牛冠状病毒S基因的序列分析及原核表达

doi:10.16431/j.cnki.1671-7236.2018.07.002

摘要/Abstract

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0.2 mmol/L IPTG诱导5 h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58 ku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。

关键词: 牛冠状病毒(BCoV); S基因; 序列分析; 原核表达

Abstract:

In order to understand the variation of bovine coronavirus (BCoV) S gene 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 S and S1 genes were supplemented by PCR.The target fragment S1 was lighted into the expression vector pET-32a(+) 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 S gene of CD and WD isolates was 98.4%,and the isolates had the highest nucleotide homology with the reference strain BCoV-ENT,which were 98.4% 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-32a-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.

Key words: bovine coronavirus (BCoV); S gene; sequence analysis; prokaryotic expression

中图分类号:

S852.65

高国强, 王梦心, 刘明明, 于仁冬, 侯喜林, 周玉龙, 武瑞, 张国华, 刘琳珊, 任德强. 牛冠状病毒S基因的序列分析及原核表达[J]. 《中国畜牧兽医》, 2018, 45(7): 1740-1749.

GAO Guoqiang, WANG Mengxin, LIU Mingming, YU Rendong, HOU Xilin, ZHOU Yulong, WU Rui, ZHANG Guohua, LIU Linshan, REN Deqiang. Sequence Analysis and Prokaryotic Expression of Bovine Coronavirus S Gene[J]. , 2018, 45(7): 1740-1749.

参考文献

[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)
[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 Green Ⅰ Real-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 domainⅡ of 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 4 provinces 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 large-scale 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 S gene 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.Evolutionarycharacteristics on S gene 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 S gene[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 S gene 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 S1 gene 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 protein-loaded chitosan microspheres[J].Chinese Journal ofPreventive Veterinary Medicine,2009,31(11):882-886.(in Chinese)