马疱疹病毒1型主要毒力基因分析及TK基因缺失载体的构建
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摘要
为了解新疆马疱疹病毒1型(EHV-1)主要毒力基因遗传进化情况并构建TK基因缺失株,本研究以EHV-1XJ2015株DNA为模板,对其主要毒力基因TK、gI和gE全长进行克隆、测序及生物信息学分析,并扩增TK基因左右重组臂TKL和TKR,构建质粒pUC-TKLR,将扩增后的增强绿色荧光蛋白(EGFP,含有CMV+polyA)插入pUC-TKLR质粒,构建TK基因缺失打靶质粒。TK、gI和gE基因同源性分析结果显示,XJ2015株与国外EHV-1分离株TK、gI和gE基因同源性均较高,分别为99.8%~100.0%、99.6%~100.0%和99.9%~100.0%;与EHV-3分离株同源性均最低,分别为72.9%、59.4%和62.1%;遗传进化分析显示,3个基因均与国外EHV-1同属于一个遗传进化分支,与EHV-9和EHV-4进化关系较近,但与EHV-3进化关系较远,表明XJ2015毒株与国外EHV-1毒株TK、gI、gE基因核苷酸上差异不明显,没有明显的地域性特征,功能基因保守且进化缓慢,同源基因功能相同或相近;经PCR扩增、酶切、测序及转染鉴定,本试验成功构建了用于TK基因缺失的打靶质粒pUC-TKLR-EGFP。通过对EHV-1主要毒力基因的分析及TK基因缺失打靶载体的构建,为新疆地区马鼻肺炎流行病学调查分析、TK基因缺失株的构建提供理论依据。
In order to understand the genetic evolution of the major virulence genes of equine herpesvirus 1(EHV-1)of Xinjiang(XJ2015 strain)and construct TK gene-deleted strain,we cloned,sequenced and bioinformatics analyzed the full-length of TK,gI and gEgenes of XJ2015 strain in this study.The DNA of the XJ2015 strain was used as a template,the TKgene recombination arms TKL and TKR were amplified to construct plasmid pUC-TKLR,and then inserted the amplified EGFP(CMV+polyA)into the pUC-TKLR plasmid.The results showed that it had higher homology between TK,gI and gE genes of XJ2015 strain and foreign EHV-1 isolates,which were 99.8%to 100.0%,99.6%to 100.0% and 99.9%to 100.0%,respectively,and had the lowest homology with foreign EHV-3 isolate,which were 72.9%,59.4%and 62.1%,respec-tively;Genetic evolutionary analysis showed that all three genes were genetically related to foreign EHV-1 isolates,the evolutionary relationship with EHV-3 was far away,but it was closely related to EHV-9 and EHV-4.There were no significant difference,no obvious regional features,the functional genes were conserved,the evolution was slow and homologous genes had the same or similar functions of TK,gI and gEgenes of XJ2015 strain between foreign EHV-1 strains.After enzyme digestion,sequencing and transfection,the plasmid pUC-TKLR-EGFP for TK gene deletion targeting was successfully constructed.Through the analysis of EHV-1 main virulence genes and the construction of TK gene deletion targeting vector,it provided a theoretical basis for the epidemiological investigation and analysis of the TKgene deletion strain in Xinjiang.
In order to understand the genetic evolution of the major virulence genes of equine herpesvirus 1(EHV-1)of Xinjiang(XJ2015 strain)and construct TK gene-deleted strain,we cloned,sequenced and bioinformatics analyzed the full-length of TK,gI and gEgenes of XJ2015 strain in this study.The DNA of the XJ2015 strain was used as a template,the TKgene recombination arms TKL and TKR were amplified to construct plasmid pUC-TKLR,and then inserted the amplified EGFP(CMV+polyA)into the pUC-TKLR plasmid.The results showed that it had higher homology between TK,gI and gE genes of XJ2015 strain and foreign EHV-1 isolates,which were 99.8%to 100.0%,99.6%to 100.0% and 99.9%to 100.0%,respectively,and had the lowest homology with foreign EHV-3 isolate,which were 72.9%,59.4%and 62.1%,respec-tively;Genetic evolutionary analysis showed that all three genes were genetically related to foreign EHV-1 isolates,the evolutionary relationship with EHV-3 was far away,but it was closely related to EHV-9 and EHV-4.There were no significant difference,no obvious regional features,the functional genes were conserved,the evolution was slow and homologous genes had the same or similar functions of TK,gI and gEgenes of XJ2015 strain between foreign EHV-1 strains.After enzyme digestion,sequencing and transfection,the plasmid pUC-TKLR-EGFP for TK gene deletion targeting was successfully constructed.Through the analysis of EHV-1 main virulence genes and the construction of TK gene deletion targeting vector,it provided a theoretical basis for the epidemiological investigation and analysis of the TKgene deletion strain in Xinjiang.
