慢病毒介导稳定表达增强小反刍兽疫病毒复制的山羊Vero/SLAM细胞系的建立
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摘要
利用Gateway技术和慢病毒表达系统将山羊淋巴细胞信号活化因子SLAM稳定整合在Vero细胞基因组染色体上,建立稳定表达可增强小反刍兽疫病毒(PPRV)复制的SLAM的Vero阳性细胞亚克隆,并验证阳性细胞亚克隆Vero/SLAM表达SLAM的活性、遗传稳定性及其对PPRV复制的增殖效果。分离山羊外周血淋巴细胞,提取基因组Total RNA,一步RT-PCR获得完整ORF的SLAM基因,采用BP及LR位点的基因重组技术,构建入门载体pDONR/SLAM并获得表达骨架pDEST/SLAM,与Packaging Mix pLP1、pLP2及VSV-G共转染293-FT细胞,获得SLAM复制缺陷型慢病毒样粒子,用其感染Vero细胞,杀稻瘟菌素抗性筛选获得阳性细胞克隆,通过靶标基因扩增、间接免疫荧光、激光扫描共聚焦显微技术、Western blot免疫印迹检测技术、致细胞病变效应及Realtime RT-PCR等技术分别验证SLAM基因组整合、转录,SLAM蛋白表达、反应活性以及PPRV在表达SLAM阳性细胞亚克隆上的增殖效果。结果显示:SLAM受体基因被稳定整合在Vero细胞基因组染色体上,该受体蛋白表达于细胞周质,建立的细胞连续传代不丢失,接种病毒后致细胞病变时间由4～7d缩短为3.5d,TCID_(50)·0.1mL~(-1)由4.25增加为5.67,相对于正常Vero细胞,整合有SLAM的Vero细胞,由于表达了PPRV特异的细胞受体使PPRV对其易感性显著增强。成功建立一株稳定表达靶向增强PPRV复制的Vero/SLAM细胞:该细胞表达的SLAM具有明显增强PPRV复制的功能,不仅可以从细胞模型水平阐明增强PPRV复制的关键靶基因,也为PPRV感染引起宿主细胞的变化以及对机体的致病机制等提供研究工具。
By the Gateway technology and the lentiviral expression system,goat signaling lymphocyte activation molecule(SLAM,also known as CD150)was stably integrated into the genome chromosome of Vero cell,to establish a Vero/g-SLAM positive cell clones,with which the ability of replication of peste des petits ruminants virus(PPRV)can be improved.On the other hand,to verify the activity,genetic stability and proliferation of PPRV expressing SLAM of Vero/g-SLAM.Peripheral blood lymphocytes were isolated from goat,of which genomic total RNA was extracted,SLAM gene with complete ORF were obtained by one step RT-PCR.Using of the BP and LR loci gene recombination technology to construct the entry clone vector pDONR/SLAM and expression skeleton of pDEST/SLAM,with packaging plasmids pLP1,pLP2 and VSV-G to co-transfect 293-FT cells to produce a lentiviral stock,use the lentiviral stock to infect Vero cell.The Vero cells with SLAM were named Vero/SLAM,and were resistant to the blasticidin(3.5μg·mL~(-1)).By the target gene amplification,Confocal laser scanning microscope,indirect immunofluorescence,Western blot and cytopathic effect,the SLAM genome integration,transcription,expression and reaction activity of SLAM protein,and PPRV proliferation effect in Vero/SLAM were verified respectively.Results were as follows:SLAM was stably integrated in the genome of Vero cells;The receptor protein was expressed in the periplasmic part of the cell;After continuous subpassages of the cells,SLAM are not lost;Vero/SLAM were inoculated virus The cytopathic time was shortened from 4-7dto 3.5d,and TCID_(50)·0.1mL~(-1) increased from4.25 to 5.67.Compared with the normal Vero cells,Vero cells that integrate SLAMhave significantly enhanced their susceptibility to PPRV because of expression of PPRV specific cell receptors.In this study,a new cell line,Vero/SLAM cells was established successfully.The SLAM expressed by this cell has the obvious function of enhancing PPRV replication,the critical target genes that enhance PPRV replication can be elucidated at the cellular model level.It also provides information for the study of host cell changes caused by PPRV infection and the pathogenesis of the organism.
