鸡传染性法氏囊病病毒rVP2蛋白高效组装亚病毒颗粒的鉴定

doi:10.16431/j.cnki.1671-7236.2020.06.005

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (6): 1677-1684.doi: 10.16431/j.cnki.1671-7236.2020.06.005

鸡传染性法氏囊病病毒rVP2蛋白高效组装亚病毒颗粒的鉴定

王彦伟, 张素玲, 吴芃, 耿笑林, 黄玉欣, 李鹏昊, 王孟月, 李向东, 逄文强, 田克恭

国家兽用药品工程技术研究中心, 洛阳 471000

收稿日期:2019-11-11 出版日期:2020-06-20 发布日期:2020-06-20
通讯作者: 逄文强, 田克恭 E-mail:pangwq123@hotmail.com;tiankg@263.net
作者简介:王彦伟(1991-),男,河南洛阳人,硕士,研究方向:蛋白表达与纯化,E-mail:wangyw2017@foxmail.com;张素玲(1989-),女,河南鹤壁人,硕士,研究方向:蛋白表达与纯化,E-mail:zhangsuling@tju.edu.cn
基金资助:
郑洛新国家自主创新示范区创新引领型产业集群专项（181200211700）

Identification of Subviral Particles Efficiently Self-assembled by Infectious Bursal Disease Virus rVP2 Protein

WANG Yanwei, ZHANG Suling, WU Peng, GENG Xiaolin, HUANG Yuxin, LI Penghao, WANG Mengyue, LI Xiangdong, PANG Wenqiang, TIAN Kegong

National Research Center for Veterinary Medicine, Luoyang 471000, China

Received:2019-11-11 Online:2020-06-20 Published:2020-06-20

摘要/Abstract

摘要： 为获得高纯度的鸡传染性法氏囊病病毒（infectious bursal disease virus，IBDV）VP2蛋白自组装的亚病毒颗粒（subviral particles，SVPs），并对重组VP2蛋白（rVP2）SVPs的组装效率和均一性进行评价，本试验利用大肠杆菌表达IBDV rVP2，通过SDS-PAGE和Western blotting对蛋白表达情况和免疫反应性进行鉴定，通过阴离子交换层析和切向流超滤浓缩系统纯化rVP2蛋白，并通过透射电镜（TEM）、高效液相尺寸排阻色谱（HPSEC）、蔗糖密度梯度离心及粒径和表面电位（Zeta potential）等多种检测技术对单个SVP组装形态和整体组装情况进行分析检测，初步建立了IBDV rVP2 SVPs组装效率的系统性检测方法。结果显示，在大肠杆菌中实现IBDV rVP2的大部分可溶性表达，且能与鸡IBDV阳性血清有明显的免疫反应性，纯化后rVP2纯度提高66.9倍，回收率达到93.3%。TEM观察rVP2自组装成直径约25 nm的SVPs，视野内SVPs大小均一，均匀分散。HPSEC检测结果显示，rVP2 SVPs的分子质量约为2 482 ku，与20个VP2三聚体形成的T=1二十面体SVPs分子质量一致。蔗糖密度梯度离心检测结果显示，rVP2 SVPs体积分布均一，组装效率高。粒径和Zeta电位检测结果表明，rVP2 SVPs颗粒分散度较好、体系稳定，有利于rVP2 SVPs的长期保存。本试验通过多种检测技术对rVP2 SVPs的组装情况进行检测分析，实现了rVP2 SVPs的高效组装，为IBDV SVPs疫苗的质量控制提供了有力支撑。

关键词: 传染性法氏囊病病毒(IBDV); rVP2蛋白; 亚病毒颗粒(SVPs); 高效液相尺寸排阻色谱(HPSEC)

Abstract: In order to obtain highly purified subviral particles (SVPs) of VP2 protein of infectious bursal disease virus (IBDV),and evaluate the assembly efficiency and homogeneity of rVP2 SVPs,IBDV rVP2 was expressed in Escherichia coli (E.coli).The protein expression and immunoreactivity were identified by SDS-PAGE and Western blotting.rVP2 was purified by anion exchange chromatography and tangential flow ultrafiltration concentration system.The assembly morphology of single SVP and the overall assembly of rVP2 SVPs were analyzed by a variety of detection technologies,such as transmission electron microscopy (TEM),high performance liquid size exclusion chromatography (HPSEC),sucrose density gradient centrifugation,particle size and Zeta potential,and systematic methods for measuring the assembly efficiency of IBDV rVP2 SVPs were preliminarily established.The results showed most of the rVP2 was expressed as soluble protein in E.coli,and had obvious immunoreactivity with IBDV positive serum.After purification,the purity of rVP2 was increased 66.9 times and the recovery was 93.3%.The observation by TEM showed rVP2 self-assembled into SVPs with a diameter of about 25 nm,and the size of SVPs in the field of vision was uniform and evenly dispersed.The molecular weight of rVP2 SVPs detected by HPSEC was about 2 482 ku,which was consistent with the molecular weight of T=1 SVPs assembled by 20 VP2 trimers.The results of sucrose density gradient centrifugation showed rVP2 protein was efficiently self-assembled into SVPs with a uniform volume distribution.The results of particle size distribution and Zeta potential showed that the system of rVP2 SVPs was stable with a good particle dispersion,and was beneficial to the long-term preservation of rVP2 SVPs.The results of the assembly of rVP2 SVPs analyzed by various detection technologies showed that rVP2 SVPs were self-assembled efficiently,and provided a strong support for the quality control of IBDV SVPs vaccine.

Key words: infectious bursal disease virus (IBDV); rVP2 protein; subviral particles (SVPs); high performance liquid size exclusion chromatography (HPSEC)

中图分类号:

S852.65

王彦伟, 张素玲, 吴芃, 耿笑林, 黄玉欣, 李鹏昊, 王孟月, 李向东, 逄文强, 田克恭. 鸡传染性法氏囊病病毒rVP2蛋白高效组装亚病毒颗粒的鉴定[J]. 中国畜牧兽医, 2020, 47(6): 1677-1684.

WANG Yanwei, ZHANG Suling, WU Peng, GENG Xiaolin, HUANG Yuxin, LI Penghao, WANG Mengyue, LI Xiangdong, PANG Wenqiang, TIAN Kegong. Identification of Subviral Particles Efficiently Self-assembled by Infectious Bursal Disease Virus rVP2 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(6): 1677-1684.

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鸡传染性法氏囊病病毒rVP2蛋白高效组装亚病毒颗粒的鉴定

Identification of Subviral Particles Efficiently Self-assembled by Infectious Bursal Disease Virus rVP2 Protein

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