传染性法氏囊病病毒超强毒株衣壳蛋白的可溶性表达、纯化与活性分析

doi:10.16431/j.cnki.1671-7236.2017.07.027

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (7): 2096-2102.doi: 10.16431/j.cnki.1671-7236.2017.07.027

传染性法氏囊病病毒超强毒株衣壳蛋白的可溶性表达、纯化与活性分析

高祥¹, 李辉¹, 张礼洲¹, 卢珍¹, 王永强¹, 高立¹, 吴甜甜¹, 高玉龙¹, 刘长军¹, 王笑梅^1,2, 祁小乐¹

1. 中国农业科学院哈尔滨兽医研究所, 兽医生物技术国家重点实验室, 禽传染病研究室, 哈尔滨 150069;
2. 江苏省动物重要疫病与人畜共患病协同创新中心, 扬州 225009

收稿日期:2016-12-09 出版日期:2017-07-20 发布日期:2017-07-22
通讯作者: 王笑梅, 祁小乐 E-mail:xmw@hvri.ac.cn;qxl@hvri.ac.cn
作者简介:高祥(1990-),男,河南新密人,硕士生,研究方向:动物病毒学,E-mail:caasgx@126.com
基金资助:
国家自然科学基金重点项目（31430087）；哈尔滨市应用技术研究与开发项目（2014AB3AN058）；现代农业产业技术体系建设专项基金（CARS-42-G07）；哈尔滨市科技创新人才项目（2014RFQYJ129）

Soluble Expression, Purification and Activity Analysis of Capsid Protein of Very Virulent Infectious Bursal Disease Virus

GAO Xiang¹, LI Hui¹, ZHANG Li-zhou¹, LU Zhen¹, WANG Yong-qiang¹, GAO Li¹, WU Tian-tian¹, GAO Yu-long¹, LIU Chang-jun¹, WANG Xiao-mei^1,2, QI Xiao-le¹

1. Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agriculture Sciences, Harbin 150069, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China

Received:2016-12-09 Online:2017-07-20 Published:2017-07-22

摘要/Abstract

摘要：

为获得高质量的病毒衣壳蛋白VP2，本研究克隆了传染性法氏囊病病毒（infectious bursal disease virus，IBDV）超强毒株Gx的VP2基因，并亚克隆至载体pCold-Ⅰ，进而获得重组原核表达质粒pCold-Ⅰ-GxVP2，在Transetta（DE3）工程菌中优化条件进行IBDV衣壳蛋白VP2的可溶性表达，运用Ni-NTA亲和层析和凝胶过滤两种技术串联的方法进行蛋白纯化，运用单克隆抗体介导的Western blotting技术鉴定纯化蛋白的特异性，免疫SPF鸡鉴定纯化蛋白的免疫活性。结果显示，在冷休克条件下，IBDV衣壳蛋白VP2在Transetta（DE3）工程菌中实现了可溶性表达；通过纯化获得了高纯度的VP2，浓度为542 μg/mL；该蛋白不仅能与VP2单克隆抗体特异性反应，还能刺激鸡产生特异性免疫应答，具有良好的免疫活性。本研究在IPTG为1 mmol/L，15℃、120 r/min，诱导时间为24 h的条件下，实现了有免疫活性的IBDV衣壳蛋白VP2的可溶性表达和纯化。高纯度、可溶、具有功能活性的衣壳蛋白的制备，为深入开展IBDV致病机制研究奠定了基础。

关键词: 传染性法氏囊病病毒; 衣壳蛋白; 可溶性表达; 纯化

Abstract:

To obtain the capsid VP2 with high quality, VP2 gene of very virulent infectious bursal disease virus (vvIBDV) Gx was cloned and inserted into pCold-Ⅰ and the prokaryotic expression plasmid pCold-Ⅰ-GxVP2 was constructed. In engineering bacteria Transetta(DE3), the induction conditions of protein VP2 expression were optimized.With affinity chromatography and gel filtration, protein VP2 was purified. With the monoclonal antibody directed Western blotting, protein VP2 was identified. Using SPF chicken, immunocompetence of VP2 was evaluated. The results showed that the dissoluble protein VP2 was expressed successfully in Transetta(DE3) in cold-shock conditions; Protein VP2 was purified and the concentration was 542 μg/mL; The purified protein VP2 not only reacted with the monoclonal antibody against protein VP2, but also induced specific immune response in immunized chickens. In general, with 15℃ of cold-shock condition, 120 r/min of shaking culture, 1 mmol/L of IPTG,inducting for 24 h, soluble capsid VP2 of IBDV with immunocompetence was successfully expressed and purified.The preparation of highly purified, soluble capsid protein with functional activity laid the foundation for further researches on the pathogenic mechanism.

Key words: infectious bursal disease virus (IBDV); capsid protein; soluble expression; purification

中图分类号:

S858.31

高祥, 李辉, 张礼洲, 卢珍, 王永强, 高立, 吴甜甜, 高玉龙, 刘长军, 王笑梅, 祁小乐. 传染性法氏囊病病毒超强毒株衣壳蛋白的可溶性表达、纯化与活性分析[J]. 《中国畜牧兽医》, 2017, 44(7): 2096-2102.

