不同代次缺失meq基因的重组马立克病毒SC9-1株主要基因序列同源性分析
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摘要
分析缺失meq基因的重组马立克病病毒SC9-1原代、10代及40代主要基因序列的同源性。提取缺失meq基因的重组马立克病病毒SC9-1原代、10代及40代DNA,设计引物PCR扩增其与致瘤相关pp38基因、缺失meq基因后残余的FRT位点序列及囊膜蛋白基因gB、gC、gD、gE、gH、gI、gK,连接pMD18-T载体,转化DH5α感受态细胞,挑取阳性克隆进行测序,DNAStar软件对3代次各基因片段进行同源性比对。不同代次SC9-1病毒株的致瘤相关pp38基因、残留FRT位点序列及囊膜蛋白基因gB、gC、gD、gE、gH、gI、gK的核苷酸序列同源性均为100%,核苷酸序列差异性分析结果显示序列完全一致,无位点上的变化。缺失meq基因的重组马立克病病毒SC9-1在鸡胚成纤维细胞(CEF)上的传代过程中其致瘤相关pp38基因、残留FRT位点序列及囊膜蛋白基因gB、gC、gD、gE、gH、gI、gK没有发生变异,侧面反映了SC9-1在CEF细胞传代过程中具有很好的遗传稳定性。
To analyze the homology of the major gene sequences of the meq gene-deleted recombinant Marek's disease virus strain SC9-1in the original,the tenth and the forty generations,the DNA of the three different generations of the meq gene-deleted recombinant Marek's disease virus strain SC9-1was extracted,and the tumor associated gene pp38 and residual FRT sequence after the meq gene deleted,and envelope protein gene gB,gC,gD,gE,gH,gI,gK were amplified by PCR,then inserted into pMD18-T vector and transformed into DH5α,positive clones were sequenced and the three generations of each gene fragment were analyzed for homologous comparison by DNAStar software.Nucleotide sequence homology of the tumor associated gene pp38,residual FRT sequence and envelope protein gene gB,gC,gD,gE,gH,gI,gKin different generations of SC9-1strains was 100%,nucleotide sequence difference analysis showed that the sequence was completely consistent with each other and no changed in any sites.The tumor associated gene pp38,residual FRT sequence and envelope protein gene gB,gC,gD,gE,gH,gI,gK of the meq gene-deleted recombinant Marek's disease virus strain SC9-1have no sign of genetic mutation in the process of CEF cell passaging.It reflects that the SC9-1has good genetic stability during CEF cell passaging from a side.
To analyze the homology of the major gene sequences of the meq gene-deleted recombinant Marek's disease virus strain SC9-1in the original,the tenth and the forty generations,the DNA of the three different generations of the meq gene-deleted recombinant Marek's disease virus strain SC9-1was extracted,and the tumor associated gene pp38 and residual FRT sequence after the meq gene deleted,and envelope protein gene gB,gC,gD,gE,gH,gI,gK were amplified by PCR,then inserted into pMD18-T vector and transformed into DH5α,positive clones were sequenced and the three generations of each gene fragment were analyzed for homologous comparison by DNAStar software.Nucleotide sequence homology of the tumor associated gene pp38,residual FRT sequence and envelope protein gene gB,gC,gD,gE,gH,gI,gKin different generations of SC9-1strains was 100%,nucleotide sequence difference analysis showed that the sequence was completely consistent with each other and no changed in any sites.The tumor associated gene pp38,residual FRT sequence and envelope protein gene gB,gC,gD,gE,gH,gI,gK of the meq gene-deleted recombinant Marek's disease virus strain SC9-1have no sign of genetic mutation in the process of CEF cell passaging.It reflects that the SC9-1has good genetic stability during CEF cell passaging from a side.
引文
[1]BIGGS P M,PAYNE L N.Studies on Marek's disease.I.Experimental transmission[J].J Natl Cancer Inst,1967,39(2):267-280.
[2]左天荣,赵子轶,韦平,等.一株具有急性致瘤特性的马立克病病毒的分离与鉴定[J].病毒学报,2007,23(3):218-223.
