表达PEDV中国变异株S基因重组猪伪狂犬病病毒的构建和纯化
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摘要
为了研制出能够同时预防猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)和猪伪狂犬病病毒(pseudorabies virus,PRV)感染的二联活疫苗,本研究将增强型绿色荧光蛋白(EGFP)和PEDV中国变异株的纤突蛋白免疫决定簇区域(S1)基因分别克隆至含有PRV胸苷激酶(TK)基因上、下游同源臂的穿梭载体pTK中,构建重组PRV转移质粒pTK-EGFP和pTK-S1。将重组质粒pTK-EGFP与PRV疫苗毒株Bartha-K61基因组DNA共转染Vero细胞,经绿色荧光蚀斑纯化得到重组病毒rPRV-EGFP。随后将重组质粒pTK-S1与rPRV-EGFP基因组DNA共转染Vero细胞,通过反向筛选EGFP阴性蚀斑,蚀斑纯化得到表达PEDV S蛋白的重组伪狂犬病病毒rPRV-S1。本研究将为更有效防控猪伪狂犬病和猪流行性腹泻提供辅助工具。
In order to develop a combined live vaccine capable of simultaneously preventing porcine epidemic diarrhea virus(PEDV)and porcine pseudorabies virus(PRV)infection,enhanced green fluorescent protein(EGFP)gene and immunodominant region of spike gene(S1)of PEDV Chinese epidemic strain were cloned into shuttle vector pTK which contained upstream and downstream sequences of the PRV thymidine kinase(TK)gene.The resulted recombinant plasmid was designed as pTK-EGFP and pTK-S1,respectively.The recombinant plasmid pTK-EGFP was co-transfected into Vero cells with PRV vaccine strain Bartha-K61 genomic DNA and the recombinant virus rPRV-EGFP was screened by selecting green fluorescent plaque and plaque purification.The recombinant plasmid pTK-S1 was co-transfected with rPRV-EGFP genomic DNA into Vero cells.The recombinant rPRV-S1 expressing PEDV S protein was obtained by reverse screening of EGFP-negative plaques and plaque purification.This study will provide additional means for more effective control of pseudorabies and porcine epidemic diarrhea.
In order to develop a combined live vaccine capable of simultaneously preventing porcine epidemic diarrhea virus(PEDV)and porcine pseudorabies virus(PRV)infection,enhanced green fluorescent protein(EGFP)gene and immunodominant region of spike gene(S1)of PEDV Chinese epidemic strain were cloned into shuttle vector pTK which contained upstream and downstream sequences of the PRV thymidine kinase(TK)gene.The resulted recombinant plasmid was designed as pTK-EGFP and pTK-S1,respectively.The recombinant plasmid pTK-EGFP was co-transfected into Vero cells with PRV vaccine strain Bartha-K61 genomic DNA and the recombinant virus rPRV-EGFP was screened by selecting green fluorescent plaque and plaque purification.The recombinant plasmid pTK-S1 was co-transfected with rPRV-EGFP genomic DNA into Vero cells.The recombinant rPRV-S1 expressing PEDV S protein was obtained by reverse screening of EGFP-negative plaques and plaque purification.This study will provide additional means for more effective control of pseudorabies and porcine epidemic diarrhea.
引文
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[18]PARK S J,SONG D S,HA G W,et al.Cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus DR13[J].Virus Genes,2007,35(1):55-64.
[19]SATO T,TAKEYAMA N,KATSUMATA A,et al.Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo[J].Virus Genes,2011,43(1):72-78.
[20]陈静,杨盼盼,雷喜梅,等.PEDV中国流行株和疫苗株S蛋白免疫反应性差异分析[J].中国兽医学报,2015,35(10):1573-1577.
[2]周如月,刘琪,甘振磊,等.1株猪流行性腹泻病毒S基因的克隆及序列分析[J].中国兽医学报,2017,37(12):2294-2299.
[3]STEVENSON G W,HOANG H,SCHWARTZ K J,et al.Emergence of porcine epidemic diarrhea virus in the United States:clinical signs,lesions,and viral genomic sequences[J].J Vet Diagn Invest,2013,25:649-654.
[4]王明月,鲁海冰,刘亚静,等.猪流行性腹泻病毒病S蛋白单抗制备及夹心ELISA检测PEDV方法的建立与应用[J].中国兽医学报,2017,37(11):2033-2037.
[5]CRUZ D J,KIM C J,SHIN H J.The GPRLQPY motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus[J].Virus Res,2008,132(1/2):192-196.
[6]LEE D K,CHA S Y,LEE C.The N-terminal region of the porcine epidemic diarrhea virus spike protein is important for the receptor binding[J].Korean J Microbiol Biotechnol,2011,39(2):140-145.
[7]丁光明,王彬,肖鹏,等.猪流行性腹泻病毒YT1401株分离鉴定及分子进化分析[J].中国兽医学报,2017,37(10):1841-1846.
[8]WANG X M,NIU B B,YAN H,et al.Genetic properties of endemic Chinese porcine epidemic diarrhea virus strains isolated since 2010[J].Arch Virol,2013,158(12):2487-2494.
[9]王伟,张双翔,金志强,等.猪流行性腹泻病毒RTLAMP检测方法的建立与应用[J].中国兽医学报,2017,37(7):1220-1224.
[10]付朋飞,乔涵,杨兴武,等.重组猪细小病毒VP2基因的猪伪狂犬病病毒rPRV-VP2株的构建及纯化[J].中国兽医学报,2016,36(9):1476-1483.
[11]付朋飞,乔涵,张宇,等.表达猪细小病毒VP2蛋白的猪伪狂犬病病毒rPRV-VP2株的生物学特性[J].中国兽医学报,2017,37(1):11-17.
[12]王林青,付朋飞,乔涵,等.表达猪细小病毒VP2蛋白和猪IL-18重组伪狂犬病毒的构建[J].中国兽医学报,2015,35(9):1422-1428.
[13]王建科,刘辉哲,林鹏,等.犬细小病毒新2b型分离株的分离鉴定[J].中国兽医学报,2016,36(5):734-738.
[14]赵武,肖少波,方六荣,等.共表达与牛疱疹病毒1型VP22融合的猪繁殖与呼吸综合征病毒E及M蛋白的重组伪狂犬病毒的构建及其免疫原性观察[J].畜牧兽医学报,2006(4):401-407.
[15]江云波,方六荣,肖少波,等.表达猪繁殖与呼吸综合征病毒修饰型GP5m蛋白的重组伪狂犬病毒的构建及其在小鼠的免疫反应[J].生物工程学报,2005(6):6-12.
[16]方六荣,肖少波,江云波,等.表达猪繁殖与呼吸综合征病毒(PRRSV)GP5的重组伪狂犬病毒TK-/gG-/GP5+的构建及其生物学特性初步探讨[J].病毒学报,2004(3):249-254.
[17]CHANG S H,BAE J L,KANG T J,et al.Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus[J].Mol Cells,2002,14(2):295-299.
[18]PARK S J,SONG D S,HA G W,et al.Cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus DR13[J].Virus Genes,2007,35(1):55-64.
[19]SATO T,TAKEYAMA N,KATSUMATA A,et al.Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo[J].Virus Genes,2011,43(1):72-78.
[20]陈静,杨盼盼,雷喜梅,等.PEDV中国流行株和疫苗株S蛋白免疫反应性差异分析[J].中国兽医学报,2015,35(10):1573-1577.