猪δ冠状病毒N蛋白的原核表达和间接ELISA检测方法的建立
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摘要
【目的】制备猪δ冠状病毒(porcine deltacoronavirus,PDCoV)N蛋白及其多克隆抗体,建立检测PDCoV N蛋白抗体的间接ELISA方法。【方法】从PDCoV CH-01毒株中扩增N全基因,克隆至原核表达载体pET-28a中,构建重组表达质粒pET-28a-PDCoV-N,转入BL21(DE3),用IPTG诱导表达重组N蛋白,并对该蛋白的反应原性进行鉴定。诱导表达的N蛋白经镍柱纯化后免疫新西兰大白兔制备多克隆抗体。同时用镍柱纯化后的N蛋白作为包被抗原,建立PDCoV的间接ELISA检测方法,对该方法的特异性、重复性和稳定性进行检测,并利用该方法对河南部分地区猪场送检的165份临床猪血清样本进行PDCoV抗体检测。【结果】成功克隆了1 026 bp的N基因,构建了重组表达载体pET-28a-PDCoV-N,经诱导表达后获得了重组N蛋白,其相对分子质量约42.9 ku,Western blot分析表明所表达的蛋白可与抗PDCoV全病毒猪阳性血清发生反应。制备的PDCoV N蛋白多克隆抗体经Western blot检测表明,该抗体具有特异性;间接免疫荧光试验测定该多克隆抗体在ST细胞上的最佳使用稀释度为1∶200。以N蛋白作为包被抗原建立了间接ELISA方法,确定最佳的反应条件如下:抗原2μg/mL在4℃过夜包被,10 g/L BSA在37℃下封闭2 h,血清(1∶80稀释)37℃孵育1 h,酶标二抗(1∶6 000稀释)37℃孵育1 h,TMB在37℃下显色15 min;待检血清OD_(450)值≥0.272判定为阳性。特异性试验结果显示,该包被抗原与其他猪常见病毒的阳性血清均无交叉反应,批内和批间重复试验变异系数均小于5%。用间接ELISA法对来自河南不同地区的165份血清样本进行检测,结果表明各地样本PDCoV抗体阳性率平均为53.94%。【结论】成功表达了PDCoV N蛋白,制备了其多克隆抗体,建立了PDCoV间接ELISA检测方法,该方法具有良好的特异性、重复性和稳定性,可用于临床猪血清中PDCoV抗体的检测。
【Objective】 This study prepared N protein and polyclonal antibody of porcine deltacoronavirus(PDCoV),and developed an indirect ELISA to detect PDCoV antibody of clinical pig serum samples from pig farms.【Method】 The N gene was amplified from the PDCoV CH-01 strain and cloned into pET-28 a vector.The recombinant protein was expressed in E. coli BL21 by induction with IPTG.The recombinant N protein purified from the nickel column was emulsified with Freund's adjuvant,and the polyclonal antibody of anti-PDCoV-N protein was prepared by immunizing rabbits.An indirect ELISA method for detection of PDCoV antibody was established using N protein.After the specificity,repeatability and stability were detected,it was used for clinical detection of 165 clinical swine serum samples from pig farms in Henan.【Result】 The N gene was cloned successfully,and the recombinant expression vector pET-28 a-PDCoV-N was constructed.After induced expression,N protein was obtained and the protein molecular mass was approximately 42.9 ku.The recombinant N protein can react specifically with PDCoV positive serum.The specificity of polyclonal antibody was detected by Western blot.The titers of polyclonal antibodies were 200 by indirect immunofluorescence test in ST cell.This protein was used to establish an indirect ELISA method,the optimal antigen coating concentration was 2 μg/mL at 4 ℃ overnight and blocked at 37 ℃ for 2 h with 10 g/L BSA.The optimal serum dilution was 1∶80 and incubated at 37 ℃ for 1 h.The optimal dilution of the secondary antibody was 1∶6 000 and incubated at 37 ℃ for 1 h,and TMB color at 37 ℃ for 15 min.The serum was determined to be positive when the OD_(450) value was ≥0.272.The specific test results showed that there were no cross reactions of the coated antigen with the positive sera of other porcine viruses.Intra-and inter-assay variation coefficients were less than 5%.The ELISA method was used to detect 165 clinical swine serum samples and the positive rate of PDCoV antibody average was 53.94%.【Conclusion】 This study expressed the PDCoV N protein,prepared rabbit polyclonal antibody and established the indirect ELISA method.The method had good specificity,reproducibility and stability,and can be used for the detection of PDCoV antibody in clinic.
