共表达TGEV AD蛋白和PEDV S蛋白CS区重组伪狂犬病病毒的构建及纯化  

Construction and purification of a recombinant porcine pseudorabies virus co-expressing the AD protein of the TGEV and the CS region of the PEDV S protein

作　　者：赵丽[1] 吕玉金[1] 徐通 许瑞勤 金钺[2] 陈红英[2] ZHAO Li;LYU Yujin;XU Tong;XU Ruiqin;JIN Yue;CHEN Hongying(College of Veterinary Medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南牧业经济学院动物医药学院,河南郑州450046 [2]河南农业大学动物医学院,河南郑州450046

出　　处：《中国兽医学报》2024年第2期237-243,共7页Chinese Journal of Veterinary Science

基　　金：河南省科技攻关基金资助项目(222102110332,222102110252);河南牧业经学院博士启动基金资助项目(2020HNUAHEDF010)。

摘　　要：根据猪流行性腹泻病毒(PEDV)S蛋白的CS中和表位区(499~789 aa)序列设计1对特异性引物,在其5′端分别引入BamHⅠ和HindⅢ,PCR扩增PEDV CS区。将扩增的CS片段插入至pBApo-EF1α_Pur_DNA真核表达载体的BamHⅠ和HindⅢ位点,然后扩增含CS区表达盒,将其插入至含猪传染性胃肠炎病毒(TGEV)AD基因的PRV转移质粒pG-AD-EGFP中,构建转移质粒pG-AD-CS-EGFP。利用转染试剂ZLip2000将伪狂犬病病毒(PRV)三基因缺失毒株rPRV NY-gE-/gI-/TK-基因组与转移质粒pG-AD-CS-EGFP共转染ST细胞,获得携带有TGEV AD、PEDV CS区和绿色荧光蛋白标记基因的重组病毒rPRV-AD-CS-EGFP。扩增重组病毒rPRV-AD-CS的表达盒,测序后进行遗传稳定性评价,同时将重组病毒rPRV-AD-CS、亲本株Bartha-K61和PRV强毒株分别接种小鼠,进行安全性评价。将该重组病毒与CRISPR/Cas9-EGFP敲除质粒共转染ST细胞,经3轮病毒空斑纯化获得无绿色荧光标记的重组病毒rPRV-AD-CS。经Western blot和IFA证实rPRV-AD-CS在ST细胞中能表达CS外源蛋白,具有良好的遗传稳定性和安全性。结果表明,成功构建共表达TGEV AD蛋白和PEDV S蛋白CS区的重组病毒株rPRV-AD-CS,该重组病毒具有遗传稳定性和安全性,为开发安全、有效的预防TGEV、PEDV和PRV感染的三联苗奠定基础。To construct a recombinant virus strain rPRV-AD-CS co-expressing the CS region of TGEV AD protein and PEDV S protein,and to lay the groundwork for the development cof a safe and effective triple vaccine for the prevention of TGEV,PEDV and PRV infections.A pair of specific primers were designed based on the sequence of CS neutralization epitope region(499-789 aa)of porcine epidemic diarrhea virus(PEDV)S protein,and the PEDV CS region was PCR amplified by introducing BamHI and HindⅢat its 5 end,respectively.The amplified GS fragment was inserted into the BamHI and HindⅢsites of the pBApo-EF1αDNA eukaryotic expression vector,and then the CS region-containing expression cassette was amplified and inserted into the PRV transfer plasmid pG-AD-EGFP containing the AD gene of transmissible gastroenteritis virus(TGEV)to construct the transfer plasmid pG-AD-CS-EGFP.The PRV triple gene deletion strain rPRV NY-gE~-/gI~-/TK~-genome was cotransfected with the transfer plasmid pG-AD-CS-EGFP using transfection reagent ZLip2000 to obtain the recombinant virus rPRV-AD-CSEGFP carrying TGEV AD,PEDV CS region and green fluorescent protein marker genes.The expression cassette of recombinant virus rPRV-AD-CS,the genetic stability evaluation was carried out after sequencing,and the recombinant virus rPRV-AD-CS,the parental strain Bartha-K61 and the PRV strong strain were inoculated in mice for safety evaluation.The recombinant virus was cotransfected with CRISPR/Cas9-EGFP knockout plasmid in ST cells,and the recombinant virus rPRV-AD-CS without green fluorescent marker was obtained after three rounds of virus null spot purification.rPRV-AD-CS was confirmed to express CS exogenous protein in ST cells by Western blot and IFA.It had good genetic stability and safety.In conclusion,recombinant virus rPRV-ADCS was successful constructed,which was genetically stable and safe.

关 键 词：PEDV CS中和表位区 TGEV AD抗原表位区 PRV 

分 类 号：S852.65[农业科学—基础兽医学]