伪狂犬病毒gE、gI和US9三基因缺失株的构建

doi:10.3969/j.issn.1004-1524.2017.04.04

浙江农业学报 ›› 2017, Vol. 29 ›› Issue (4): 542-547.DOI: 10.3969/j.issn.1004-1524.2017.04.04

伪狂犬病毒gE、gI和US9三基因缺失株的构建

樊毅¹, 李碧¹, 郭万柱¹, 李萍¹, 黄剑波¹, 杨凡¹, 姜子义¹, 赵军¹, 许思遥¹, 邓益超¹, 殷玥¹, 毛汐语¹, 吕雯婷¹, 徐志文^{1, 2}, 朱玲^{1, 2, *}

1.四川农业大学动物医学院,四川温江 611130;
2.四川农业大学动物疫病与人类健康四川省重点实验室,四川温江 611130

收稿日期:2016-11-13 出版日期:2017-04-20 发布日期:2017-04-27
通讯作者: 朱玲,E-mail:abtcxzw@126.com
作者简介:樊毅(1990—),男,四川资阳人,硕士研究生,研究方向为动物传染病病原分子生物学。E-mail:348840733@qq.com
基金资助:
国家“十二五”科技支撑计划项目(2015BAD12B04); 四川省科技支撑计划(2014NZ0043,2017NZ0038); 长江学者与创新团队发展计划(IRT13083)

Constructing gE, gI and US9 gene deletion strain of pseudorabies virus

FAN Yi¹, LI Bi¹, GUO Wanzhu¹, LI Ping¹, HUANG Jianbo¹, YANG Fan¹, JIANG Ziyi¹, ZHAO Jun¹, XU Siyao¹, DENG Yichao¹, YIN Yue¹, MAO Xiyu¹, LYU Wenting¹, XU Zhiwen^{1, 2}, ZHU Ling^{1, 2, *}

1. Veterinary Medicine College of Sichuan Agricultural University, Chengdu 611130, China;
2. Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China

Received:2016-11-13 Online:2017-04-20 Published:2017-04-27

摘要/Abstract

摘要： 伪狂犬病毒(pseudorabies virus,PRV)是一种疱疹病毒,在自然界具有极广泛的宿主类群,是中国养猪业发展主要威胁之一。为了研究病毒的神经传导机制,实验采用Lipofectamine^® 3 000转染试剂,将PP63质粒与伪狂犬病毒Fa株基因组DNA共转染BHK-21细胞,空斑筛选得到gI/gE/US9重组缺失病毒,并命名为SA215-T。采用PCR、基因测序、Western Blot、电镜检查和生长曲线测定等方法检测重组病毒PRV-SA215-T。研究结果显示,Western Blot未发现gE基因的表达,SA215-T株的电镜形态与野毒株无明显差异,SA215-T株与亲本毒株Fa株在细胞中的生长曲线差异不明显且均达到了较高的病毒滴度。

关键词: 伪狂犬, gE/gI/US9, 重组病毒, 基因缺失

Abstract: Pseudorabies virus (PRV), a member of Herpes virus has an extremely broad range in nature and threatens the pig-industry development in our country. To explore the mechanism of nerve conduction of PRV, a virus mutant with a deletion in gE, gI and US9 genes was constructed. Plasmid of PP63 and pseudorabies virus Fa strain genomic DNA were co-transfected into BHK-21 cells by using Lipofectamine 3000 transfection reagent, and a recombinant virus with the deletion of gI/gE/US9 , named SA215-T, was screened and purified by plaque assay. PCR, gene sequencing. Western Blot, electron microscope and growth curve were used for identification of the deletion of genes, and features of the recombinant. The results showed that SA215-T was with effective deletion of gI and gE gene by PCR,and was absent in the expression of gE gene by Western Blot. There were no obvious differences in the morphology and growth curve of the recombinant virus as compared to its parental virus, they both achieved a high viral titer in cells.

Key words: pseudorabies virus, gI/gE/US9 gene, recombinant virus, gene deletion

中图分类号:

S852.65

樊毅, 李碧, 郭万柱, 李萍, 黄剑波, 杨凡, 姜子义, 赵军, 许思遥, 邓益超, 殷玥, 毛汐语, 吕雯婷, 徐志文, 朱玲. 伪狂犬病毒gE、gI和US9三基因缺失株的构建[J]. 浙江农业学报, 2017, 29(4): 542-547.

FAN Yi, LI Bi, GUO Wanzhu, LI Ping, HUANG Jianbo, YANG Fan, JIANG Ziyi, ZHAO Jun, XU Siyao, DENG Yichao, YIN Yue, MAO Xiyu, LYU Wenting, XU Zhiwen, ZHU Ling. Constructing gE, gI and US9 gene deletion strain of pseudorabies virus[J]. , 2017, 29(4): 542-547.

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伪狂犬病毒gE、gI和US9三基因缺失株的构建

Constructing gE, gI and US9 gene deletion strain of pseudorabies virus

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