[1]王安平,朱善元,王永娟,等.Ⅰ型鸭甲型肝炎病毒VP3基因在昆虫细胞中的表达与鉴定[J].江苏农业学报,2017,(03):649-653.[doi:doi:10.3969/j.issn.1000-4440.2017.03.024]
　WANG An-ping,ZHU Shan-yuan,WANG Yong-juan,et al.Expression and identification of VP3 genes of duck hepatitis A virus type-I in insect cells[J].,2017,(03):649-653.[doi:doi:10.3969/j.issn.1000-4440.2017.03.024]
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Ⅰ型鸭甲型肝炎病毒VP3基因在昆虫细胞中的表达与鉴定()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2017年03期
页码:
649-653
栏目:
畜牧兽医·水产养殖
出版日期:
2017-06-30

文章信息/Info

Title:
Expression and identification of VP3 genes of duck hepatitis A virus type-I in insect cells
作者:
王安平; 朱善元; 王永娟; 吴双; 左伟勇; 洪伟鸣
（江苏农牧科技职业学院/江苏省兽用生物制药高技术研究重点实验室，江苏泰州225300）
Author(s):
WANG An-ping; ZHU Shan-yuan; WANG Yong-juan; WU Shuang; ZUO Wei-yong; HONG Wei-ming
(Jiangsu Agri-animal Husbandry Vocational College，Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals，Taizhou 225300, China)
关键词:
I型鸭甲型肝炎病毒; VP3基因; 昆虫细胞; 表达; 鉴定
Keywords:
duck hepatitis A virus type-I; VP3 gene; insect cell; expression; identification
分类号:
S855.3
DOI:
doi:10.3969/j.issn.1000-4440.2017.03.024
文献标志码:
A
摘要:
为在昆虫细胞中表达Ⅰ型鸭甲型肝炎病毒(Duck hepatitis A virus type-I，DHAV-I) SH株的主要结构蛋白VP3，首先根据DHAV-I SH株VP3基因序列设计1对引物，RT-PCR方法扩增出VP3基因，克隆至杆状病毒表达载体pFastBac1，获得重组杆状病毒转移载体pFB-VP3，将其转化到DH10Bac感受态细胞中，经抗性和蓝白斑筛选，获得重组穿梭质粒rBacmid-VP3，在脂质体介导下转染昆虫细胞Sf9，获得重组杆状病毒rBac-VP3。间接免疫荧光结果显示重组蛋白获得了正确表达，能与鸭抗全病毒阳性血清发生特异性反应。Western blot结果显示表达的重组蛋白分子量约为27 000。表明，DHAV-I SH株的主要结构蛋白VP3在昆虫细胞中获得了成功表达，为VP3结构蛋白的功能研究及相关亚单位疫苗的研制奠定了基础。
Abstract:
In order to express the main structural protein VP3 of duck hepatitis A virus type-I (DHAV-I) in insect cells, one pair of specific primers were designed according to the published genome sequences of DHAV-I to amplify VP3 genes by PCR, and the amplified fragment was cloned into baculovirus expression vector pFastBac1. The recombinant vector pFB-VP3 was transformed into DH10Bac Escherichia coli, and the positive recombinant bacmid rBacmid-VP3 was selected through resistance and blue-white plague screening. The recombinant bacmid rBacmid-VP3 was then transfected into the Sf9 insect cells by liposome. Indirect immunofluorescence analysis revealed that the recombinant protein was expressed, which could be recognized by the positive anti-virus serum, and the protein was about 27 000 in molecular weight. The successful expression of protein VP3 of DHAV-I in insect cells lays a foundation of function study of VP3 protein.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期：2017-01-02 基金项目：国家自然科学基金项目（31302096）；江苏省农业科技支撑项目（BE2013415）；江苏省六大人才高峰项目（NY-009） 作者简介：王安平（1980-），女，江苏泰州人，博士，副教授，主要从事兽用生物制药研究。（Tel）15189910087;（E-mail）wap4017@163.com 通讯作者：朱善元，（Tel）13775665658
更新日期/Last Update: 2017-06-29