[1]张 瑶,任超琪,于蒙蒙,等.J亚群禽白血病病毒gp85蛋白的真核表达纯化及其生物学活性分析[J].中国预防兽医学报,2017,(10):815-819.[doi:10.3969/j.issn.1008-0425.201701003]
 ZHANG Yao,REN Chao-qi,YU Meng-meng,et al.Eukaryotic expression purification and biologic activity analysis of avian leukosis virus gp85[J].Chinese journal of preventive veterinary medicine,2017,(10):815-819.[doi:10.3969/j.issn.1008-0425.201701003]
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J亚群禽白血病病毒gp85蛋白的真核表达纯化及其生物学活性分析()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2017年10
页码:
815-819
栏目:
免疫学
出版日期:
2017-10-15

文章信息/Info

Title:
Eukaryotic expression purification and biologic activity analysis of avian leukosis virus gp85
文章编号:
1008-0589(2017)10-0815-05
作者:
 

张 瑶任超琪于蒙蒙高 祥管晓璐祁小乐王永强刘长军王笑梅高玉龙*

 (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/禽免疫抑制病创新团队, 黑龙江 哈尔滨 150069)
Author(s):
 

ZHANG Yao REN Chao-qi YU Meng-meng GAO Xiang GUAN Xiao-lu QI Xiao-leWANG Yong-qiang LIU Chang-jun WANG Xiao-mei GAO Yu-long*

 (State Key Laboratory of Veterinary Biotechnology/Division of Avian Immunosuppressive Diseases,
Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China)
关键词:
J亚群禽白血病病毒gp85表达纯化生物学活性
Keywords:
 ALV-J  gp85  expression  purification  biologic activity
分类号:
S852.65
DOI:
10.3969/j.issn.1008-0425.201701003
文献标志码:
A
摘要:
gp85蛋白是禽白血病病毒(ALV)的囊膜表面蛋白,含有病毒-受体决定簇,通过识别和结合受体介导病毒侵入宿主细胞。为表达具有正确构象和生物学活性的J亚群ALV (ALV-J) gp85蛋白并对其生物活性进行分析,本研究以pCAGGS为载体,构建N端带有信号肽编码序列、C端融合Fc编码序列的gp85重组表达质粒pCAGGS-s-gp85-Fc,将其转染293T细胞进行瞬时表达,收集细胞培养液。SDS-PAGE结果表明,gp85-Fc蛋白高效表达,并且经Protein A亲和层析纯化得到高纯度的gp85-Fc蛋白。利用HRV 3C蛋白酶切除Fc标签并且进行分子筛纯化,得到纯度高于90 %的gp85蛋白单体。经过流式细胞仪检测,表达的可溶性gp85蛋白能够与ALV-J受体特异性结合。病毒感染阻断试验结果显示,gp85蛋白能够通过封闭受体阻断ALV-J进入DF-1细胞,并且呈现剂量依赖性,在100 μg/mL时仍能阻断70 %以上的病毒侵入。本研究可溶性的、具有生物学活性的gp85蛋白的制备为体外研究病毒感染宿主细胞机制奠定了的基础。
  
Abstract:
gp85 is the surface unit (SU) of avian leukosis virus (ALV) envelop which mediates ALV infecting cells by interacting with specific receptor. To express soluble ALV-J gp85 and analysis its biological activity, we constructed a combined gp85 plasmid pCAGGS-s-gp85-Fc that fused a signal peptide coding-sequences on N terminal and Fc coding-sequences on C terminal. After transfected 293T cells with the pCAGGS-s-gp85-Fc plasmid, the supernatant was collected and the expression of gp85-Fc was detected with SDS-PAGE. By affinity chromatography of protein A, we obtained gp85-Fc protein with high purity. After cut the Fc tag with HRV 3C protease, gp85 monomer with more than 90% purity was obtained. By flow cytometry detected, we identified that the pure gp85 protein could bind with the receptor specially. Meanwhile, gp85 could block ALV-J enter into DF-1 cells by sealing viral receptor in a dose-dependent manner. When the concentration of gp85 was 100 μg/mL, over 70% virus were blocked. These results indicated that soluble gp85 protein was successfully expressed with biologic activity, which would lay the foundation of studying the mechanism of ALV-J infecting cells in vitro.
 

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                                             (本文编辑:彭永刚)

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更新日期/Last Update: 2017-11-06