含EGFP基因的马疱疹病毒1型新疆株gE基因缺失株的构建

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。

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

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.