牛冠状病毒S基因的序列分析及原核表达
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摘要
为了解牛冠状病毒(bovine coronavirus,BCoV)的S基因变异情况并建立ELISA检测方法,本研究对采自不同牛场的新生犊牛腹泻(CD)和成年牛冬痢(WD)腹泻样本提取总RNA,反转录合成cDNA,利用PCR扩增S全基因和S1基因。将S1基因目的片段连接表达载体pET-32a(+),并转化大肠杆菌BL21(DE3)感受态细胞,经PCR、双酶切及测序验证正确后,进行IPTG诱导表达。结果显示,CD与WD分离株S基因核苷酸为98.4%,CD和WD分离株与参考毒株BCoV-ENT株核苷酸同源性最高,分别为98.4%和98.5%,CD分离株与参考毒株SUN5株的同源性最低,为97.5%,WD分离株与FRA/EPI/Caen/2003/13同源性最低,为97.3%。通过比对可知,分离株与已知毒株之间存在较大差异,为疫苗候选毒株筛选提供依据。本试验同时构建了pET-32a-S1表达载体,在0.2mmol/L IPTG诱导5h时,重组菌能在大肠杆菌BL21感受态细胞中产生大量的S1融合蛋白,获得约58ku表达产物。本试验成功表达了S1蛋白,并对BCoV进行了核苷酸进化分析,为疫苗免疫效果评价方法的建立奠定了基础。
In order to understand the variation of bovine coronavirus(BCoV)Sgene and establish an ELISA detection method,total RNA was extracted from neonatal calf diarrhea(CD)and adult cattle winter dysentery(WD)diarrhea samples from different farms.cDNA was synthesized,and Sand S1 genes were supplemented by PCR.The target fragment S1 was lighted into the expression vector pET-32 a(+)and transformed into E.coli BL21(DE3).The recombinant plasmid was induced by IPTG after being verified by PCR,double enzyme digestion and sequencing.The results showed that the nucleotide identitie of Sgene of CD and WD isolates was 98.4%,and the isolates had the highest nucleotide homology with the reference strain BCoV-ENT,which were98.4% and 98.5%,respectively.The homology of CD isolate with the reference strain SUN5 was the lowest,which was 97.5%,and the homology of WD isolate with strain FRA/EPI/Caen/2004/13 was the lowest,which was 97.3%.The comparison showed that there were a big difference between the isolated strains and the known strains,and provided a basis for screening the vaccine candidate strains.At the same time,pET-32 a-S1 expression vector was constructed.The recombinant bacteria could produce a large amount of fusion S1 protein in E.coli BL21 and obtain 58 ku expression product,induced by 0.2 mmol/L IPTG for 5 h.In this study,S1 protein was successfully expressed,and nucleotide evolution analysis of BCoV were carried out,which laid the foundation for the establishment of the vaccine immune effect evaluation method.
In order to understand the variation of bovine coronavirus(BCoV)Sgene and establish an ELISA detection method,total RNA was extracted from neonatal calf diarrhea(CD)and adult cattle winter dysentery(WD)diarrhea samples from different farms.cDNA was synthesized,and Sand S1 genes were supplemented by PCR.The target fragment S1 was lighted into the expression vector pET-32 a(+)and transformed into E.coli BL21(DE3).The recombinant plasmid was induced by IPTG after being verified by PCR,double enzyme digestion and sequencing.The results showed that the nucleotide identitie of Sgene of CD and WD isolates was 98.4%,and the isolates had the highest nucleotide homology with the reference strain BCoV-ENT,which were98.4% and 98.5%,respectively.The homology of CD isolate with the reference strain SUN5 was the lowest,which was 97.5%,and the homology of WD isolate with strain FRA/EPI/Caen/2004/13 was the lowest,which was 97.3%.The comparison showed that there were a big difference between the isolated strains and the known strains,and provided a basis for screening the vaccine candidate strains.At the same time,pET-32 a-S1 expression vector was constructed.The recombinant bacteria could produce a large amount of fusion S1 protein in E.coli BL21 and obtain 58 ku expression product,induced by 0.2 mmol/L IPTG for 5 h.In this study,S1 protein was successfully expressed,and nucleotide evolution analysis of BCoV were carried out,which laid the foundation for the establishment of the vaccine immune effect evaluation method.
