基于PCV3-Cap蛋白间接ELISA检测方法的建立及临床应用
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摘要
为了建立一种敏感和特异的猪圆环病毒3型(PCV3)抗体检测方法,对PCV3-Cap蛋白抗原表位预测发现其抗原表位多聚集在C端(羧基端),而N端前33氨基酸为核定位序列。以截断N端前120个氨基酸后的PCV3ORF2序列为靶基因,设计引物。以PCV3阳性病料为模板,PCR扩增截短的ORF2基因,并将其克隆至pET-30a载体构建重组质粒,并转染至大肠杆菌E.coli BL21感受态细胞,获得重组菌后选择最佳诱导表达条件,采用Ni-NTA亲和层析柱纯化表达产物。以纯化后的重组Cap蛋白作为包被抗原,建立PCV3间接ELISA (indirectELISA)诊断方法,并初步用于临床样品检测。结果:从阳性病料中扩增出大小为285bp的PCV3ORF2基因片段,重组质粒经双酶切和测序鉴定构建成功。采用1mmol·L-1 IPTG诱导,在37℃条件下培养6h重组菌,重组蛋白获最佳表达。Western blot结果表明,该重组蛋白与PCV3阳性血清具有较好的反应原性。ELISA的最佳包被抗原质量浓度为1μg·mL-1,待检血清最佳稀释度为1∶20,酶标抗体最佳工作浓度为1∶5 000。阳性判定值为S/P≥0.273。批内和批间系数均小于10%,表明该方法具有较好的重复性。PCV2阳性血清用本方法检测为阴性,表明该方法有较好的特异性。检测采集的439份临床猪血清,PCV3抗体阳性检出率为60.59%(266/439)。结果表明,本研究建立了一种快速、简便、敏感、特异的猪圆环病毒3型(PCV3)抗体检测方法。
The aim of this study was to establish a sensitive and specific method for detection of antibody against porcine circovirus type 3(PCV3).The bioinformatical prediction shows that most antigen epitopes of PCV3 Cap protein are clustered at the C terminal(carboxyl terminal)of PCV3-Cap protein,while first 33 amino acids in the N terminal of Cap protein are the nuclear localization sequences.Primers were designed to amplify apart of ORF2 gene which does not contain first 120 amino acids of N terminal and PCV3 positive samples were used as templates for PCR amplification.The recombinant plasmid was constructed by cloning the PCR product into pET-30 avector.The recombinant plasmid was transfected into E.coli BL21 competent cells,and the best induction conditions were determined.The expression protein was purified by NiNTA affinity chromatography.Finally,using the Cap protein as a coating protein,an indirect ELISA for detection of antibody against PCV3 was established and then primary applied to clinical detection.As a result:PCV3 ORF2 gene fragment with size of 285 bp was amplified from positive sample.The recombinant plasmid was constructed by inserting the amplicon into pET-30 a,followed by enzymatical digestion and sequencing.Expression of the recombinant protein was induced by adding 1 mmol·L-1 IPTG,incubating at 37 ℃ for 6 h.Western blot result showed that the recombinant protein possessed good reactivity with positive serum against PCV3.The optimal antigen concentration,dilution of testing serum,working dilution of enzyme conjugated antibody of ELISA were 1μg·mL-1,1∶20 and 1∶5 000,respectively.The positive value was 0.273 and the coefficients of inter-and intra-batches were less than 10%,indicating the good repeatability of the method.PCV2 positive serum was negative in this method,indicating its high specificity.Finally,439 clinical serum samples were detected through this ELISA method and 60.59%(266/439)of them were identified as positive.In summary,a rapid,easy to use,sensitive and specific method for detection of antibody against porcine circovirus type 3(PCV3)was established.