By the Gateway technology and the lentiviral expression system,goat signaling lymphocyte activation molecule(SLAM,also known as CD150)was stably integrated into the genome chromosome of Vero cell,to establish a Vero/g-SLAM positive cell clones,with which the ability of replication of peste des petits ruminants virus(PPRV)can be improved.On the other hand,to verify the activity,genetic stability and proliferation of PPRV expressing SLAM of Vero/g-SLAM.Peripheral blood lymphocytes were isolated from goat,of which genomic total RNA was extracted,SLAM gene with complete ORF were obtained by one step RT-PCR.Using of the BP and LR loci gene recombination technology to construct the entry clone vector pDONR/SLAM and expression skeleton of pDEST/SLAM,with packaging plasmids pLP1,pLP2 and VSV-G to co-transfect 293-FT cells to produce a lentiviral stock,use the lentiviral stock to infect Vero cell.The Vero cells with SLAM were named Vero/SLAM,and were resistant to the blasticidin(3.5μg·mL~(-1)).By the target gene amplification,Confocal laser scanning microscope,indirect immunofluorescence,Western blot and cytopathic effect,the SLAM genome integration,transcription,expression and reaction activity of SLAM protein,and PPRV proliferation effect in Vero/SLAM were verified respectively.Results were as follows:SLAM was stably integrated in the genome of Vero cells;The receptor protein was expressed in the periplasmic part of the cell;After continuous subpassages of the cells,SLAM are not lost;Vero/SLAM were inoculated virus The cytopathic time was shortened from 4-7dto 3.5d,and TCID_(50)·0.1mL~(-1) increased from4.25 to 5.67.Compared with the normal Vero cells,Vero cells that integrate SLAMhave significantly enhanced their susceptibility to PPRV because of expression of PPRV specific cell receptors.In this study,a new cell line,Vero/SLAM cells was established successfully.The SLAM expressed by this cell has the obvious function of enhancing PPRV replication,the critical target genes that enhance PPRV replication can be elucidated at the cellular model level.It also provides information for the study of host cell changes caused by PPRV infection and the pathogenesis of the organism.
引文
[1]陈溥言.兽医传染病学[M].5版.北京:中国农业出版社,2006:294-295.CHEN P Y.Veterinary lemology[M].5th ed.Beijing:China Agriculture Press,2006:294-295.(in Chinese)
[2]ABU ELZEIN E M,HASSANIEN M M,AL-AFALEQ A I,et al.Isolation of peste des petits ruminants from goats in Saudi Arabia[J].Vet Rec,1990,127(12):309-310.
[3]TAYLOR W P,AL BUSAIDY S,BARRETT T.The epidemiology of peste des petits ruminants in the Sultanate of Oman[J].Vet Microbiol,1990,22(4):341-352.
[4]OBI T U,ROWE L W,TAYLOR W P.Serological studies with Peste des petits ruminants and rinderpest viruses in Nigeria[J].Trop Anim Health Prod,1984,16(2):115-118.