引文
[1]侯佩莉,王洪梅,何洪彬.牛病毒性腹泻病毒、牛冠状病毒和牛肠道病毒多重RT-PCR检测方法的建立及初步应用[J].中国兽医学报,2016,36(10):1672-1675.HOU P L,WANG H M,HE H B.Establishment and application of a multiple RT-PCR for detection of BVDV,BCoV and BEV[J].Chinese Journal of Veterinary Science,2016,36(10):1672-1675.(in Chinese)
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[5]YOO D,DEREGT D.A single amino acid change within antigenic domainⅡof the spike protein of bovine coronavirus confers resistance to virus neutralization[J].Clinical and Diagnostic Laboratory Immunology,2001,8(2):297-302.
[6]姚火春,杜念兴,徐为燕,等.牛冠状病毒的血清流行病学调查[J].南京农业大学学报,1990,13(1):117-121.YAO H C,DU N X,XU W Y,et al.Seroepizootiological survey of bovine coronavirus in cattle,human and other animals[J].Journal of Nanjing Agricultural University,1990,13(1):117-121.(in Chinese)
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[10]蒋静,李健,胡永强,等.上海等4省市动物冠状病毒的流行病学调查[J].畜牧与兽医,2007,39(12):50-51.JIANG J,LI J,HU Y Q,et al.Epidemiological investigation of animal coronavirus in 4provinces and cities,including Shanghai[J].Animal Husbandry&Veterinary Medicine,2007,39(12):50-51.(in Chinese)
[11]刘合义,孙留霞,王进轶,等.牛冠状病毒重组N蛋白间接ELISA检测方法的建立[J].中国预防兽医学报,2009,31(8):618-622.LIU H Y,SUN L X,WANG J Y,et al.Development of an indirect ELISA for the detection of bovine coronavirus using recombinant N protein[J].Chinese Journal of Preventive Veterinary Medicine,2009,31(8):618-622.(in Chinese)
[12]傅彩霞,靳兴军,郑瑞峰,等.北京地区规模化奶牛场三种病毒性腹泻病的血清学调查[J].动物医学进展,2012,33(5):85-88.FU C X,JIN X J,ZHENG R F,et al.Serological survey of three viral diarrheal diseases in Beijing largescale dairy cow farms[J].Progress in Veterinary Medicine,2012,33(5):85-88.(in Chinese)
[13]张坤,剡根强,王静梅,等.新疆北疆部分地区致犊牛腹泻冠状病毒的分子流行病学调查[J].中国兽医科学,2015,45(12):1270-1276.ZHANG K,YAN G Q,WANG J M,et al.Molecular epidemiological investigation of calf diarrhea coronavirus in parts of Northern Xinjiang[J].Chinese Veterinary Science,2015,45(12):1270-1276.(in Chinese)
[14]THEIL K W,MCCLOSKEY C M.Rotavirus shedding in feces of gnotobiotic calves orally inoculated with a commercial rotavirus-coronavirus vaccine[J].Journal of Veterinary Diagnostic Investigation,1995,7(4):427-432.
[15]柳强,侯喜林,车车,等.牛冠状病毒和轮状病毒的双重RT-PCR检测方法的建立及应用[J].中国预防兽医学报,2009,31(9):701-704.LIU Q,HOU X L,CHE C,et al.The establishment and application of a double PCR for bovine coronavirus and bovine rotavirus[J].Chinese Journal of Preventive Veterinary Medicine,2009,31(9):701-704.(in Chinese)
[16]LANZ U F,KAUFMANN T,SAGER H,et al.Prevalence of four enteropathogens in the faeces of young diarrhoeic dairy calves in Switzerland[J].Veterinary Record,2008,163(12):362-366.
[17]AMMAR S S M,MOKHTARIA K,TAHAR B B,et al.Prevalence of rotavirus(GARV)and coronavirus(BCoV)associated with neonatal diarrhea in calves in Western Algeria[J].Asian Pacific Journal of Tropical Biomedicine,2014,4(1):318-322.
