about 45 ku, which correspond to the anticipated result, and the rVP6 reacted with the positive pig antiserum against PoRVA. These results indicated the rVP6 expressed in baculovirus expression system could keep the effective reactionogenicity. This study provided experimental bases for developing antibody detection method against PoRVA.

参考文献/References:

[1] Zimmerman Jeffery J. 猪病学[M]. 第9版. 赵德明,等. 北京:中国农业大学出版社,2008.
[2]Desselberger U. Rotaviruses [J]. Virus Res, 2014, 190: 75-96.
[3]潘小霞,张顺,陈元鼎. 轮状病毒 VP6 蛋白免疫学研究进展[J]. 中国疫苗和免疫,2011,17(2):169-172.
[4]Vlasova A N, Amimo J O, Saif L J. Porcine Rotaviruses: Epidemiology, immune responses and control strategies [J]. Viruses, 2017, 9(48): 1-27.
[5]Varghese V, Das S, Singh N B, et al. Molecular characterization of a human rotavirus reveals porcine characteristics in most of the genes including VP6 and NSP4 [J]. Arch Virol, 2004, 149(1): 155-172.
[6]Memon A M,  Bhuyan A A, Chen Fang-zhou, et al. Development and validation of monoclonal antibody-based antigen capture ELISA for detection of group A Porcine rotavirus [J]. Viral Immunol, 2017, 30(4): 1-7.
[7]陈淑华,库旭钢,闫贵伟,等. 猪A群轮状病毒VP6的表达及IgG抗体ELISA检测方法的建立与初步应用[J]. 动物医学进展,2014,35(7):1-6.
[8]Yuan Jing, Zhang Xin, Shi Hong-yan, et al. The interaction of Rotavirus A pig/China/NMTL/2008/G9P[23] VP6 with cellular beta-actin is required for optimal RV replication and infectivity [J]. Vet Microbiol, 2016, 197: 111-121.
[9]袁静,潘小霞,滕玉梅,等. 轮状病毒结构蛋白VP6 的原核表达及其作为检测抗原在病毒检测中的应用[J]. 中国生物制品学杂志,2012,25(6):730-733.
[10]刘晓,熊新宇,陈元鼎,等. 轮状病毒亚单位疫苗研究进展[J]. 国外医学预防诊断治疗用生物制品分册,2004,27(6):254-258.
[11]Crawford S E, Labbe M, Cohen J, et al. Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells [J]. J Virol, 1994, 68: 5945-5952.
[12]原霖,韩焘,倪建强,等. 猪A群轮状病毒BJ株的分离与鉴定[J]. 中国畜牧兽医,2016,12:3306-3313.
[13]Burns J W, Siadat-Pajouh M, Krishnaney A A, et al. Protective effect of rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity [J]. Science, 1996, 272(5258): 104- 107.
[14]曹志亮,文喻呤,陈元鼎,等. A组人轮状病毒VP6基因克隆及在大肠杆菌中的高效表达[J]. 中国生物工程杂志,2006,26(3):37-41.
[15]余海燕. 轮状病毒分子生物学研究进展[J]. 畜牧与饲料科学,2014,35(10):103-105.
[16]Da S J H, Da S E M J S, de Mendon?觭a M C, et al. Comparison of two eukaryotic systems for the expression of VP6 protein of rotavirus specie A: transient gene expression in HEK293-T cells and insect cell-baculovirus system [J]. Biotechnol Lett, 2012, 34: 1623-1627.
[17]Blazevic V, Lappalainen S, Nurminen K, et al. Norovirus VLPs and rotavirus VP6 protein as combined vaccine for childhood gastroenteritis [J]. Vaccine, 2011, 29: 8126-8133.
[18]Feng N, Lawton JA, Gilbert J, et al. Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA Mab [J]. J Clin Invest, 2002, 109(9): 1203-1213.
[19]Burns J W, Siadat-Pajouh M, Krishananey A A, et al. Protective effect ofrotavirus VP6-specific IgA monoclonal antibodies that lack neut ralizing activity [J]. Science, 1996, 272: 104-107.
(本文编辑:李 爽)

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更新日期/Last Update: 2018-07-11