【Objective】 This study prepared N protein and polyclonal antibody of porcine deltacoronavirus(PDCoV),and developed an indirect ELISA to detect PDCoV antibody of clinical pig serum samples from pig farms.【Method】 The N gene was amplified from the PDCoV CH-01 strain and cloned into pET-28 a vector.The recombinant protein was expressed in E. coli BL21 by induction with IPTG.The recombinant N protein purified from the nickel column was emulsified with Freund's adjuvant,and the polyclonal antibody of anti-PDCoV-N protein was prepared by immunizing rabbits.An indirect ELISA method for detection of PDCoV antibody was established using N protein.After the specificity,repeatability and stability were detected,it was used for clinical detection of 165 clinical swine serum samples from pig farms in Henan.【Result】 The N gene was cloned successfully,and the recombinant expression vector pET-28 a-PDCoV-N was constructed.After induced expression,N protein was obtained and the protein molecular mass was approximately 42.9 ku.The recombinant N protein can react specifically with PDCoV positive serum.The specificity of polyclonal antibody was detected by Western blot.The titers of polyclonal antibodies were 200 by indirect immunofluorescence test in ST cell.This protein was used to establish an indirect ELISA method,the optimal antigen coating concentration was 2 μg/mL at 4 ℃ overnight and blocked at 37 ℃ for 2 h with 10 g/L BSA.The optimal serum dilution was 1∶80 and incubated at 37 ℃ for 1 h.The optimal dilution of the secondary antibody was 1∶6 000 and incubated at 37 ℃ for 1 h,and TMB color at 37 ℃ for 15 min.The serum was determined to be positive when the OD_(450) value was ≥0.272.The specific test results showed that there were no cross reactions of the coated antigen with the positive sera of other porcine viruses.Intra-and inter-assay variation coefficients were less than 5%.The ELISA method was used to detect 165 clinical swine serum samples and the positive rate of PDCoV antibody average was 53.94%.【Conclusion】 This study expressed the PDCoV N protein,prepared rabbit polyclonal antibody and established the indirect ELISA method.The method had good specificity,reproducibility and stability,and can be used for the detection of PDCoV antibody in clinic.
引文
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[14] Le V P,Song S,An B H,et al.A novel strain of porcine deltacoronavirus in Vietnam [J].Arch Virol,2018,163(1):203-207.
[15] 韦学雷,梁青青,曹贝贝,等.猪Delta冠状病毒、猪传染性胃肠炎病毒和猪流行性腹泻病毒多重RT-PCR检测方法的建立及应用 [J].中国兽医学报,2018(1):11-16.Wei X L,Liang Q Q,Cao B B,et al.Establishment and application of a multiplex RT-PCR assay for simultaneous detection of porcine delta coronavirus,porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus [J].Chinese Journal of Veterinary Science,2018(1):11-16.
[16] 张帆帆,宋德平,郭楠楠,等.以原核表达的猪δ冠状病毒N蛋白为包被抗原的间接ELISA方法的建立 [J].中国预防兽医学报,2016,38(10):795-799.Zhang F F,Song D P,Guo N N,et al.Establishment of a recombinant nucleoprotein-based ELISA for detection of antibodies against newly emerged porcine deltacoronavirus [J].Chinese Journal of Preventive Veterinary Medicine,2016,38(10):795-799.
[17] Kapust R B,Waugh D S.Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused [J].Protein Sci,2010,8(8):1668-1674.
[18] Terpe K.Overview of tag protein fusions:from molecular and biochemical fundamentals to commercial systems [J].Appl Microbiol Biotechnol,2003,60(5):523-533.
[19] Marblestone J G,Edavettal S C,Lim Y,et al.Comparison of SUMO fusion technology with traditional gene fusion systems:enhanced expression and solubility with SUMO [J].Protein Sci,2010,15(1):182-189.
[20] Ma Y M,Zhang Y,Liang X Y,et al.Origin,evolution,and virulence of porcine deltacoronaviruses in the United States [J].M Bio,2015,6(2):e00064.
[21] 郑景生,吕蓓.PCR技术及实用方法 [J].分子植物育种,2003,1(3):381-394.Zheng J S,Lü B.PCR technique and its practical methods [J].Molecular Plant Breeding,2003,1(3):381-394.
[22] Hu H,Jung K,Vlasova A N,et al.Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States [J].J Clin Microbiol,2015,53(5):1537-1548.
[23] Hu H,Jung K,Vlasova A N,et al.Experimental infection of gnotobiotic pigs with the cell-culture-adapted porcine deltacoronavirus train OH-FD22 [J].Arch Virol,2016,161(12):3421-3434.
[24] 舒静超.猪δ冠状病毒E和S1蛋白的原核表达及多克隆抗体的制备 [D].郑州:河南农业大学,2016.Shu J C.The prokaryotic expression and polyclonal antibodies preparation of porcine deltacoronaviruses protein E and S1 [D].Zhengzhou:Henan Agriculture University,2016.
[25] 方谱县,方六荣,董楠,等.猪δ冠状病毒的研究进展 [J].病毒学报,2016,32(2):243-248.Fang P X,Fang L R,Dong N,et al.Research advances in the porcine deltacoronavirus [J].Chinese Journal of Virology,2016,32(2):243-248.
[2] Homwong N,Jarvis M C,Lam H C,et al.Characterization and evolution of porcine deltacoronavirus in the United States [J].Prev Vet Med,2016,123(2):168-174.