GAO Xiang, LI Hui, ZHANG Li-zhou, LU Zhen, WANG Yong-qiang, GAO Li, WU Tian-tian, GAO Yu-long, LIU Chang-jun, WANG Xiao-mei, QI Xiao-le. Soluble Expression, Purification and Activity Analysis of Capsid Protein of Very Virulent Infectious Bursal Disease Virus[J]. , 2017, 44(7): 2096-2102.

参考文献

[1] Lasher H N, Shane S M. Infectious bursal disease[J]. Worlds Poult Sci J, 1994,50(2):133-166.
[2] Becht H, Muller H. Infectious bursal disease-B cell dependent immunodeficiency syndrome in chickens[J]. Behring Inst Mitt,1991,89:217-225.
[3] Calnek B W主编,高福,苏敬良主译. 禽病学[M].第10版.北京:中国农业出版社,1999.
[4] Muller H, Islam M R, Raue R. Research on infectious bursal disease——The past, the present and the future[J].Vet Microbiol,2003,97(1-2):153-165.
[5] Gao Y C, Yeung W S, Law M, et al. Molecular characterization of seven Chinese isolates of infectious bursal disease virus:Classical, very virulent, and variant strains[J].Avian Dis,1998,42(2):340-351.
[6] Ona A, Luque D, Abaitua F,et al. The C-terminal domain of the pVP2 precursor is essential for the interaction between VP2 and VP3,the capsid ploypepetides of inectious bursal disease virus[J].Virology,2004,322(1):135-142.
[7] Qi X, Zhang L, Chen Y, et al. Mutations of residues 249 and 256 in VP2 are involved in the replication and virulence of infectious bursal disease virus[J]. PLoS One, 2013, 8(7):e70982.
[8] Qi X, Gao H, Gao Y, et al. Naturally occurring mutations at residues 253 and 284 in VP2 contribute to the cell tropism and virulence of very virulent infectious bursal disease[J]. Antiviral Res, 2009, 84(3):225-233.
[9] 陈玉明,张礼洲,任宪刚,等. 不同毒力传染性法氏囊病病毒衣壳蛋白酵母双杂交诱饵载体的构建及鉴定[J]. 中国畜牧兽医,2014,41(1):30-34.
[10] Luo J, Zhang H, Teng M, et al. Surface IgM on DT40 cells may be a component of the putative receptor complex responsible for the binding of infectious bursal disease virus[J]. Avian Pathol, 2010, 39(5):359-365.
[11] Lin T W, Lo C W, Lai S Y, et al. Chicken heat shock protein 90 is a component of the putative cellular receptor complex of infectious bursal disease virus[J]. J Virol, 2007, 81(16):8730-8741.
[12] Ye C, Han X, Yu Z, et al. Infectious bursal disease virus activates c-Src to promote α4β1 integrin-dependent viral entry via modulating downstream Akt-RhoAGTPase-actin rearrangement cascade[J]. J Virol, 2017, 91(3):e01891-16.
[13] Ren X G, Zhang L Z, Gao Y L, et al. Binding chicken Anx2 is beneficial for infection with infectious bursal disease virus[J]. Virus Res, 2015, 210:232-240.
[14] Zhang L, Ren X, Chen Y, et al. Chondroitin sulfate N-acetylgalactosaminyltransferase-2 contributes to the replication of infectious bursal disease virus via interaction with the capsid protein VP2[J]. Viruses, 2015, 7(3):1474-1491.
[15] Wang X M,Wang M P,Gao H L,et al. Molecular and antigenic characterization of very virulent infectious bursal disease virus strain Gx isolated in China[J].Agricultural Sciences in China, 2003,2(5):566-572.
[16] Tsukamoto K, Kojima C, Komori Y, et al. Protection of chickens against very virulent infectious bursal disease virus (IBDV) and Marek's disease virus (MDV) with a recombinant MDV expressing IBDV VP2[J]. Virology, 1999, 257(2):352-362.
[17] Taghavian O, Spiegel H, Hauck R, et al. Protective oral vaccination against infectious bursal disease virus using the major viral antigenic protein VP2 produced in Pichia pastoris[J]. PLoS One, 2013, 8(12):e83210.
[18] 梅敏敏,梁国智,黄雯晶,等.新型鸭呼肠孤病毒σB蛋白的原核表达及其多克隆抗体制备[J].中国畜牧兽医,2016,43(12):3149-3155.
[19] 孙芬芬,宇文延青,高宏雷,等.鸡传染性法氏囊病病毒VP2基因的融合表达及兔抗血清的制备[J]. 中国动物传染病学报,2009,17(4):11-14.
[20] Jiang D,Liu Y,Wang A,et al. High level soluble expression and one-step purificationof IBDV VP2 protein in Escherichia coli[J]. Biotechnol Lett, 2016,38(6):901-908.
[21] 王晓龙,卢天成,王秀然,等. 鹿结核分枝杆菌MPB70蛋白的表达与免疫原性鉴定[J]. 中国畜牧兽医,2016,43(12):3135-3140.
[22] 郑仲华,杨佩,赵明,等. 猪传染性胃肠炎病毒截短S基因原核表达及免疫原性初步检测[J]. 中国畜牧兽医,2016,43(12):2541-2546.

传染性法氏囊病病毒超强毒株衣壳蛋白的可溶性表达、纯化与活性分析

Soluble Expression, Purification and Activity Analysis of Capsid Protein of Very Virulent Infectious Bursal Disease Virus

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