含EGFP基因的马疱疹病毒1型新疆株gE基因缺失株的构建

doi:10.16431/j.cnki.1671-7236.2020.08.035

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (8): 2652-2659.doi: 10.16431/j.cnki.1671-7236.2020.08.035

含EGFP基因的马疱疹病毒1型新疆株gE基因缺失株的构建

范斌^1,2, 鲍子磊^1,2, 贾钦瑞², 刘建华², 贺笋¹, 冉多良²

1. 天康生物股份有限公司, 乌鲁木齐 830052;
2. 新疆农业大学动物医学学院, 乌鲁木齐 830052

收稿日期:2019-10-31 出版日期:2020-08-20 发布日期:2020-08-15
通讯作者: 贺笋, 冉多良 E-mail:hesun@tecon-bio.com;xjrdl7@163.com
作者简介:范斌(1993-),男,陕西汉中人,硕士,研究方向:动物传染病诊断与防治,E-mail:944327928@qq.com;鲍子磊(1992-),男,新疆昌吉人,硕士,研究方向:动物传染病诊断与防治,E-mail:821249346@qq.com
基金资助:
新疆维吾尔自治区重大科技专项项目（2017A01002）

Construction of gE Gene Deletion Strain of Equine Herpesvirus Type 1 Xinjiang Strain Containing EGFP Gene

FAN Bin^1,2, BAO Zilei^1,2, JIA Qinrui², LIU Jianhua², HE Sun¹, RAN Duoliang²

1. TECON Biology Co., Ltd., Urumqi 830052, China;
2. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China

Received:2019-10-31 Online:2020-08-20 Published:2020-08-15

摘要/Abstract

摘要： 为筛选马鼻肺炎（equine rhinopneumonitis，ER）基因缺失减毒活疫苗的候选毒株，本研究参考GenBank中EHV-1（登录号：KF644579.1）目的基因序列设计同源臂引物，以本地区流行株XJ2015株DNA为模板PCR扩增gE基因同源臂gEH1、gEH1，以EGFP表达盒（CMV+EGFP+polyA）为标记基因，酶切后依次连接至载体pUC-19，成功构建重组质粒pUC-gEH1H2-EGFP。将XJ2015基因组与质粒pUC-gEH1H2-EGFP共转染至RK-13细胞进行同源重组，以EGFP为标记进行gE基因缺失毒株的筛选及纯化，并测定重组毒株效价。结果显示：经5轮荧光噬斑纯化、PCR及测序鉴定，成功获取一株携带EGFP基因的重组毒株XJ2015-△gE-EGFP，且重组毒株效价（10^7.1TCID₅₀/0.1 mL）较原毒株（10^8.8TCID₅₀/0.1 mL）下降约10^1.7TCID₅₀/0.1 mL。采用同源重组技术成功构建了1株马疱疹病毒1型流行株gE基因缺失突变株，为未来筛选马鼻肺炎基因缺失弱毒疫苗奠定了基础。

关键词: 马鼻肺炎; 马疱疹病毒1型(EHV-1); gE基因缺失

Abstract: In order to select the candidate strain of live attenuated vaccine with gene deletion for equine rhinopneumonitis(ER).The primers were designd according to the target gene sequence in GenBank of EHV-1 (accession No.:KF644579.1),using the DNA of the popular strain XJ2015 in this region as a template,the left and right homology arms gEH1,gEH1 of gE gene were amplified by PCR,EGFP expression cassette (CMV+EGFP+polyA) as the marker gene,after enzyme digestion,the genes were ligated to the vector pUC-19 in turn,and the plasmid pUC-gEH1H2-EGFP was successfully constructed.Co-transfection of XJ2015 genome and plasmid pUC-gEH1H2-EGFP into RK-13 cells for homologous recombination,screening for gE-deletion strains with a markered gene EGFP,and then determining the titer of recombinant strain after purification.The results showed that,a recombinant strain XJ2015-△gE-EGFP with EGFP gene was successfully obtained after 5 rounds of fluorescent plaque purification,PCR and sequencing identification,and the titer of the recombinant strain (10^7.1TCID₅₀/0.1 mL) decreased by about 10^1.7TCID₅₀/0.1 mL compared with the original strain (10^8.8TCID₅₀/0.1 mL).A gE gene deletion mutant of equine herpesvirus type 1 strain was successfully constructed with homologous recombination technology which provided a foundation for future screening of weak virus vaccine with gene deletion in equine rhinopneumonitis.

Key words: equine rhinopneumonitis (ER); equine herpesvirus type 1 (EHV-1); gE gene deletion

中图分类号:

S852.65⁺2

范斌, 鲍子磊, 贾钦瑞, 刘建华, 贺笋, 冉多良. 含EGFP基因的马疱疹病毒1型新疆株gE基因缺失株的构建[J]. 中国畜牧兽医, 2020, 47(8): 2652-2659.

FAN Bin, BAO Zilei, JIA Qinrui, LIU Jianhua, HE Sun, RAN Duoliang. Construction of gE Gene Deletion Strain of Equine Herpesvirus Type 1 Xinjiang Strain Containing EGFP Gene[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(8): 2652-2659.

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含EGFP基因的马疱疹病毒1型新疆株gE基因缺失株的构建

Construction of gE Gene Deletion Strain of Equine Herpesvirus Type 1 Xinjiang Strain Containing EGFP Gene

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