[3]WITTER R L.Increased virulence of Marek's disease virus field isolates[J].Avian Dis,1997,41(1):149-163.
[4]SCHAT K A,XING Z.Specific and nonspecific immune responses to Marek's disease virus[J].Dev Comp Immunol,2000,24(2-3):201-221.
[5]REDDY S M,LUPIANI B,GIMENO M,et al.Rescue of a pathogenic Marek's disease virus with overlapping cosmid DNAs:use of a pp38 mutant to validate the technology for the study of gene function[J].Proc Natl Acad Sci U S A,2002,99(10):7054-7059.
[6]CUIX P,LEE L F,HUNT H D,et al.A Marek's disease virus vIL-8deletion mutant has attenuated virulence and confers protection against challenge with a very virulent plus strain[J].Avian Dis,2005,49(2):199-206.
[7]CUIX P,LEE L F,REED W M,et al.Marek's disease virus-encoded vIL-8gene is involved in early cytolytic infection but dispensable for establishment of latency[J].J Virol,2004,78(9):4753-4760.
[8]LUPIANI B,LEE L F,CUIX P,et al.Marek's disease virus-encoded Meq gene is involved in transformation of lymphocytes but is dispensable for replication[J].Proc Natl Acad Sci U S A,2004,101(32):11815-11820.
[9]SCHUMACHER D,TISCHER B K,FUCHS W,et al.Reconstitution of Marek's disease virus serotype 1(MDV-1)from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein Bnegative MDV-1 mutant[J].J Virol,2000,74(23):11088-11098.
[10]PETHERBRIDGE L,HOWES K,BAIGENT S J,et al.Replication-competent bacterial artificial chromosomes of Marek's disease virus:novel tools for generation of molecularly defined herpesv irus vaccines[J].J Virol,2003,77(16):8712-8718.
[11]LEE L F,LUPIANI B,SILVAR F,et al.Recombinant Marek's disease virus(MDV)lacking the Meq oncogene confers protection against challenge with a very virulent plus strain of MDV[J].Vaccine,2008,26(15):1887-1892.
[12]张志,崔治中.整合进禽反转录病毒基因组片段的鸡马立克病病毒重组野毒株的发现[J].中国科学,2004,34(4):317-324.
[13]许晓云,孙爱军,崔言顺,等.带有REV-LTR片段的马立克病病毒重组野毒株与超强毒株致病性和横向传播性比较[J].微生物学报,2009,49(4):540-543.
[14]崔治中.禽反转录病毒与DNA病毒间的基因重组及其流行病学意义[J].病毒学报,2006,22(2):150-154.
[15]孙爱军,PETHERBRIDGE L,ZHAO Y G,等.带有REV-LTR片段的MDV野毒株GX0101BAC克隆的构建及拯救病毒的致病性分析[J].科学通报,2009,54(11):1541-1546.
[16]苏帅,李延鹏,孙爱军,等.敲除meq的鸡马立克病毒强毒株对超强毒的免疫保护作用[J].微生物学报,2010,50(3):380-386.
[17]苏帅.重组马立克病毒及其突变株生物学活性的比较研究[D].山东泰安:山东农业大学,2013.
[18]CHURCHILL A E.Herpes-type virus isolated in cell culture from tumors of chickens with Marek's disease.I.Studies in cell culture[J].J Natl Cancer Inst,1968,41(4):939-950.
[19]施维松,刘长军,张艳萍,等.4株鸡马立克病病毒国内分离株Meq基因的克隆与序列分析[J].病毒学报,2008,24(2):117-125.
[20]付本懂,王鲁,韦旭斌.马立克病病毒致瘤相关基因的研究进展[J].中国兽医科学,2006,36(7):592-596.
[21]LEVY A M,IZUMIYA Y,BRUNOVSKIS P,et al.Characterization of the chromosomal binding sites and dimerization partners of the viral oncoprotein Meq in Marek's disease virus-transformed T cells[J].J Virol,2003,77(23):12841-12851.
[22]AJITHDOSS D K,REDDY S M,SUCHODOLSKI P F,et al.In vitro characterization of the Meq proteins of Marek's disease virus vaccine strain CVI988[J].Virus Res,2009,142(1-2):57-67.