[3] Wang L Y,Hayes J,Sarver C,et al.Porcine deltacoronavirus:histological lesions and genetic characterization [J].Arch Virol,2015,161(1):171-175.
[4] Jung K,Hu H,Eyerly B,et al.Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs [J].Emerg Infect Dis,2015,21(4):650-654.
[5] Li G W,Chen Q,Harmon K M,et al.Full-length genome sequence of porcine deltacoronavirus strain USA/IA/2014/8734 [J].Genome Announc,2014,2(2):e00278.
[6] Martmaler D,Jiang Y,Collins J E,et al.Complete genome sequence of strain SDCV/USA/Illinois121/2014,a porcine deltacoronavirus from the United States [J].Genome Announc,2014,2(2):e00218.
[7] Woo P C Y,Lau S K P,Lam C S F,et al.Discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacor navirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavir [J].J Virol,2012,86(7):3995-4008.
[8] Marthater D,Raymond L,Jiang Y,et al.Rapid detection,complete genome sequencing,and phylogenetic analysis of porcine deltacoronavirus [J].Emerg Infect Dis,2014,20(8):1347-1350.
[9] Sinha A,Gauger P,Zhang J Q,et al.PCR-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in US swine [J].Vet Microbiol,2015,179(3/4):296-298.
[10] 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.
[11] Dong N,Fang L R,Zeng S L,et al.Porcine deltacoronavirus in mainland China [J].Emerg Infect Dis,2015,21(12):2254-2255.
[12] Wang Y W,Yue H,Fang W H,et al.Complete genome sequence of porcine deltacoronavirus strain CH/Sichuan/S27/2012 from mainland China [J].Genome Announc,2015,3(5):e00945.
[13] Madapong A,Saeng-chuto K,LorsirIgool A,et al.Complete genome sequence of porcine deltacoronavirus isolated in Thailand in 2015 [J].Genome Announc,2016,4(3):e00408.
[14] Le V P,Song S,An B H,et al.A novel strain of porcine deltacoronavirus in Vietnam [J].Arch Virol,2018,163(1):203-207.
[15] 韦学雷,梁青青,曹贝贝,等.猪Delta冠状病毒、猪传染性胃肠炎病毒和猪流行性腹泻病毒多重RT-PCR检测方法的建立及应用 [J].中国兽医学报,2018(1):11-16.Wei X L,Liang Q Q,Cao B B,et al.Establishment and application of a multiplex RT-PCR assay for simultaneous detection of porcine delta coronavirus,porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus [J].Chinese Journal of Veterinary Science,2018(1):11-16.
[16] 张帆帆,宋德平,郭楠楠,等.以原核表达的猪δ冠状病毒N蛋白为包被抗原的间接ELISA方法的建立 [J].中国预防兽医学报,2016,38(10):795-799.Zhang F F,Song D P,Guo N N,et al.Establishment of a recombinant nucleoprotein-based ELISA for detection of antibodies against newly emerged porcine deltacoronavirus [J].Chinese Journal of Preventive Veterinary Medicine,2016,38(10):795-799.
[17] Kapust R B,Waugh D S.Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused [J].Protein Sci,2010,8(8):1668-1674.
[18] Terpe K.Overview of tag protein fusions:from molecular and biochemical fundamentals to commercial systems [J].Appl Microbiol Biotechnol,2003,60(5):523-533.
[19] Marblestone J G,Edavettal S C,Lim Y,et al.Comparison of SUMO fusion technology with traditional gene fusion systems:enhanced expression and solubility with SUMO [J].Protein Sci,2010,15(1):182-189.
[20] Ma Y M,Zhang Y,Liang X Y,et al.Origin,evolution,and virulence of porcine deltacoronaviruses in the United States [J].M Bio,2015,6(2):e00064.
[21] 郑景生,吕蓓.PCR技术及实用方法 [J].分子植物育种,2003,1(3):381-394.Zheng J S,Lü B.PCR technique and its practical methods [J].Molecular Plant Breeding,2003,1(3):381-394.
[22] Hu H,Jung K,Vlasova A N,et al.Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States [J].J Clin Microbiol,2015,53(5):1537-1548.
[23] Hu H,Jung K,Vlasova A N,et al.Experimental infection of gnotobiotic pigs with the cell-culture-adapted porcine deltacoronavirus train OH-FD22 [J].Arch Virol,2016,161(12):3421-3434.
[24] 舒静超.猪δ冠状病毒E和S1蛋白的原核表达及多克隆抗体的制备 [D].郑州:河南农业大学,2016.Shu J C.The prokaryotic expression and polyclonal antibodies preparation of porcine deltacoronaviruses protein E and S1 [D].Zhengzhou:Henan Agriculture University,2016.
[25] 方谱县,方六荣,董楠,等.猪δ冠状病毒的研究进展 [J].病毒学报,2016,32(2):243-248.Fang P X,Fang L R,Dong N,et al.Research advances in the porcine deltacoronavirus [J].Chinese Journal of Virology,2016,32(2):243-248.
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