[5]王志亮,包静月,吴晓东,等.我国首例小反刍兽疫诊断报告[J].中国动物检疫,2007,24(8):24-26.WANG Z L,BAO J Y,WU X D,et al.Diagnosis of the first outbreak of peste des petits ruminants in Tibet[J].Chinese Journal of Animal Health Inspection,2007,24(8):24-26.(in Chinese)
[6]王乐元,次真,吴国珍,等.中国西藏小反刍兽疫的发生状况与防控[J].畜牧兽医学报,2011,42(5):717-720.WANG L Y,TSE D,WU G Z,et al.Occurrence and control strategy of peste des petits ruminants in Tibet autonomous region of China[J].Acta Veterinaria et Zootechnica Sinica,2011,42(5):717-720.(in Chinese)
[7]吴锦艳,尚佑军,田宏,等.2007-2014年国内外小反刍兽疫流行现状及分析[J].中国兽医学报,2016,36(4):687-693.WU J Y,SHANG Y J,TIAN H,et al.The epidemic situation and analysis of peste des petits ruminants in China and worldwide from 2007to 2014[J].Chinese Journal of Veterinary Science,2016,36(4):687-693.(in Chinese)
[8]中华人民共和国农业部.10月16日全国小反刍兽疫确诊疫情情况[EB/OL].(2016-10-18)[2018-03-20].http://www.syj.moa.gov.cn/dwyqdt/qt/201610/t20161018_5308509.htm.Ministry of Agriculture of PRC.Diagnosis of epidemic situation of peste des petits ruminants in China on October 16[EB/OL].(2016-10-18)[2018-03-20].http://www.syj.moa.gov.cn/dwyqdt/qt/201610/t20161018_5308509.htm.(in Chinese)
[9]TATSUO H,ONO N,TANAKA K,et al.SLAM(CDw150)is a cellular receptor for measles virus[J].Nature,2000,406(6798):893-897.
[10]BARON M D.Wild-type Rinderpest virus uses SLAM(CD150)as its receptor[J].J Gen Virol,2005,86(6):1753-1757.
[11]TATSUO H,ONO N,YANAGI Y.Morbilliviruses use signaling lymphocyte activation molecules(CD150)as cellular receptors[J].J Virol,2001,75(13):5842-5850.
[12]ZHANG J L,LIU W X,CHEN W Y,et al.Development of an immunoperoxidase monolayer assay for the detection of antibodies against peste des petits ruminants virus based on BHK-21cell line stably expressing the goat signaling lymphocyte activation molecule[J].PLoS One,2016,11(10):e0165088.
[13]ADOMBI C M,LELENTA M,LAMIEN C E,et al.Monkey CV1 cell line expressing the sheep-goat SLAM protein:a highly sensitive cell line for the isolation of peste des petits ruminants virus from pathological specimens[J].J Virol Methods,2011,173(2):306-313.
[14]NAKANO H,KAMEO Y,ANDOH K,et al.Establishment of canine and feline cells expressing canine signaling lymphocyte activation molecule for canine distemper virus study[J].Vet Microbiol,2009,133(1-2):179-183.
[15]SEKI F,ONO N,YAMAGUCHI R,et al.Efficient isolation of wild strains of canine distemper virus in Vero cells expressing canine SLAM(CD150)and their adaptability to marmoset B95acells[J].J Virol,2003,77(18):9943-9950.
[16]朱颖,刘文鑫,赵姝静,等.稳定表达犬瘟热病毒细胞受体SLAM的BHK-21/SLAM细胞系的建立[J].中国预防兽医学报,2014,36(6):440-443.ZHU Y,LIU W X,ZHAO S J,et al.Establishment of BHK-21cell line stably expressing caine distemper virus recepter of signaling lymphocyte activation molecule[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(6):440-443.(in Chinese)
[17]PAWAR R M,RAJ G D,KUMAR T M A S,et al.Effect of siRNA mediated suppression of signaling lymphocyte activation molecule on replication of peste des petits ruminants virus in vitro[J].Virus Res,2008,136(1-2):118-123.
[18]刘湘涛,吴锦艳,田宏,等.一种用于检测小反刍兽疫病毒的Taqman Real-time RT-PCR试剂盒:中国,201410745664.0[P].2016-03-16.LIU X T,WU J Y,TIAN H,et al.Reagent kit used for testing Taqman Real-time RT-PCR(reverse transcription-polymerase chain reaction)of peste des petits ruminants virus:CN,201410745664.0[P].2016-03-16.(in Chinese)
[19]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the method[J].Methods,2001,25(4):402-408.
