猪丁型冠状病毒S1基因的克隆、原核表达及多克隆抗体制备
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摘要
本研究旨在克隆猪丁型冠状病毒(PDCoV)S1基因(1—1 566bp),体外表达S1基因抗原表位预测区Sa(106—1 290bp)蛋白并制备抗该蛋白质的多克隆抗体。运用生物信息学软件分析S1核苷酸和编码氨基酸序列特征,将S1基因抗原表位预测区Sa克隆至原核表达载体pET22b(+),构建重组表达载体pET22b-Sa,转化Rosetta(DE3)菌株,IPTG诱导表达、超滤柱浓缩纯化重组蛋白、Ni柱亲和层析,Western blot检测Sa蛋白的活性,将Sa蛋白免疫新西兰兔制备多克隆抗体。结果表明,CHN-2015毒株S1基因开放型阅读框大小为1 566bp(GenBank No.KY398009),编码522个氨基酸,是具有亲水性的非跨膜蛋白,存在15个潜在的B细胞抗原表位;原核表达Sa重组蛋白,该蛋白质在37℃下用终浓度为0.8mmol·L~(-1)IPTG诱导5h后,蛋白质主要以可溶性形式存在,免疫印迹显示表达的Sa蛋白在上清与包涵体中都有良好的反应性;用纯化的Sa蛋白四次免疫家兔,获得的兔抗Sa蛋白抗体效价可达1∶10 240。本研究克隆了S1基因,原核表达了Sa蛋白和制备了高效价的兔抗Sa蛋白抗体,具有良好的免疫原性,为后续深入开展S蛋白的功能研究奠定了基础。
In order to obtain polyclonal antibody against porcine deltacoronavirus(PDCoV)spike-1(S1)protein,we cloned the gene(1-1 566 bp),and then expressed the Sa protein(106-1 290 bp)of S1 gene epitope prediction region successfully.The S1 gene epitope prediction region Sa(106-1 290 bp)was cloned into the prokaryotic expression vector pET22 b(+)to construct recombinant plasmid pET22 b-Sa by using the bioinformatics software to analyze the S1 nucleotide and the amino acid sequence.The recombinant expression vector was transformed into Rosetta(DE3)competent strain,and the recombinant protein was acquired by induction of IPTG.The recombinant protein was purified by ultrafiltration column concentration and immobilized Ni ion affinity chromatography.The activity of the protein Sa obtained was examined by Western blot.New Zealand rabbits were immunized with the Sa protein 4 times to prepare polyclonal antibody.Theresults showed that the open reading frame of S1 gene of CHN-2015 strain was 1 566 bp(GenBank No.:KY398009),encoding 522 amino acids.It is a non-transmembrane protein with hydrophilicity and 15 potential B cell epitopes Bit;The recombinant protein was expressed in prokaryotic expression.The protein was mainly expressed in soluble form at 37 ℃for 5 hat the final concentration of 0.8 mmol·L~(-1) IPTG.Immunoblotting results showed that the expressed Sa protein had good immunogenicity in the supernatant and inclusion bodies.The rabbit anti-Sa antibody titer was up to 1:10 240.The results showed that the expressed Sa protein contained antigen epitope recognition region of porcine Deltacoronavirus,and had a good immunogenicity.
In order to obtain polyclonal antibody against porcine deltacoronavirus(PDCoV)spike-1(S1)protein,we cloned the gene(1-1 566 bp),and then expressed the Sa protein(106-1 290 bp)of S1 gene epitope prediction region successfully.The S1 gene epitope prediction region Sa(106-1 290 bp)was cloned into the prokaryotic expression vector pET22 b(+)to construct recombinant plasmid pET22 b-Sa by using the bioinformatics software to analyze the S1 nucleotide and the amino acid sequence.The recombinant expression vector was transformed into Rosetta(DE3)competent strain,and the recombinant protein was acquired by induction of IPTG.The recombinant protein was purified by ultrafiltration column concentration and immobilized Ni ion affinity chromatography.The activity of the protein Sa obtained was examined by Western blot.New Zealand rabbits were immunized with the Sa protein 4 times to prepare polyclonal antibody.Theresults showed that the open reading frame of S1 gene of CHN-2015 strain was 1 566 bp(GenBank No.:KY398009),encoding 522 amino acids.It is a non-transmembrane protein with hydrophilicity and 15 potential B cell epitopes Bit;The recombinant protein was expressed in prokaryotic expression.The protein was mainly expressed in soluble form at 37 ℃for 5 hat the final concentration of 0.8 mmol·L~(-1) IPTG.Immunoblotting results showed that the expressed Sa protein had good immunogenicity in the supernatant and inclusion bodies.The rabbit anti-Sa antibody titer was up to 1:10 240.The results showed that the expressed Sa protein contained antigen epitope recognition region of porcine Deltacoronavirus,and had a good immunogenicity.