[23]韦平,崔治中,LEE L F.马立克病病毒meq基因功能研究[J].广西科学,2003,10(1):52-62.
[24]GIMENO I M,WITTER R L,HUNT H D,et al.The pp38gene of Marek's disease virus(MDV)is necessary for cytolytic infection of B cells and maintenance of the transformed state but not for cytolytic infection of the feather follicle epithelium and horizontal spread of MDV[J].J Virol,2005,79(7):4545-4549.
[25]张纪元,丁家波,崔治中,等.不同致病型马立克病病毒1.8kb基因家族序列比较[J].中国病毒学,2005,20(3):277-282.
[26]韦平.马立克病病毒致瘤基因meq功能研究的概述[J].广西畜牧兽医,2003,19(2):51-52.
[27]崔治中,张志,秦爱建,等.马立克病病毒38ku磷蛋白H19-和T65-抗原表位分析及鸡对该抗原表位点突变病毒的免疫反应[J].中国科学C辑(生命科学),2003,33(3):263-272.
[28]CUI Z Z,QIN A J,LEE L F.Expression and processing of Marek's disease virus pp38gene insert cells and immunological characteriation of the gene product[A].Proceedings of 19th World's Poultry Congress[C].Dutch Branch:World's Poultry Science Association,1992,123-126.
[29]马诚泰.不同启动子和插入位点表达AIV-H9 HA抗原的重组马立克病病毒的构建和比较研究[D].山东泰安:山东农业大学,2014.
[30]张振杰.共表达不同外源基因的重组弱毒Ι型马立克病毒的构建和生物学特性比较[D].山东泰安:山东农业大学,2014.
[31]SU S,CUI N,ZHOU Y,et al.A recombinant field strain of Marek's disease(MD)virus with reticuloendotheliosis virus long terminal repeat insert lacking the meq gene as a vaccine against MD[J].Vaccine,2015,33(5):596-603.
[2]左天荣,赵子轶,韦平,等.一株具有急性致瘤特性的马立克病病毒的分离与鉴定[J].病毒学报,2007,23(3):218-223.
[3]WITTER R L.Increased virulence of Marek's disease virus field isolates[J].Avian Dis,1997,41(1):149-163.
[4]SCHAT K A,XING Z.Specific and nonspecific immune responses to Marek's disease virus[J].Dev Comp Immunol,2000,24(2-3):201-221.
[5]REDDY S M,LUPIANI B,GIMENO M,et al.Rescue of a pathogenic Marek's disease virus with overlapping cosmid DNAs:use of a pp38 mutant to validate the technology for the study of gene function[J].Proc Natl Acad Sci U S A,2002,99(10):7054-7059.
[6]CUIX P,LEE L F,HUNT H D,et al.A Marek's disease virus vIL-8deletion mutant has attenuated virulence and confers protection against challenge with a very virulent plus strain[J].Avian Dis,2005,49(2):199-206.
[7]CUIX P,LEE L F,REED W M,et al.Marek's disease virus-encoded vIL-8gene is involved in early cytolytic infection but dispensable for establishment of latency[J].J Virol,2004,78(9):4753-4760.
[8]LUPIANI B,LEE L F,CUIX P,et al.Marek's disease virus-encoded Meq gene is involved in transformation of lymphocytes but is dispensable for replication[J].Proc Natl Acad Sci U S A,2004,101(32):11815-11820.
[9]SCHUMACHER D,TISCHER B K,FUCHS W,et al.Reconstitution of Marek's disease virus serotype 1(MDV-1)from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein Bnegative MDV-1 mutant[J].J Virol,2000,74(23):11088-11098.
[10]PETHERBRIDGE L,HOWES K,BAIGENT S J,et al.Replication-competent bacterial artificial chromosomes of Marek's disease virus:novel tools for generation of molecularly defined herpesv irus vaccines[J].J Virol,2003,77(16):8712-8718.
[11]LEE L F,LUPIANI B,SILVAR F,et al.Recombinant Marek's disease virus(MDV)lacking the Meq oncogene confers protection against challenge with a very virulent plus strain of MDV[J].Vaccine,2008,26(15):1887-1892.
