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

共表达TGEV AD蛋白和PEDVS蛋白CS区重组伪狂犬病病毒的构建及纯化

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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BamH Ⅰ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BamH Ⅰ和Hind Ⅲ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.
Construction and purification of a recombinant porcine pseudorabies virus co-ex-pressing the AD protein of the TGEV and the CS region of the PEDV S protein
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 of a safe and effective triple vaccine for the prevention of TGEV,PEDV and PRV infections.A pair of spe-cific 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 HindⅢ at its 5'end,respectively.The amplified CS fragment was in-serted into the BamHI and HindⅢ sites of the pBApo-EF1α DNA 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-CS-EGFP carrying TGEV AD,PEDV CS region and green fluorescent protein marker genes.The ex-pression 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 co-transfected 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-AD-CS was successful constructed,which was genetically stable and safe.

PEDVCS neutralizing epitope regionTGEVAD epitope regionPRV

赵丽、吕玉金、徐通、许瑞勤、金钺、陈红英

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河南牧业经济学院动物医药学院,河南 郑州 450046

河南农业大学动物医学院,河南郑州 450046

PEDV CS中和表位区 TGEV AD抗原表位区 PRV

河南省科技攻关计划河南省科技攻关计划河南牧业经学院博士启动基金

2221021103322221021102522020HNUAHEDF010

2024

中国兽医学报
吉林大学

中国兽医学报

CSTPCD北大核心
影响因子:0.702
ISSN:1005-4545
年,卷(期):2024.44(2)
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