引文
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[20]THACHIL A,GERBER P F,XIAO C T,et al.Development and application of an ELISA for the detection of porcine deltacoronavirus IgG antibodies[J].PLoS One,2015,10(4):e124363.
[21]ZHAI S L,WEI W K,LI X P,et al.Occurrence and sequence analysis of porcine deltacoronaviruses in southern China[J].Virol J,2016,13:136.
[22]MAHY B W J.Virology:Molecular biology of the coronaviruses[J].Nature,1983,305(5934):474-475.
[23]BRIAN D A,BARIC R S.Coronavirus genome structure and replication[M]//ENJUANES L.Coronavirus Replication and Reverse Genetics.Berlin Heidelberg:Springer,2005.
[24]LEE H K,YEO S G.Cloning and sequence analysis of the nucleocapsid gene of porcine epidemic diarrhea virus Chinju99[J].Virus Genes,2003,26(2):207-212.
[25]CHEN Q,LI G W,STASKO J,et al.Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States[J].J Clin Microbiol,2014,52(1):234-243.
[26]SUN D B,FENG L,SHI H Y,et al.Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein[J].Vet Microbiol,2008,131(1-2):73-81.
[27]解庭波.大肠杆菌表达系统的研究进展[J].长江大学学报:自然科学版,2008,5(3):77-82.XIE T B.Research progress on E.coli expression system[J].Journal of Yangtze University:Natural Science Edition,2008,5(3):77-82.(in Chinese)
[28]苏鹏,龚国利.优化大肠杆菌表达外源蛋白的研究进展[J].生物技术通报,2017,33(2):16-23.SU P,GONG G L.Research progress on optimizing the expression of exogenous proteins in Escherichia coli[J].Biotechnology Bulletin,2017,33(2):16-23.(in Chinese)
[2]SU M J,LI C Q,GUO D H,et al.A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus[J].J Vet Med Sci,2016,78(4):601-606.
[3]ZHANG J Q.Porcine deltacoronavirus:Overview of infection dynamics,diagnostic methods,prevalence and genetic evolution[J].Virus Res,2016,226:71-84.
[4]WOO P C Y,HUANG Y,LAU S K P,et al.Coronavirus genomics and bioinformatics analysis[J].Viruses,2010,2(8):1804-1820.
[5]JUNG K,HU H,EYERLY B,et al.Pathogenicity of2porcine deltacoronavirus strains in gnotobiotic pigs[J].Emerg Infect Dis,2015,21(4):650-654.
[6]MCCLUSKEY B J,HALEY C,ROVIRA A,et al.Retrospective testing and case series study of porcine delta coronavirus in U.S.swine herds[J].Prev Vet Med,2016,123:185-191.
[7]MA Y M,ZHANG Y,LIANG X Y,et al.Origin,evolution,and virulence of porcine deltacoronaviruses in the United States[J].mBio,2015,6(2):e00064.
[8]LEE J H,CHUNG H C,NGUYEN V G,et al.Detection and phylogenetic analysis of porcine deltacoronavirus in Korean swine farms,2015[J].Transbound Emerg Dis,2016,63(3):248-252.
[9]LORSIRIGOOL A,SAENG-CHUTO K,TEMEEYASEN G,et al.The first detection and full-length genome sequence of porcine deltacoronavirus isolated in Lao PDR[J].Arch Virol,2016,161(10):2909-2911.