[18]张弦.上海地区奶牛四种病毒性疾病血清学调查和牛α、γ干扰素的原核表达[D].南京:南京农业大学,2015.ZHANG X.Serosurvey of four bovine viral disease in Shanghai dairy herds and expression,purification of recombinant bovine interferon-α,γ[D].Nanjing:Nanjing Agricultural University,2015.(in Chinese)
[19]KANNO T,KAMIYOSHI T,ISHIHARA R,et al.Phylogenetic studies of bovine coronaviruses isolated in Japan[J].Journal of Veterinary Medical Science,2009,71(1):83-86.
[20]GUNN L,COLLINS P J,O'Connell M J,et al.Phylogenetic investigation of enteric bovine coronavirus in Ireland reveals partitioning between European and global strains[J].Irish Veterinary Journal,2015,68(1):31.
[21]孙秋艳,郭洪梅,沈美艳,等.猪传染性胃肠炎病毒的分离鉴定及S基因进化分析[J].动物医学进展,2015,36(7):7-12.SUN Q Y,GUO H M,SHEN M Y,et al.Isolation and identification of transmissible gastroenteritis virus and phylogenetic analysis of S gene[J].Progress in Veterinary Medicine,2015,36(7):7-12.(in Chinese)
[22]乔涵,李辉,赵丽,等.猪流行性腹泻病毒河南流行株S基因的克隆与遗传进化分析[J].中国兽医学报,2017,37(5):781-786.QIAO H,LI H,ZHAO L,et al.Cloning and phylogenetic analysis of Sgene of porcine epidemic diarrhea virus prevailing in Henan province[J].Chinese Journal of Veterinary Science,2017,37(5):781-786.(in Chinese)
[23]黄平,宋怀东,邹丽容,等.2004年SARS-CoV毒株S基因分子进化特征[J].中国人兽共患病学报,2006,22(10):957-960.HUANG P,SONG H D,ZOU L R,et al.Evolutionary characteristics on Sgene of SARS-CoVs in 2004[J].Chinese Journal of Zoonoses,2006,22(10):957-960.(in Chinese)
[24]沈美艳,孙秋艳,李舫,等.上海株犬冠状病毒的分离鉴定及S基因的遗传进化分析[J].中国动物检疫,2017,34(3):97-101.SHEN M Y,SUN Q Y,LI H,et al.Isolation and identification of canine coronavirus Shanghai strain and phylogenetic analysis of Sgene[J].China Animal Health Inspection,2017,34(3):97-101.(in Chinese)
[25]王玉东,刘俊辉,郑增忍,等.鸡腺胃型传染性支气管炎病毒山东分离株S基因的克隆及序列分析[J].动物医学进展,2015,36(6):104-110.WANG Y D,LIU J H,ZHENG Z R,et al.Cloning and sequence analysis of Sgene of Shandong isolates of proventriculus type avian infectious bronchitis virus[J].Progress in Veterinary Medicine,2015,36(6):104-110.(in Chinese)
[26]周国辉,于力,魏萍.表达牛冠状病毒S1基因的重组人腺病毒的构建及鉴定[J].中国预防兽医学报,2010,32(11):834-838.ZHOU G H,YU L,WEI P.Construction and identification of recombinant adenovirus expressing S1gene of bovine coronavirus[J].Chinese Journal of Preventive Veterinary Medicine,2010,32(11):834-838.(in Chinese)
[27]孙庆申,李鑫,车小琼,等.载牛冠状病毒N蛋白壳聚糖微球的免疫效果评价[J].中国预防兽医学报,2009,31(11):882-886.SUN Q S,LI X,CHE X Q,et al.Evaluation on the immune efficiency of bovine coronavirus N proteinloaded chitosan microspheres[J].Chinese Journal of Preventive Veterinary Medicine,2009,31(11):882-886.(in Chinese)
[2]沈付娆.牛冠状病毒实时荧光定量PCR检测方法的建立及应用[D].济南:山东师范大学,2015.SHEN F R.Development and application of a SYBR GreenⅠReal-time PCR assay for detection of bovine coronavirus[D].Jinan:Shandong Normal University,2015.(in Chinese)
[3]刘振格,侯喜林,余丽芸,等.牛冠状病毒DQ株感染BALB/c小鼠的敏感性研究[J].中国预防兽医学报,2014,36(1):15-18.LIU Z G,HOU X L,YU L Y,et al.Study on the sensitivity of BALB/c mice to bovine coronavirus[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(1):15-18.(in Chinese)
[4]NASIRI K,NASSIRI M,TAHMOORESPUR M,et al.Design and construction of chimeric VP8-S2antigen for bovine rotavirus and bovine coronavirus[J].Advanced Pharmaceutical Bulletin,2016,6(1):91-98.
