山羊痘病毒TK基因功能缺失对其增殖的影响
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摘要
为了研究山羊痘病毒TK基因功能缺失对其增殖的影响,同源重组构建TK基因缺失型重组山羊痘病毒,检测其增殖特性。试验在通用转移质粒pTKfpgigp内插入不同长度的X1~X4片段,构建了4个重组转移质粒;重组质粒转染已感染山羊痘病毒的羔羊睾丸细胞,经同源重组产生4株TK基因缺失型重组山羊痘病毒rGPV/tk-,其TK基因内分别插入了长度约为2 800、4 000、5 200、7 700bp的外源DNA片段;筛选纯化重组毒株,测定rGPV/tk-病毒滴度。结果显示,TK基因失活后,rGPV/tk-病毒滴度明显降低;随着插入DNA片段的延长,重组毒株的病毒滴度下降程度更加明显。研究表明,TK基因功能的缺失对山羊痘病毒的增殖具有一定的抑制作用,并与外源DNA的长度具有相关性。
In this study,the proliferation of goat pox virus(GPV)was researched using the recombinant GPV of TK gene insertion inactivation.Different length DNA fragments X1,X2,X3 and X4were inserted into the transfer plasmid pTKfpgigp to constructed the recombinant transfer plasmids and these plasmids were transfected into lamb testis cells infected GPV.Recombinant GPVs of TK gene inactivation by inserting foreign DNA fragment of 2 800,4 000,5 200and7 700 bp,respectively,were generated via homologous recombination.Four recombinant GPVs(rGPV/tk-)were obtained by selective culture and the virus titer were mensurated by 50% tissue culture infective dose.The results showed that the rGPV/tk-virus titers were decreased obviosuly which was comparable with the wild type GPV AV41.Furthermore,the virus titer of the recombinant virus was decreased more obviously with the extension of the foreign DNA fragment.The result indicated that the proliferation of the GPV was depressed when GPV TK gene was deficient.
In this study,the proliferation of goat pox virus(GPV)was researched using the recombinant GPV of TK gene insertion inactivation.Different length DNA fragments X1,X2,X3 and X4were inserted into the transfer plasmid pTKfpgigp to constructed the recombinant transfer plasmids and these plasmids were transfected into lamb testis cells infected GPV.Recombinant GPVs of TK gene inactivation by inserting foreign DNA fragment of 2 800,4 000,5 200and7 700 bp,respectively,were generated via homologous recombination.Four recombinant GPVs(rGPV/tk-)were obtained by selective culture and the virus titer were mensurated by 50% tissue culture infective dose.The results showed that the rGPV/tk-virus titers were decreased obviosuly which was comparable with the wild type GPV AV41.Furthermore,the virus titer of the recombinant virus was decreased more obviously with the extension of the foreign DNA fragment.The result indicated that the proliferation of the GPV was depressed when GPV TK gene was deficient.
引文
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[15]Sebastian S,Gilbert S C.Recombinant modified vaccinia virus Ankara-based malaria vaccines[J].Expert Rev Vaccines,2016,15(1):91-103.
[16]Caufour P,Rufael T,Lamien C E,et al.Protective efficacy of a single immunization with capripoxvirusvectored recombinant peste des petits ruminants vaccines in presence of pre-existing immunity[J].Vaccine,2014,32(30):3772-3779.
[17]Gari G,Abie G,Gizaw D,et al.Evaluation of the safety,immunogenicity and efficacy of three capripoxvirus vaccine strains against lumpy skin disease virus[J].Vaccine,2015,33(28):3256-3261.
[18]Backes S,Jager C,Dembek C J,et al.Proteinprime/modified vaccinia virus Ankara vector-boost vaccination overcomes tolerance in high-antigenemic HBV-transgenic mice[J].Vaccine,2016,34(7):923-932.
[19]郑敏,梁晟,郑彦梓,等.TK基因重组缺陷山羊痘病毒构建及其生物学特性鉴定[J].南方农业学报,2013,44(9):1552-1557.
[20]Boshra H,Cao J,Babiuk S.Generation of recombinant capripoxvirus vectors for vaccines and gene knockout function studies[J].Methods Mol Biol,2016,1349:151-161.
[21]Gelaye E,Lamien C E,Silber R,et al.Development of a cost-effective method for capripoxvirus genotyping using snapback primer and dsDNA intercalating dye[J].PLoS One,2013,8(10):e75971.