[12]张志,崔治中.整合进禽反转录病毒基因组片段的鸡马立克病病毒重组野毒株的发现[J].中国科学,2004,34(4):317-324.
[13]许晓云,孙爱军,崔言顺,等.带有REV-LTR片段的马立克病病毒重组野毒株与超强毒株致病性和横向传播性比较[J].微生物学报,2009,49(4):540-543.
[14]崔治中.禽反转录病毒与DNA病毒间的基因重组及其流行病学意义[J].病毒学报,2006,22(2):150-154.
[15]孙爱军,PETHERBRIDGE L,ZHAO Y G,等.带有REV-LTR片段的MDV野毒株GX0101BAC克隆的构建及拯救病毒的致病性分析[J].科学通报,2009,54(11):1541-1546.
[16]苏帅,李延鹏,孙爱军,等.敲除meq的鸡马立克病毒强毒株对超强毒的免疫保护作用[J].微生物学报,2010,50(3):380-386.
[17]苏帅.重组马立克病毒及其突变株生物学活性的比较研究[D].山东泰安:山东农业大学,2013.
[18]CHURCHILL A E.Herpes-type virus isolated in cell culture from tumors of chickens with Marek's disease.I.Studies in cell culture[J].J Natl Cancer Inst,1968,41(4):939-950.
[19]施维松,刘长军,张艳萍,等.4株鸡马立克病病毒国内分离株Meq基因的克隆与序列分析[J].病毒学报,2008,24(2):117-125.
[20]付本懂,王鲁,韦旭斌.马立克病病毒致瘤相关基因的研究进展[J].中国兽医科学,2006,36(7):592-596.
[21]LEVY A M,IZUMIYA Y,BRUNOVSKIS P,et al.Characterization of the chromosomal binding sites and dimerization partners of the viral oncoprotein Meq in Marek's disease virus-transformed T cells[J].J Virol,2003,77(23):12841-12851.
[22]AJITHDOSS D K,REDDY S M,SUCHODOLSKI P F,et al.In vitro characterization of the Meq proteins of Marek's disease virus vaccine strain CVI988[J].Virus Res,2009,142(1-2):57-67.
[23]韦平,崔治中,LEE L F.马立克病病毒meq基因功能研究[J].广西科学,2003,10(1):52-62.
[24]GIMENO I M,WITTER R L,HUNT H D,et al.The pp38gene of Marek's disease virus(MDV)is necessary for cytolytic infection of B cells and maintenance of the transformed state but not for cytolytic infection of the feather follicle epithelium and horizontal spread of MDV[J].J Virol,2005,79(7):4545-4549.
[25]张纪元,丁家波,崔治中,等.不同致病型马立克病病毒1.8kb基因家族序列比较[J].中国病毒学,2005,20(3):277-282.
[26]韦平.马立克病病毒致瘤基因meq功能研究的概述[J].广西畜牧兽医,2003,19(2):51-52.
[27]崔治中,张志,秦爱建,等.马立克病病毒38ku磷蛋白H19-和T65-抗原表位分析及鸡对该抗原表位点突变病毒的免疫反应[J].中国科学C辑(生命科学),2003,33(3):263-272.
[28]CUI Z Z,QIN A J,LEE L F.Expression and processing of Marek's disease virus pp38gene insert cells and immunological characteriation of the gene product[A].Proceedings of 19th World's Poultry Congress[C].Dutch Branch:World's Poultry Science Association,1992,123-126.
[29]马诚泰.不同启动子和插入位点表达AIV-H9 HA抗原的重组马立克病病毒的构建和比较研究[D].山东泰安:山东农业大学,2014.
[30]张振杰.共表达不同外源基因的重组弱毒Ι型马立克病毒的构建和生物学特性比较[D].山东泰安:山东农业大学,2014.
[31]SU S,CUI N,ZHOU Y,et al.A recombinant field strain of Marek's disease(MD)virus with reticuloendotheliosis virus long terminal repeat insert lacking the meq gene as a vaccine against MD[J].Vaccine,2015,33(5):596-603.