[5]YOO D,DEREGT D.A single amino acid change within antigenic domainⅡof the spike protein of bovine coronavirus confers resistance to virus neutralization[J].Clinical and Diagnostic Laboratory Immunology,2001,8(2):297-302.
[6]姚火春,杜念兴,徐为燕,等.牛冠状病毒的血清流行病学调查[J].南京农业大学学报,1990,13(1):117-121.YAO H C,DU N X,XU W Y,et al.Seroepizootiological survey of bovine coronavirus in cattle,human and other animals[J].Journal of Nanjing Agricultural University,1990,13(1):117-121.(in Chinese)
[7]BOILEAU M J,KAPIL S.Bovine coronavirus associated syndromes[J].Veterinary Clinics of North America Food Animal Practice,2010,26:123-146.
[8]孙留霞,朱平军,李文华,等.牛冠状病毒检测方法的研究进展[J].上海畜牧兽医通讯,2014,5:20-22.SUN L X,ZHU P J,LI W H,et al.Research progress in detection of bovine coronavirus[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2014,5:20-22.(in Chinese)
[9]胡传伟,贾赞,简中友,等.东北三省冠状病毒流行病学调查[J].黑龙江畜牧兽医,2006,12:88-89.HU C W,JIA Z,JIAN Z Y,et al.Epidemiological survey of coronavirus in three provinces in Northeast China[J].Heilongjiang Animal Science and Veterinary Medicine,2006,12:88-89.(in Chinese)
[10]蒋静,李健,胡永强,等.上海等4省市动物冠状病毒的流行病学调查[J].畜牧与兽医,2007,39(12):50-51.JIANG J,LI J,HU Y Q,et al.Epidemiological investigation of animal coronavirus in 4provinces and cities,including Shanghai[J].Animal Husbandry&Veterinary Medicine,2007,39(12):50-51.(in Chinese)
[11]刘合义,孙留霞,王进轶,等.牛冠状病毒重组N蛋白间接ELISA检测方法的建立[J].中国预防兽医学报,2009,31(8):618-622.LIU H Y,SUN L X,WANG J Y,et al.Development of an indirect ELISA for the detection of bovine coronavirus using recombinant N protein[J].Chinese Journal of Preventive Veterinary Medicine,2009,31(8):618-622.(in Chinese)
[12]傅彩霞,靳兴军,郑瑞峰,等.北京地区规模化奶牛场三种病毒性腹泻病的血清学调查[J].动物医学进展,2012,33(5):85-88.FU C X,JIN X J,ZHENG R F,et al.Serological survey of three viral diarrheal diseases in Beijing largescale dairy cow farms[J].Progress in Veterinary Medicine,2012,33(5):85-88.(in Chinese)
[13]张坤,剡根强,王静梅,等.新疆北疆部分地区致犊牛腹泻冠状病毒的分子流行病学调查[J].中国兽医科学,2015,45(12):1270-1276.ZHANG K,YAN G Q,WANG J M,et al.Molecular epidemiological investigation of calf diarrhea coronavirus in parts of Northern Xinjiang[J].Chinese Veterinary Science,2015,45(12):1270-1276.(in Chinese)
[14]THEIL K W,MCCLOSKEY C M.Rotavirus shedding in feces of gnotobiotic calves orally inoculated with a commercial rotavirus-coronavirus vaccine[J].Journal of Veterinary Diagnostic Investigation,1995,7(4):427-432.
[15]柳强,侯喜林,车车,等.牛冠状病毒和轮状病毒的双重RT-PCR检测方法的建立及应用[J].中国预防兽医学报,2009,31(9):701-704.LIU Q,HOU X L,CHE C,et al.The establishment and application of a double PCR for bovine coronavirus and bovine rotavirus[J].Chinese Journal of Preventive Veterinary Medicine,2009,31(9):701-704.(in Chinese)
[16]LANZ U F,KAUFMANN T,SAGER H,et al.Prevalence of four enteropathogens in the faeces of young diarrhoeic dairy calves in Switzerland[J].Veterinary Record,2008,163(12):362-366.
