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

作　　者：范斌 鲍子磊[1,2] 贾钦瑞 刘建华 贺笋[1] 冉多良 FAN Bin;BAO Zilei;JIA Qinrui;LIU Jianhua;HE Sun;RAN Duoliang(TECON Biology Co.,Ltd., Urumqi 830052,China;College of Veterinary Medicine,Xinjiang Agricultural University, Urumqi 830052,China)

机构地区：[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

出　　处：《中国畜牧兽医》2020年第8期2652-2659,共8页China Animal Husbandry & Veterinary Medicine

基　　金：新疆维吾尔自治区重大科技专项项目(2017A01002)。

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(107.1TCID50/0.1 mL)较原毒株(108.8TCID50/0.1 mL)下降约101.7TCID50/0.1 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。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(107.1TCID50/0.1 mL)decreased by about 101.7TCID50/0.1 mL compared with the original strain(108.8TCID50/0.1 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.

关 键 词：马鼻肺炎 马疱疹病毒1型(EHV-1) gE基因缺失 

分 类 号：S852.652[农业科学—基础兽医学]