表达新城疫病毒血凝素神经氨酸酶(HN)基因重组鸭肠炎病毒的构建

doi:10.3969/j.issn.1674-5817.2015.03.004

实验动物与比较医学 ›› 2015, Vol. 35 ›› Issue (3): 195-201.DOI: 10.3969/j.issn.1674-5817.2015.03.004

表达新城疫病毒血凝素神经氨酸酶(HN)基因重组鸭肠炎病毒的构建

梁殊林¹, 李慧昕², 陈洪岩³, 刘胜旺^1,2

1.东北农业大学生命科学学院, 哈尔滨 150030;
2.中国农业科学院哈尔滨兽医研究所禽呼吸道病创新团队, 哈尔滨 150001;
3.中国农业科学院哈尔滨兽医研究所实验动物中心, 哈尔滨 150001

收稿日期:2014-12-26 出版日期:2015-06-25 发布日期:2015-06-25
作者简介:梁殊林(1988-), 女, 硕士, 动物学,E-mail: liangshulin2010@163.com

Construction of Recombinant Duck Enteritis Virus with HN Gene of Newcastle Disease Virus

LIANG Shu-lin¹, LI Hui-xin², CHEN Hong-yan³, LIU Sheng-wang^1,2

1. College of Life Science, Northeast Agricultural University, Harbin 150030, China;
2. Innovation Team of Disease of Avian Respiratory Tract, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China;
3. Experimental Animal Center, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China

Received:2014-12-26 Online:2015-06-25 Published:2015-06-25

摘要/Abstract

摘要： 目的构建表达新城疫病毒血凝素神经氨酸酶(HN)基因的重组鸭肠炎病毒(DEV)。方法利用同源重组技术,在DEV Clone-03基因组中胸苷激酶(TK)基因内部插入新城疫病毒HN基因。以本实验室已构建的rDEV TK-EGFP株为亲本病毒, 将病毒基因组与转移载体共转染及蚀斑纯化, 获得表达新城疫病毒HN基因的重组鸭肠炎病毒。结果重组病毒经基因水平和蛋白水平的鉴定, 证明新城疫病毒HN基因正确插入DEV基因组内部且有效表达; 复制动力学和遗传稳定性检测, 证明重组病毒rDEV TK-HN滴度略有下降, 但复制趋势与亲本病毒一致; 在鸡胚成纤维细胞(CEF)和SPF鸡胚连续传代20代, 表明HN基因随病毒传代而稳定遗传、表达。结论利用同源重组技术, 对鸭肠炎病毒基因组进行修饰, 构建了TK基因缺失表达新城疫病毒HN基因的重组鸭肠炎病毒, 该病毒能够携带HN基因稳定遗传和表达。

关键词: 鸭肠炎病毒(DEV), 新城疫病毒(NDV), 血凝素神经氨酸酶(HN)基因, 重组病毒, 基因修饰

Abstract: Objective To construct the recombinant duck enteritis virus (DEV) expressing the HN gene of Newcastle disease virus (NDV). Methods The HN gene of NDV was inserted into TK gene of DEV Clone-03 by homologous recombination technology. rDEV TK-EGFP which was constructed as parent virus, by means of cotransfection of the viral genome and transfer vector and plaque purification, the recombinant duck enteritis virus was obtained expression the HN gene of Newcastle disease virus. Results The HN gene of NDV was inserted into DEV genome correctly and expressed effectively on gene and protein level. Replication kinetics and the genetic stability detection of the recombinant virus rDEV TK-HN proved that the titer of rDEV TK-HN decreased slightly, but the trend was consistent with the parent virus replication. After 20 passages in chicken embryo fibroblast and SPF chicken embryos, it showed that the HN gene was of good genetic and expression stability with the passage of the virus. Conclusion By homologous recombination technology and modifying duck enteritis virus genome, the recombinant virus which could carry the HN gene of NDV was constructed and the virus was of good genetic and expression stability.

Key words: Duck enteritis virus (DEV), Newcastle disease virus (NDV), HN gene, Recombinant virus, Genetic modification

中图分类号:

Q95-33

梁殊林, 李慧昕, 陈洪岩, 刘胜旺. 表达新城疫病毒血凝素神经氨酸酶(HN)基因重组鸭肠炎病毒的构建[J]. 实验动物与比较医学, 2015, 35(3): 195-201.

LIANG Shu-lin, LI Hui-xin, CHEN Hong-yan, LIU Sheng-wang. Construction of Recombinant Duck Enteritis Virus with HN Gene of Newcastle Disease Virus[J]. Laboratory Animal and Comparative Medicine, 2015, 35(3): 195-201.

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表达新城疫病毒血凝素神经氨酸酶(HN)基因重组鸭肠炎病毒的构建

Construction of Recombinant Duck Enteritis Virus with HN Gene of Newcastle Disease Virus

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