猪伪狂犬病毒EP0基因生物信息学分析及TK蛋白的同源模建
摘要
将四川农业大学动物生物技术中心保存FA株、疫苗株783株、Bartha株和SA215株,四川野毒株SN株、SS株、SQ株、SL株和SCZ株,以及由韶关学院英东生物工程学院娄高明惠赠的北京株BJ株、广东株DG、湖北株HS株共计12株伪狂犬病病毒株,应用PCR技术扩增得到了PRV完整的EP0基因的片段12条,同时以FA株为材料,PCR扩增得到PRV完整的TK基因片段。回收PCR产物,成功连接转化到感受态细胞E.coli DH_(5a)中。通过菌落PCR鉴定正确后,送上海英俊生物公司进行测序。
将所有的12条序列连同GenBank中登录的3条EP0基因全序列共15条基因序列,使用生物软件对它们的基因序列的同源性、突变区域的定位、遗传进化关系、CpG岛分析、密码子偏爱性,氨基酸序列的同源性、蛋白质亲水性、抗原表位分析、二级结构等生物信息学的内容进行预测和分析。同时,对猪伪狂犬病病毒FA株、疱疹病毒属的单纯疱疹病毒I型、牛疱疹病毒I型、马疱疹病毒I型、马雷克(氏)病疱疹病毒I型以及水痘带状疱疹病毒的早期蛋白的同源性和进化关系的分析。结果表明:PRV-EP0基因的开放阅读框的核苷酸长度在1230～1233bp,氨基酸长度在409～410个之间,核酸同源性为97.6%～99.9%,氨基酸的同源性在96.6%～100%之间,在EP0基因核酸序列最大的差异是在684～688位之间存在一个AGG缺失突变区,不同毒株基因均对GC极为偏爱。毒株间基因蛋白质亲水性、抗原表位和蛋白质二级结构预测等内容的分析结果十分相似,说明PRV-EP0基因具有很高的保守性。疱疹病毒属间的核苷酸同源性在1.4%～41.4%之间,氨基酸同源性在5.9%～42.0%之间。
以PRVFA为材料,克隆测序胸苷激酶(TK)基因,用FASTA程序在蛋白质结构数据库(Brookhaven protein data bank,PDB)中搜索同源蛋白,通过同源模建和分子动力学模拟建立胸苷激酶的3D模型,模型的可靠性经Ramachandran图和Profile-3D图验证。采用InsightⅡ/Binding site和Delphi方法准确定位了胸苷激酶的活性位点,在此基础上,设计出胸苷激酶抑制小分子(N-苯基-N'-甲基脲),通过柔性分子对接方法阐明了胸苷激酶抑制剂与靶酶活性位点的相互作用模式。
该研究从病原、分子水平、生物信息等方面对PRV-EP0基因进行了研究,摸清该基因的遗传背景,为以后更进一步研究该基因对PRV潜伏感染状态的建立、维持和再激活的作用。同时同源模建TK蛋白的3D模型,预测该蛋白的活性区域,同时设计了配体,讨论了配体对TK蛋白抑制作用。为研究对猪伪狂犬病新的治疗方案(实现先治疗后预防的新的防制方案)奠定基础。
There are 12 pseudorabies virus strain,SS、SQ and FA、783strain、Bartha、(purchased from China Institue of Veterinary Drug Control) and SA215(purchased from China Animal Husbandry Industry CO.LTD)and SN、SL、SCZ(Isolated by Xu Zhiwen associate professor in Sichuan Agriculture University Animal biotechnology center in2000) and BJ、DG、HS (gived by Lou Gaoming professor in Shaoguan University College of Yingdong Biorngineering).Designing a pair of primer according to PRV EP0 gene,we received 12 EP0 complete gene by PCR.Using pseudorabies virus strain FA,We received a TK gene by PCR.The TK gene was transformed into E.coli DH5αwith TaKaRa's kit to retrieve gel and to ligate.After identified by COL-PCR and RE,they were sequenced by TaKaRa and GeneCore.
12 sequenced EP0 genes of different PRV strains,as well as 3 EP0 genes which downloaded from GenBank were analyzed by bioinformatics software,the analysis and prediction of these genes including nucleotide homology,codons bias,location of mutation area,phylogenetic tree,amino acids homology,protein hydrophilicity and epitope. Meanwhile,the nucleotide homology,amino acids homology and the protein domain of EP0 genes of pseudorabies virus strain FA,Herpesvirus I,BHV I,EHV I,MDV I and varicella-herpes zoster virus were analysed.The results showed that the ORF length of PRV-EP0 gene is 1230-1233nt,amino acids length is 409-410,nucleotide homology is 97.6%-99.9%,and amino acids homology is 96.6%-100%.One hypermutation replicated plot is located in 684～688nt.Different strains were prefer to GC basi-.The results of different strains of enzyme site,protein hydrophilicity and epitope were very similar.The nucleotide and amino acids homology of Herpesvirus was 1.4%～41.4%and 5.9%～42% respectively.
With PRV FA as subject,TK gene has been cloned and sequence analysed.We searched for homology protein through FASTA procedure on Brookhaven protein data bank(PDB) and then established its 3D model by the method of homology model establishment and molecular dynamic analysis.Validity of such model was verified by Ramachandran and Profile-3D procedure.The acive site of TK had been accurately located with InsightⅡ/Binding site and Delphi procedures.Based on this 3D profile,we designed inhibitor(N-phenyl-N'- methylurea) of TK,and clarified the interactive mode between the active site and its inhibitor.