[20]赵建军,闫如勋,张海玲,等.稳定表达貉犬瘟热病毒细胞受体SLAM的Vero细胞系的建立及应用[J].微生物学报,2012,52(12):1515-1523.ZHAO J J,YAN R X,ZHANG H L,et al.Establishment and application of a Vero cell line stably expressing raccoon dog SLAM,the cellular receptor of canine distemper virus[J].Acta Microbiologica Sinica,2012,52(12):1515-1523.(in Chinese)
[21]闫如勋.稳定表达不同动物犬瘟热病毒受体SLAM的Vero细胞系的建立[D].镇江:江苏科技大学,2011.YAN R X.Establishment of Vero cell line stably expressing CDV SLAM receptors of different animals[D].Zhenjiang:Jiangsu University of Science and Technology,2011.(in Chinese)
[22]张家林,李翠翠,谢梅梅,等.稳定表达山羊SLAM受体的BHK-21细胞系的建立及应用[J].中国预防兽医学报,2015,37(7):506-509.ZHANG J L,LI C C,XIE M M,et al.The establishment and application of BHK-21expressing goat SLAM[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(7):506-509.(in Chinese)
[23]吴锦艳,田宏,尚佑军,等.慢病毒介导稳定表达抑制PRRSV复制的shRNA细胞系的建立[J].畜牧兽医学报,2016,47(11):2280-2287.WU J Y,TIAN H,SHANG Y J,et al.Establishment of cell lines with stable expression of shRNA to inhibit porcine reproductive and respiratory syndrome virus proliferation using the lentiviral expression system[J].Acta Veterinaria et Zootechnica Sinica,2016,47(11):2280-2287.(in Chinese)
[24]齐兴财,秦晓东,甘晓丽,等.慢病毒介导siRNA抑制O型口蹄疫病毒ON株病毒复制[J].畜牧兽医学报,2018,49(2):360-367.QI X C,QIN X D,GAN X L,et al.Inhibition of type O foot-and-mouth disease virus ON strain replication by lentivirus-mediated siRNA[J].Acta Veterinaria et Zootechnica Sinica,2018,49(2):360-367.(in Chinese)
[25]HARTLEY J L,TEMPLE G F,BRASCH M A.DNA cloning using in vitro site-specific recombination[J].Genome Res,2000,10(11):1788-1795.
[26]赵容,荣齐仙,刘玉忠,等.利用Gateway克隆技术构建丹参SmNAC1转录因子的RNAi表达载体[J].中国中药杂志,2014,39(9):1569-1573.ZHAO R,RONG Q X,LIU Y Z,et al.Construction of RNAi vectors for SmNAC1transcription factors of Salvia miltiorrhiza using Gateway cloning technology[J].China Journal of Chinese Materia Medica,2014,39(9):1569-1573.(in Chinese)
[27]周洁,王栩鸣,陈斌,等.基于Gateway技术的低成本植物双分子荧光互补分析系统[J].浙江农业学报,2013,25(5):1024-1030.ZHOU J,WANG X M,CHEN B,et al.Low-cost Gateway-compatible bimolecular fluorescence complementation assay system[J].Acta Agriculturae Zhejiangensis,2013,25(5):1024-1030.(in Chinese)
[28]刘健.利用Gateway技术构建植物表达载体与植物筛选标记研究[D].呼和浩特:内蒙古师范大学,2014.LIU J.The construction of plant expression vector by Gateway technology and studies on selection markers of plant[D].Hohhot:Inner Mongolia Normal University,2014.(in Chinese)
[2]ABU ELZEIN E M,HASSANIEN M M,AL-AFALEQ A I,et al.Isolation of peste des petits ruminants from goats in Saudi Arabia[J].Vet Rec,1990,127(12):309-310.
[3]TAYLOR W P,AL BUSAIDY S,BARRETT T.The epidemiology of peste des petits ruminants in the Sultanate of Oman[J].Vet Microbiol,1990,22(4):341-352.
[4]OBI T U,ROWE L W,TAYLOR W P.Serological studies with Peste des petits ruminants and rinderpest viruses in Nigeria[J].Trop Anim Health Prod,1984,16(2):115-118.