引文
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[14] MATSUMURA T,KONDO T,SUGITA S,et al.An equine herpesvirus type 1recombinant with a deletion in the gE and gI genes is avirulent in young horses[J].Virology,1998,242(1):68-79.
[15]杨永龙,刘建华,宋焕堂,等.新疆伊犁地区马疱疹病毒1型的分离与鉴定[J].中国预防兽医学报,2016,38(7):550-553.YANG Y L,LIU J H,SONG H T,et al.Isolation and identification of equine herpesvirus 1in Xinjiang[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(7):550-553.(in Chinese)
[16] ABDELGAWAD A,DAMIANI A,HO S Y W,et al.Zebra alpha herpesviruses(EHV-1and EHV-9):Genetic diversity,latency and co-infections[J].Viruses,2016,8(9):E262.
[17]葛菡,程安春,汪铭书,等.疱疹病毒TK基因的研究进展[J].中国兽医科学,2008,38(1):86-90.GE H,CHENG A C,WANG M S,et al.Research advance in thymidine kinase gene of herpesvirus[J].Chinese Veterinary Science,2008,38(1):86-90.(in Chinese)
[18] FUJIWAKI R,HATA K,NAKAYAMA K,et al.Thymidine kinase in epithelial ovarian cancer:Relationship with the other pyrimidine pathway enzymes[J].International Journal of Cancer,2002,99(3):328-335.
[19] FRAMPTON A R,SMITH P M,ZHANG Y,et al.Meningoencephalitis in mice infected with an equineherpesvirus 1strain KyA recombinant expressing glycoprotein I and glycoprotein E[J].Virus Genes,2004,29(1):9-17.
[20] MARSHALL K R,SUN Y,BROWN S M,et al.An equine herpesvirus-1gene 71deletant is attenuated and elicits a protective immune response in mice[J].Virology,1997,231(1):20-27.
[21] GOODMAN L B,LOREGIAN A,PERKINS G A,et al.A point mutation in aherpesvirus polymerase determines neuropathogenicity[J].PLoS Pathogens,2007,3(11):e160.
[22] GR V D W,GOUPIL R,WISHON C,et al.A singlenucleotide polymorphism in a herpesvirus DNA polymerase is sufficient to cause lethal neurological disease[J].Journal of Infectious Diseases,2009,200(1):20-25.
[23]范斌,王燕,阿玛古丽·朱马汉,等.新疆伊犁地区马疱疹病毒1型ORF30基因序列分析[J].中国畜牧兽医,2018,45(1):65-70.FAN B,WANG Y,ZHUMAHAN·A,et al.Sequence analysis of ORF30gene of equid herpesvirus typeⅠin Ili of Xinjiang[J].China Animal Husbandry&Veterinary Medicine,2018,45(1):65-70.(in Chinese)
[2] MA G,AZAB W,OSTERRIEDER N.Equine herpesviruses type 1(EHV-1)and 4(EHV-4)——Masters of co-evolution and a constant threat to equids and beyond[J].Veterinary Microbiology,2013,167(1-2):123-134.
[3] SCHRENZEL M D,TUCKER T A,DONOVAN T A,et al.New hosts for equine herpesvirus 9[J].Emerging Infectious Diseases,2008,14(10):1616-1619.
[4] SPIESSCHAERT B,OSTERRIEDER N,AZAB W.Comparative analysis of glycoprotein B(gB)of equine herpesvirus type 1and type 4(EHV-1and EHV-4)in cellular tropism and cell-to-cell transmission[J].Viruses,2015,7(2):522-542.
[5] TEWARI D,GIBSON J S,SLATER J D,et al.Modulation of the serological response of specific pathogenfree(EHV-free)foals to EHV-1by previous infection with EHV-4or a TK-deletion mutant of EHV-1[J].Archives of Virology,1993,132(1-2):101-120.
[6] HUANG C Y,YAO H W,WANG L C,et al.Thymidine kinase-negative herpes simplex virus 1can efficiently establish persistent infection in neural tissues of nude mice[J].Journal of Virology,2017,91(4):1-10.
