[1]汪浩,乔绪稳,陈瑾,等.GEM技术浓缩纯化O型口蹄疫病毒灭活抗原方法的建立[J].南京农业大学学报,2018,41(1):147-153.[doi:10.7685/jnau.201701100]
 WANG Hao,QIAO Xuwen,CHEN Jin,et al.Concentration and purification of type O foot and mouth disease virus inactivated antigen with Gram-positive bacterial enhancer matrix[J].Journal of Nanjing Agricultural University,2018,41(1):147-153.[doi:10.7685/jnau.201701100]
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GEM技术浓缩纯化O型口蹄疫病毒灭活抗原方法的建立()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
41卷
期数:
2018年1期
页码:
147-153
栏目:
出版日期:
2018-01-15

文章信息/Info

Title:
Concentration and purification of type O foot and mouth disease virus inactivated antigen with Gram-positive bacterial enhancer matrix
作者:
汪浩12 乔绪稳13 陈瑾13 李图帅13 张元鹏13 侯继波13 郑其升13 孙卫东2
1. 国家兽用生物制品工程技术研究中心, 江苏 南京 210014;
2. 南京农业大学动物医学院, 江苏 南京 210095;
3. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 江苏 扬州 225009
Author(s):
WANG Hao12 QIAO Xuwen13 CHEN Jin13 LI Tushuai13 ZHANG Yuanpeng13 HOU Jibo13 ZHENG Qisheng13 SUN Weidong2
1. National Research Center of Veterinary Biological Engineering and Technology, Nanjing 210014, China;
2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;
3. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China
关键词:
GEM技术口蹄疫病毒抗原纯化
Keywords:
GEM technologyfoot and mouth disease virusantigen purification
分类号:
S852.4+3
DOI:
10.7685/jnau.201701100
摘要:
[目的]验证利用革兰氏阳性增强基质颗粒(GEM)浓缩纯化口蹄疫病毒(FMDV)抗原的可行性。[方法]将含有3个自溶素基序的锚钩蛋白基因(PA)与针对O型口蹄疫病毒特异性纳米抗体基因(VHH)串联克隆入原核表达载体pET-32a(+)中,构建原核表达载体pET-VPA,将其转化大肠杆菌宿主菌BL21(DE3),表达获得融合重组蛋白VPA。先将GEM颗粒与重组蛋白VPA在37℃孵育30 min,离心,取沉淀用适量的FMDV抗原液重悬,再经一步低速离心,获得沉淀即为浓缩纯化的口蹄疫病毒抗原。利用SDS-PAGE、Western-blot、蛋白含量测定及146S测定等方法对浓缩纯化方法的可行性进行鉴定,并通过动物免疫试验初步验证浓缩纯化后抗原的免疫原性。[结果]VPA融合蛋白能够在大肠杆菌中部分可溶性表达。作为接头蛋白,VPA既能与GEM颗粒结合,又能与FMDV抗原结合。SDS-PAGE与Western-blot结果表明:FMDV通过GEM法得到有效纯化;146S测定结果表明FMDV的回收效率达到99%;总蛋白含量测定结果显示口蹄疫抗原杂蛋白去除率达到90%;动物免疫试验结果显示GEM技术纯化后的FMDV具有良好的免疫原性。[结论]利用GEM技术浓缩纯化口蹄疫病毒抗原是可行的。
Abstract:
[Objectives]The purpose of this study is to verify the feasibility of purification of type O foot and mouth disease virus(FMDV) inactivated antigen with Gram-positive bacterial enhancer matrix (GEM) technology. [Methods]A dual functional molecular consisting of anchor protein gene and FMDV specific single domain antibody gene of type O FMDV were expressed in Escherichia coli,and the recombinant protein was named as VPA. The sedimentation named GEM-VPA was obtained with centrifugation following mixing the recombinant protein with GEM particles,and incubated at 37℃ for 30 min;GEM-VPA was mixed with FMDV antigen,and then the sedimentation was collected with low speed centrifugation after incubated at 37℃ for another 1 h,which was the purified antigen named GEM-FMDV. The feasibility for the method was evaluated with SDS-PAGE,Western-blot,protein quantification and 146S detection,and the immunogenicity for purified FMDV antigen was estimated with animal test. [Results]The dual functional protein designed in this study could be partly soluble when expressed in Escherichia coli. Moreever,this recombinant protein could bind with GEM particles and inactivated FMDV antigen at the same time. SDS-PAGE and Western-blot proved that the purified FMDV antigen existed in the sedimentation after centrifugation. Results from 146S determination indicated that the FMDV antigen recovery in this study could be above 99%,and the removal efficiency of the heterologous proteins from FMDV antigen was higher than 90%. More importantly,animals injected with the purified antigen developed more vigorous immune response and lesser side effect. [Conclusions]It is feasible for FMDV antigen concentration and purification with GEM technology.

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备注/Memo

备注/Memo:
收稿日期:2017-01-30。
基金项目:江苏省农业自主创新专项资金项目(CX(15)1026)
作者简介:汪浩,硕士研究生。
通信作者:郑其升,博士,副研究员,主要从事猪用新型疫苗研究,E-mail:njcvc1302@163.com;孙卫东,博士,教授,主要从事临床兽医学研究,E-mail:swd100@njau.edu.cn。
更新日期/Last Update: 1900-01-01