[10]龙云凤,王毅谦,姜珊,等.猪δ冠状病毒研究进展[J].亚洲兽医病例研究,2017,6(2):21-28.LONG Y F,WANG Y Q,JIANG S,et al.Research progress on porcine deltacoronavirus[J].Asian Case Reports in Veterinary Medicine,2017,6(2):21-28.(in Chinese)
[11]CHEN F Z,ZHU Y X,WU M Z,et al.Full-length genome characterization of Chinese porcine deltacoronavirus strain CH/SXD1/2015[J].Genome Announc,2015,3(5):e01284-15.
[12]WANG Y W,YUE H,FANG W H,et al.Complete genome sequence of porcine deltacoronavirus strain CH/Sichuan/S27/2012from Mainland China[J].Genome Announc,2015,3(5):e00945-15.
[13]陈建飞,王潇博,焦贺勋,等.国内首株猪德尔塔冠状病毒(Porcine deltacoronavirus)的分离鉴定[J].中国预防兽医学报,2016,38(3):171-174.CHEN J F,WANG X B,JIAO H X,et al.Isolation and identification of the first Porcinedeltacoronavirus strain in China[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(3):171-174.(in Chinese)
[14]DONG N,FANG L R,ZENG S L,et al.Porcine deltacoronavirus in Mainland China[J].Emerg Infect Dis,2015,21(12):2254-2255.
[15]吴美洲,陈芳洲,朱银杏,等.丁型冠状病毒我国猪源株的遗传变异分析[J].中国兽医科学,2016,46(6):689-694.WU M Z,CHEN F Z,ZHU Y X,et al.Genetic variation analysis of porcine deltacoronavirus in China[J].Chinese Veterinary Science,2016,46(6):689-694.(in Chinese)
[16]SONG D,ZHOU X,PENG Q,et al.Newly emerged porcine Deltacoronavirus associated with diarrhoea in swine in China:Identification,prevalence and fulllength genome sequence analysis[J].Transbound Emerg Dis,2015,62(6):575-580.
[17]WANG L Y,HAYES J,SARVER C,et al.Porcine deltacoronavirus:Histological lesions and genetic characterization[J].Arch Virol,2016,161(1):171-175.
[18]SUO S Q G W,REN Y D,LI G X,et al.Immune responses induced by DNA vaccines bearing Spike gene of PEDV combined with porcine IL-18[J].Virus Res,2012,167(2):259-266.
[19]GOMEZ N,WIGDOROVITZ A,CASTANON S,et al.Oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus[J].Arch Virol,2000,145(8):1725-1732.
[20]THACHIL A,GERBER P F,XIAO C T,et al.Development and application of an ELISA for the detection of porcine deltacoronavirus IgG antibodies[J].PLoS One,2015,10(4):e124363.
[21]ZHAI S L,WEI W K,LI X P,et al.Occurrence and sequence analysis of porcine deltacoronaviruses in southern China[J].Virol J,2016,13:136.
[22]MAHY B W J.Virology:Molecular biology of the coronaviruses[J].Nature,1983,305(5934):474-475.
[23]BRIAN D A,BARIC R S.Coronavirus genome structure and replication[M]//ENJUANES L.Coronavirus Replication and Reverse Genetics.Berlin Heidelberg:Springer,2005.
[24]LEE H K,YEO S G.Cloning and sequence analysis of the nucleocapsid gene of porcine epidemic diarrhea virus Chinju99[J].Virus Genes,2003,26(2):207-212.
[25]CHEN Q,LI G W,STASKO J,et al.Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States[J].J Clin Microbiol,2014,52(1):234-243.
[26]SUN D B,FENG L,SHI H Y,et al.Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein[J].Vet Microbiol,2008,131(1-2):73-81.
[27]解庭波.大肠杆菌表达系统的研究进展[J].长江大学学报:自然科学版,2008,5(3):77-82.XIE T B.Research progress on E.coli expression system[J].Journal of Yangtze University:Natural Science Edition,2008,5(3):77-82.(in Chinese)
[28]苏鹏,龚国利.优化大肠杆菌表达外源蛋白的研究进展[J].生物技术通报,2017,33(2):16-23.SU P,GONG G L.Research progress on optimizing the expression of exogenous proteins in Escherichia coli[J].Biotechnology Bulletin,2017,33(2):16-23.(in Chinese)