The objective of this study was to study the background of PRV-EP0 gene based on molecular level and bioinformation analysis and provide a base for further study of effects of TK gene on latent infection of PRV.With 3D model,we have predicted the active domain of TK protein and designed its ligand and discussed the inhibitory effect between the ligand and protein TK.It is possible that a new therapeutic procedure for Pseudorabies could be presented.
将所有的12条序列连同GenBank中登录的3条EP0基因全序列共15条基因序列,使用生物软件对它们的基因序列的同源性、突变区域的定位、遗传进化关系、CpG岛分析、密码子偏爱性,氨基酸序列的同源性、蛋白质亲水性、抗原表位分析、二级结构等生物信息学的内容进行预测和分析。同时,对猪伪狂犬病病毒FA株、疱疹病毒属的单纯疱疹病毒I型、牛疱疹病毒I型、马疱疹病毒I型、马雷克(氏)病疱疹病毒I型以及水痘带状疱疹病毒的早期蛋白的同源性和进化关系的分析。结果表明:PRV-EP0基因的开放阅读框的核苷酸长度在1230～1233bp,氨基酸长度在409～410个之间,核酸同源性为97.6%～99.9%,氨基酸的同源性在96.6%～100%之间,在EP0基因核酸序列最大的差异是在684～688位之间存在一个AGG缺失突变区,不同毒株基因均对GC极为偏爱。毒株间基因蛋白质亲水性、抗原表位和蛋白质二级结构预测等内容的分析结果十分相似,说明PRV-EP0基因具有很高的保守性。疱疹病毒属间的核苷酸同源性在1.4%～41.4%之间,氨基酸同源性在5.9%～42.0%之间。
以PRVFA为材料,克隆测序胸苷激酶(TK)基因,用FASTA程序在蛋白质结构数据库(Brookhaven protein data bank,PDB)中搜索同源蛋白,通过同源模建和分子动力学模拟建立胸苷激酶的3D模型,模型的可靠性经Ramachandran图和Profile-3D图验证。采用InsightⅡ/Binding site和Delphi方法准确定位了胸苷激酶的活性位点,在此基础上,设计出胸苷激酶抑制小分子(N-苯基-N'-甲基脲),通过柔性分子对接方法阐明了胸苷激酶抑制剂与靶酶活性位点的相互作用模式。
该研究从病原、分子水平、生物信息等方面对PRV-EP0基因进行了研究,摸清该基因的遗传背景,为以后更进一步研究该基因对PRV潜伏感染状态的建立、维持和再激活的作用。同时同源模建TK蛋白的3D模型,预测该蛋白的活性区域,同时设计了配体,讨论了配体对TK蛋白抑制作用。为研究对猪伪狂犬病新的治疗方案(实现先治疗后预防的新的防制方案)奠定基础。
There are 12 pseudorabies virus strain,SS、SQ and FA、783strain、Bartha、(purchased from China Institue of Veterinary Drug Control) and SA215(purchased from China Animal Husbandry Industry CO.LTD)and SN、SL、SCZ(Isolated by Xu Zhiwen associate professor in Sichuan Agriculture University Animal biotechnology center in2000) and BJ、DG、HS (gived by Lou Gaoming professor in Shaoguan University College of Yingdong Biorngineering).Designing a pair of primer according to PRV EP0 gene,we received 12 EP0 complete gene by PCR.Using pseudorabies virus strain FA,We received a TK gene by PCR.The TK gene was transformed into E.coli DH5αwith TaKaRa's kit to retrieve gel and to ligate.After identified by COL-PCR and RE,they were sequenced by TaKaRa and GeneCore.
12 sequenced EP0 genes of different PRV strains,as well as 3 EP0 genes which downloaded from GenBank were analyzed by bioinformatics software,the analysis and prediction of these genes including nucleotide homology,codons bias,location of mutation area,phylogenetic tree,amino acids homology,protein hydrophilicity and epitope. Meanwhile,the nucleotide homology,amino acids homology and the protein domain of EP0 genes of pseudorabies virus strain FA,Herpesvirus I,BHV I,EHV I,MDV I and varicella-herpes zoster virus were analysed.The results showed that the ORF length of PRV-EP0 gene is 1230-1233nt,amino acids length is 409-410,nucleotide homology is 97.6%-99.9%,and amino acids homology is 96.6%-100%.One hypermutation replicated plot is located in 684～688nt.Different strains were prefer to GC basi-.The results of different strains of enzyme site,protein hydrophilicity and epitope were very similar.The nucleotide and amino acids homology of Herpesvirus was 1.4%～41.4%and 5.9%～42% respectively.
With PRV FA as subject,TK gene has been cloned and sequence analysed.We searched for homology protein through FASTA procedure on Brookhaven protein data bank(PDB) and then established its 3D model by the method of homology model establishment and molecular dynamic analysis.Validity of such model was verified by Ramachandran and Profile-3D procedure.The acive site of TK had been accurately located with InsightⅡ/Binding site and Delphi procedures.Based on this 3D profile,we designed inhibitor(N-phenyl-N'- methylurea) of TK,and clarified the interactive mode between the active site and its inhibitor.
The objective of this study was to study the background of PRV-EP0 gene based on molecular level and bioinformation analysis and provide a base for further study of effects of TK gene on latent infection of PRV.With 3D model,we have predicted the active domain of TK protein and designed its ligand and discussed the inhibitory effect between the ligand and protein TK.It is possible that a new therapeutic procedure for Pseudorabies could be presented.
引文
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