[17]AMMAR S S M,MOKHTARIA K,TAHAR B B,et al.Prevalence of rotavirus(GARV)and coronavirus(BCoV)associated with neonatal diarrhea in calves in Western Algeria[J].Asian Pacific Journal of Tropical Biomedicine,2014,4(1):318-322.
[18]张弦.上海地区奶牛四种病毒性疾病血清学调查和牛α、γ干扰素的原核表达[D].南京:南京农业大学,2015.ZHANG X.Serosurvey of four bovine viral disease in Shanghai dairy herds and expression,purification of recombinant bovine interferon-α,γ[D].Nanjing:Nanjing Agricultural University,2015.(in Chinese)
[19]KANNO T,KAMIYOSHI T,ISHIHARA R,et al.Phylogenetic studies of bovine coronaviruses isolated in Japan[J].Journal of Veterinary Medical Science,2009,71(1):83-86.
[20]GUNN L,COLLINS P J,O'Connell M J,et al.Phylogenetic investigation of enteric bovine coronavirus in Ireland reveals partitioning between European and global strains[J].Irish Veterinary Journal,2015,68(1):31.
[21]孙秋艳,郭洪梅,沈美艳,等.猪传染性胃肠炎病毒的分离鉴定及S基因进化分析[J].动物医学进展,2015,36(7):7-12.SUN Q Y,GUO H M,SHEN M Y,et al.Isolation and identification of transmissible gastroenteritis virus and phylogenetic analysis of S gene[J].Progress in Veterinary Medicine,2015,36(7):7-12.(in Chinese)
[22]乔涵,李辉,赵丽,等.猪流行性腹泻病毒河南流行株S基因的克隆与遗传进化分析[J].中国兽医学报,2017,37(5):781-786.QIAO H,LI H,ZHAO L,et al.Cloning and phylogenetic analysis of Sgene of porcine epidemic diarrhea virus prevailing in Henan province[J].Chinese Journal of Veterinary Science,2017,37(5):781-786.(in Chinese)
[23]黄平,宋怀东,邹丽容,等.2004年SARS-CoV毒株S基因分子进化特征[J].中国人兽共患病学报,2006,22(10):957-960.HUANG P,SONG H D,ZOU L R,et al.Evolutionary characteristics on Sgene of SARS-CoVs in 2004[J].Chinese Journal of Zoonoses,2006,22(10):957-960.(in Chinese)
[24]沈美艳,孙秋艳,李舫,等.上海株犬冠状病毒的分离鉴定及S基因的遗传进化分析[J].中国动物检疫,2017,34(3):97-101.SHEN M Y,SUN Q Y,LI H,et al.Isolation and identification of canine coronavirus Shanghai strain and phylogenetic analysis of Sgene[J].China Animal Health Inspection,2017,34(3):97-101.(in Chinese)
[25]王玉东,刘俊辉,郑增忍,等.鸡腺胃型传染性支气管炎病毒山东分离株S基因的克隆及序列分析[J].动物医学进展,2015,36(6):104-110.WANG Y D,LIU J H,ZHENG Z R,et al.Cloning and sequence analysis of Sgene of Shandong isolates of proventriculus type avian infectious bronchitis virus[J].Progress in Veterinary Medicine,2015,36(6):104-110.(in Chinese)
[26]周国辉,于力,魏萍.表达牛冠状病毒S1基因的重组人腺病毒的构建及鉴定[J].中国预防兽医学报,2010,32(11):834-838.ZHOU G H,YU L,WEI P.Construction and identification of recombinant adenovirus expressing S1gene of bovine coronavirus[J].Chinese Journal of Preventive Veterinary Medicine,2010,32(11):834-838.(in Chinese)
[27]孙庆申,李鑫,车小琼,等.载牛冠状病毒N蛋白壳聚糖微球的免疫效果评价[J].中国预防兽医学报,2009,31(11):882-886.SUN Q S,LI X,CHE X Q,et al.Evaluation on the immune efficiency of bovine coronavirus N proteinloaded chitosan microspheres[J].Chinese Journal of Preventive Veterinary Medicine,2009,31(11):882-886.(in Chinese)