[7] BERARDUCCI B,RAJAMANI J,REICHELT M,et al.Deletion of the first cysteine-rich region of the varicella-zoster virus glycoprotein E ectodomain abolishes the gE and gI interaction and differentially affects cell-cell spread and viral entry[J].Journal of Virology,2009,83(1):228-240.
[8] ANDOH K,TAKASUGI M,MAHMOUD H Y A H,et al.Identification of a major immunogenic region of equine herpesvirus-1glycoprotein E and its application to enzyme-linked immunosorbentassay[J].Veterinary Microbiology,2013,164(1-2):18-26.
[9] ALMUBARAK A,SIMON J,COATS C,et al.Glycoprotein E(gE)specified by bovine herpesvirus type 5(BHV-5)enables trans-neuronal virus spread and neurovirulence without being a structural component of enveloped virions[J].Virology,2007,365(2):398-409.
[10] TSUJIMURA K,YAMANAKA T,KONDO T,et al.Pathogenicity and immunogenicity of equine herpesvirus type 1mutants defective in either gIor gEgene in murine and hamster models[J].Journal of Veterinary Medical Science,2006,68(10):1029-1038.
[11] GU Z,DONG J,WANG J,et al.A novel inactivated gE/gI deleted pseudorabies virus(PRV)vaccine completely protects pigs from an emerged variant PRV challenge[J].Virus Research,2015,195:57-63.
[12] HBNER S O,OLIVEIRA A P,FRANCO A C,et al.Experimental infection of calves with a gI,gE,US9negative bovine herpesvirus type 5[J].Comparative Immunology Microbiology&Infectious Diseases,2005,28(3):187-196.
[13] KRUGER J M,SUSSMAN M D,MAES R K.Glycoproteins gI and gE of feline herpesvirus-1are virulence genes:Safety and efficacy of a gI-gE deletion mutant in the natural host[J].Virology,1996,220(2):299-308.
[14] MATSUMURA T,KONDO T,SUGITA S,et al.An equine herpesvirus type 1recombinant with a deletion in the gE and gI genes is avirulent in young horses[J].Virology,1998,242(1):68-79.
[15]杨永龙,刘建华,宋焕堂,等.新疆伊犁地区马疱疹病毒1型的分离与鉴定[J].中国预防兽医学报,2016,38(7):550-553.YANG Y L,LIU J H,SONG H T,et al.Isolation and identification of equine herpesvirus 1in Xinjiang[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(7):550-553.(in Chinese)
[16] ABDELGAWAD A,DAMIANI A,HO S Y W,et al.Zebra alpha herpesviruses(EHV-1and EHV-9):Genetic diversity,latency and co-infections[J].Viruses,2016,8(9):E262.
[17]葛菡,程安春,汪铭书,等.疱疹病毒TK基因的研究进展[J].中国兽医科学,2008,38(1):86-90.GE H,CHENG A C,WANG M S,et al.Research advance in thymidine kinase gene of herpesvirus[J].Chinese Veterinary Science,2008,38(1):86-90.(in Chinese)
[18] FUJIWAKI R,HATA K,NAKAYAMA K,et al.Thymidine kinase in epithelial ovarian cancer:Relationship with the other pyrimidine pathway enzymes[J].International Journal of Cancer,2002,99(3):328-335.
[19] FRAMPTON A R,SMITH P M,ZHANG Y,et al.Meningoencephalitis in mice infected with an equineherpesvirus 1strain KyA recombinant expressing glycoprotein I and glycoprotein E[J].Virus Genes,2004,29(1):9-17.
[20] MARSHALL K R,SUN Y,BROWN S M,et al.An equine herpesvirus-1gene 71deletant is attenuated and elicits a protective immune response in mice[J].Virology,1997,231(1):20-27.
[21] GOODMAN L B,LOREGIAN A,PERKINS G A,et al.A point mutation in aherpesvirus polymerase determines neuropathogenicity[J].PLoS Pathogens,2007,3(11):e160.
[22] GR V D W,GOUPIL R,WISHON C,et al.A singlenucleotide polymorphism in a herpesvirus DNA polymerase is sufficient to cause lethal neurological disease[J].Journal of Infectious Diseases,2009,200(1):20-25.
[23]范斌,王燕,阿玛古丽·朱马汉,等.新疆伊犁地区马疱疹病毒1型ORF30基因序列分析[J].中国畜牧兽医,2018,45(1):65-70.FAN B,WANG Y,ZHUMAHAN·A,et al.Sequence analysis of ORF30gene of equid herpesvirus typeⅠin Ili of Xinjiang[J].China Animal Husbandry&Veterinary Medicine,2018,45(1):65-70.(in Chinese)