[5]王志亮,包静月,吴晓东,等.我国首例小反刍兽疫诊断报告[J].中国动物检疫,2007,24(8):24-26.WANG Z L,BAO J Y,WU X D,et al.Diagnosis of the first outbreak of peste des petits ruminants in Tibet[J].Chinese Journal of Animal Health Inspection,2007,24(8):24-26.(in Chinese)
[6]王乐元,次真,吴国珍,等.中国西藏小反刍兽疫的发生状况与防控[J].畜牧兽医学报,2011,42(5):717-720.WANG L Y,TSE D,WU G Z,et al.Occurrence and control strategy of peste des petits ruminants in Tibet autonomous region of China[J].Acta Veterinaria et Zootechnica Sinica,2011,42(5):717-720.(in Chinese)
[7]吴锦艳,尚佑军,田宏,等.2007-2014年国内外小反刍兽疫流行现状及分析[J].中国兽医学报,2016,36(4):687-693.WU J Y,SHANG Y J,TIAN H,et al.The epidemic situation and analysis of peste des petits ruminants in China and worldwide from 2007to 2014[J].Chinese Journal of Veterinary Science,2016,36(4):687-693.(in Chinese)
[8]中华人民共和国农业部.10月16日全国小反刍兽疫确诊疫情情况[EB/OL].(2016-10-18)[2018-03-20].http://www.syj.moa.gov.cn/dwyqdt/qt/201610/t20161018_5308509.htm.Ministry of Agriculture of PRC.Diagnosis of epidemic situation of peste des petits ruminants in China on October 16[EB/OL].(2016-10-18)[2018-03-20].http://www.syj.moa.gov.cn/dwyqdt/qt/201610/t20161018_5308509.htm.(in Chinese)
[9]TATSUO H,ONO N,TANAKA K,et al.SLAM(CDw150)is a cellular receptor for measles virus[J].Nature,2000,406(6798):893-897.
[10]BARON M D.Wild-type Rinderpest virus uses SLAM(CD150)as its receptor[J].J Gen Virol,2005,86(6):1753-1757.
[11]TATSUO H,ONO N,YANAGI Y.Morbilliviruses use signaling lymphocyte activation molecules(CD150)as cellular receptors[J].J Virol,2001,75(13):5842-5850.
[12]ZHANG J L,LIU W X,CHEN W Y,et al.Development of an immunoperoxidase monolayer assay for the detection of antibodies against peste des petits ruminants virus based on BHK-21cell line stably expressing the goat signaling lymphocyte activation molecule[J].PLoS One,2016,11(10):e0165088.
[13]ADOMBI C M,LELENTA M,LAMIEN C E,et al.Monkey CV1 cell line expressing the sheep-goat SLAM protein:a highly sensitive cell line for the isolation of peste des petits ruminants virus from pathological specimens[J].J Virol Methods,2011,173(2):306-313.
[14]NAKANO H,KAMEO Y,ANDOH K,et al.Establishment of canine and feline cells expressing canine signaling lymphocyte activation molecule for canine distemper virus study[J].Vet Microbiol,2009,133(1-2):179-183.
[15]SEKI F,ONO N,YAMAGUCHI R,et al.Efficient isolation of wild strains of canine distemper virus in Vero cells expressing canine SLAM(CD150)and their adaptability to marmoset B95acells[J].J Virol,2003,77(18):9943-9950.
[16]朱颖,刘文鑫,赵姝静,等.稳定表达犬瘟热病毒细胞受体SLAM的BHK-21/SLAM细胞系的建立[J].中国预防兽医学报,2014,36(6):440-443.ZHU Y,LIU W X,ZHAO S J,et al.Establishment of BHK-21cell line stably expressing caine distemper virus recepter of signaling lymphocyte activation molecule[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(6):440-443.(in Chinese)
[17]PAWAR R M,RAJ G D,KUMAR T M A S,et al.Effect of siRNA mediated suppression of signaling lymphocyte activation molecule on replication of peste des petits ruminants virus in vitro[J].Virus Res,2008,136(1-2):118-123.