[2]Qian C,Chen S,Ding P,et al.The immune response of a recombinant fowlpox virus coexpressing the HA gene of the H5N1highly pathogenic avian influenza virus and chicken interleukin 6gene in ducks[J].Vaccine,2012,30(44):6279-6286.
[3]Amann R,Rohde J,Wulle U,et al.A new rabies vaccine based on a recombinant ORF virus(parapoxvirus)expressing the rabies virus glycoprotein[J].J Virol,2013,87(3):1618-1630.
[4]Chen W,Hu S,Qu L,et al.A goat poxvirus-vectored peste-des-petits-ruminants vaccine induces longlasting neutralization antibody to high levels in goats and sheep[J].Vaccine,2010,28(30):4742-4750.
[5]Smith G L,Moss B.Infectious poxvirus vectors have capacity for at least 25 000base pairs of foreign DNA[J].Gene,1983,25(1):21-28.
[6]赵银龙,吴国华,朱海霞,等.山羊痘病毒A6L基因的克隆及序列分析[J].中国畜牧兽医,2015,42(8):1973-1978.
[7]Shen Y J,Shephard E,Douglass N,et al.A novel candidate HIV vaccine vector based on the replication deficient Capripoxvirus,Lumpy skin disease virus(LSDV)[J].Virol J,2011,8:265.
[8]Yuan M,Zhang W,Wang J,et al.Efficiently editing the vaccinia virus genome by using the CRISPR-Cas9system[J].J Virol,2015,89(9):5176-5179.
[9]Buller R M,Smith G L,Cremer K,et al.Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype[J].Nature,1985,317(6040):813-815.
[10]Burgers W A,Ginbot Z,Shen Y J,et al.The novel capripoxvirus vector lumpy skin disease virus efficiently boosts modified vaccinia Ankara human immunodeficiency virus responses in rhesus macaques[J].J Gen Virol,2014,95(10):2267-2272.
[11]李继东,蒙学莲,朱学亮,等.小反刍兽疫病毒H基因或F基因重组山羊痘病毒活载体疫苗的研究[J].中国兽医科学,2015,45(2):111-117.
[12]李继东,朱学亮,李辉,等.启动子p7.5/p11在山羊痘病毒和羊口疮病毒中的启动功能验证[J].中国预防兽医学报,2014,36(11):829-833.
[13]李有文,魏风,郭志儒,等.山羊痘病毒疫苗株TK基因的克隆与序列分析[J].西北农业学报,2010,19(1):1-5.
[14]Taylor G,Stott E J,Wertz G,et al.Comparison of the virulence of wild-type thymidine kinase(tk)-deficient and tk+phenotypes of vaccinia virus recombinants after intranasal inoculation of mice[J].J Gen Virol,1991,72(1):125-130.
[15]Sebastian S,Gilbert S C.Recombinant modified vaccinia virus Ankara-based malaria vaccines[J].Expert Rev Vaccines,2016,15(1):91-103.
[16]Caufour P,Rufael T,Lamien C E,et al.Protective efficacy of a single immunization with capripoxvirusvectored recombinant peste des petits ruminants vaccines in presence of pre-existing immunity[J].Vaccine,2014,32(30):3772-3779.
[17]Gari G,Abie G,Gizaw D,et al.Evaluation of the safety,immunogenicity and efficacy of three capripoxvirus vaccine strains against lumpy skin disease virus[J].Vaccine,2015,33(28):3256-3261.
[18]Backes S,Jager C,Dembek C J,et al.Proteinprime/modified vaccinia virus Ankara vector-boost vaccination overcomes tolerance in high-antigenemic HBV-transgenic mice[J].Vaccine,2016,34(7):923-932.
[19]郑敏,梁晟,郑彦梓,等.TK基因重组缺陷山羊痘病毒构建及其生物学特性鉴定[J].南方农业学报,2013,44(9):1552-1557.
[20]Boshra H,Cao J,Babiuk S.Generation of recombinant capripoxvirus vectors for vaccines and gene knockout function studies[J].Methods Mol Biol,2016,1349:151-161.
[21]Gelaye E,Lamien C E,Silber R,et al.Development of a cost-effective method for capripoxvirus genotyping using snapback primer and dsDNA intercalating dye[J].PLoS One,2013,8(10):e75971.