[18]刘湘涛,吴锦艳,田宏,等.一种用于检测小反刍兽疫病毒的Taqman Real-time RT-PCR试剂盒:中国,201410745664.0[P].2016-03-16.LIU X T,WU J Y,TIAN H,et al.Reagent kit used for testing Taqman Real-time RT-PCR(reverse transcription-polymerase chain reaction)of peste des petits ruminants virus:CN,201410745664.0[P].2016-03-16.(in Chinese)
[19]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the method[J].Methods,2001,25(4):402-408.
[20]赵建军,闫如勋,张海玲,等.稳定表达貉犬瘟热病毒细胞受体SLAM的Vero细胞系的建立及应用[J].微生物学报,2012,52(12):1515-1523.ZHAO J J,YAN R X,ZHANG H L,et al.Establishment and application of a Vero cell line stably expressing raccoon dog SLAM,the cellular receptor of canine distemper virus[J].Acta Microbiologica Sinica,2012,52(12):1515-1523.(in Chinese)
[21]闫如勋.稳定表达不同动物犬瘟热病毒受体SLAM的Vero细胞系的建立[D].镇江:江苏科技大学,2011.YAN R X.Establishment of Vero cell line stably expressing CDV SLAM receptors of different animals[D].Zhenjiang:Jiangsu University of Science and Technology,2011.(in Chinese)
[22]张家林,李翠翠,谢梅梅,等.稳定表达山羊SLAM受体的BHK-21细胞系的建立及应用[J].中国预防兽医学报,2015,37(7):506-509.ZHANG J L,LI C C,XIE M M,et al.The establishment and application of BHK-21expressing goat SLAM[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(7):506-509.(in Chinese)
[23]吴锦艳,田宏,尚佑军,等.慢病毒介导稳定表达抑制PRRSV复制的shRNA细胞系的建立[J].畜牧兽医学报,2016,47(11):2280-2287.WU J Y,TIAN H,SHANG Y J,et al.Establishment of cell lines with stable expression of shRNA to inhibit porcine reproductive and respiratory syndrome virus proliferation using the lentiviral expression system[J].Acta Veterinaria et Zootechnica Sinica,2016,47(11):2280-2287.(in Chinese)
[24]齐兴财,秦晓东,甘晓丽,等.慢病毒介导siRNA抑制O型口蹄疫病毒ON株病毒复制[J].畜牧兽医学报,2018,49(2):360-367.QI X C,QIN X D,GAN X L,et al.Inhibition of type O foot-and-mouth disease virus ON strain replication by lentivirus-mediated siRNA[J].Acta Veterinaria et Zootechnica Sinica,2018,49(2):360-367.(in Chinese)
[25]HARTLEY J L,TEMPLE G F,BRASCH M A.DNA cloning using in vitro site-specific recombination[J].Genome Res,2000,10(11):1788-1795.
[26]赵容,荣齐仙,刘玉忠,等.利用Gateway克隆技术构建丹参SmNAC1转录因子的RNAi表达载体[J].中国中药杂志,2014,39(9):1569-1573.ZHAO R,RONG Q X,LIU Y Z,et al.Construction of RNAi vectors for SmNAC1transcription factors of Salvia miltiorrhiza using Gateway cloning technology[J].China Journal of Chinese Materia Medica,2014,39(9):1569-1573.(in Chinese)
[27]周洁,王栩鸣,陈斌,等.基于Gateway技术的低成本植物双分子荧光互补分析系统[J].浙江农业学报,2013,25(5):1024-1030.ZHOU J,WANG X M,CHEN B,et al.Low-cost Gateway-compatible bimolecular fluorescence complementation assay system[J].Acta Agriculturae Zhejiangensis,2013,25(5):1024-1030.(in Chinese)
[28]刘健.利用Gateway技术构建植物表达载体与植物筛选标记研究[D].呼和浩特:内蒙古师范大学,2014.LIU J.The construction of plant expression vector by Gateway technology and studies on selection markers of plant[D].Hohhot:Inner Mongolia Normal University,2014.(in Chinese)