The aim of this study was to establish a sensitive and specific method for detection of antibody against porcine circovirus type 3(PCV3).The bioinformatical prediction shows that most antigen epitopes of PCV3 Cap protein are clustered at the C terminal(carboxyl terminal)of PCV3-Cap protein,while first 33 amino acids in the N terminal of Cap protein are the nuclear localization sequences.Primers were designed to amplify apart of ORF2 gene which does not contain first 120 amino acids of N terminal and PCV3 positive samples were used as templates for PCR amplification.The recombinant plasmid was constructed by cloning the PCR product into pET-30 avector.The recombinant plasmid was transfected into E.coli BL21 competent cells,and the best induction conditions were determined.The expression protein was purified by NiNTA affinity chromatography.Finally,using the Cap protein as a coating protein,an indirect ELISA for detection of antibody against PCV3 was established and then primary applied to clinical detection.As a result:PCV3 ORF2 gene fragment with size of 285 bp was amplified from positive sample.The recombinant plasmid was constructed by inserting the amplicon into pET-30 a,followed by enzymatical digestion and sequencing.Expression of the recombinant protein was induced by adding 1 mmol·L-1 IPTG,incubating at 37 ℃ for 6 h.Western blot result showed that the recombinant protein possessed good reactivity with positive serum against PCV3.The optimal antigen concentration,dilution of testing serum,working dilution of enzyme conjugated antibody of ELISA were 1μg·mL-1,1∶20 and 1∶5 000,respectively.The positive value was 0.273 and the coefficients of inter-and intra-batches were less than 10%,indicating the good repeatability of the method.PCV2 positive serum was negative in this method,indicating its high specificity.Finally,439 clinical serum samples were detected through this ELISA method and 60.59%(266/439)of them were identified as positive.In summary,a rapid,easy to use,sensitive and specific method for detection of antibody against porcine circovirus type 3(PCV3)was established.
引文
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[6]湛洋,王东亮,王乃东,等.猪圆环病毒3型检测及其Cap结构序列和抗原性预测分析[J].畜牧兽医学报,2017,48(6):1076-1084.ZHAN Y,WANG D L,WANG N D,et al.Survey on detection and analyses of Cap antigenicity prediction of porcine circovirus type 3isolated from partial provinces of southern China[J].Acta Veterinaria et Zootechnica Sinica,2017,48(6):1076-1084.(in Chinese)
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[8]WEN S,SUN W,LI Z,et al.The detection of porcine circovirus 3in Guangxi,China[J].Transbound Emerg Dis,2018,65(1):27-31.
[9]ZHENG S,WU X,ZHANG L,et al.The occurrence of porcine circovirus 3without clinical infection signs in Shandong Province[J].Transbound Emerg Dis,2017,64(5):1337-1341.
[10]DENG J H,LI X D,ZHENG D D,et al.Establishment and application of an indirect ELISA for porcine circovirus 3[J].Arch Virol,2018,163(2):479-482.
[11]STADEJEK T,WO Z′NIAK A,MILEK D,et al.First detection of porcine circovirus type 3on commercial pig farms in Poland[J].Transbound Emerg Dis,2017,64(5):1350-1353.
[12]KWON T,YOO S J,PARK C K,et al.Prevalence of novel porcinecircovirus 3in Korean pig populations[J].Vet Microbiol,2017,207:178-180.
[13]KU X,CHEN F,LI P,et al.Identification and genetic characterization of porcinecircovirus type 3in China[J].Transbound Emerg Dis,2017,64(3):703-708.
[14]WANG J C,ZHANG Y N,WANG J F,et al.Development of a TaqMan-based real-time PCR assay for the specific detection of porcine circovirus 3[J].JVirol Methods,2017,248:177-180.
[15]KIM H R,PARK Y R,LIM D R,et al.Multiplex real-time polymerase chain reaction for the differential detection of porcinecircovirus 2and 3[J].J Virol Methods,2017,250:11-16.
[16]PARK Y R,KIM H R,KIM S H,et al.Loop-mediated isothermal amplification assay for the rapid and visual detection of novel porcine circovirus 3[J].JVirol Methods,2018,253:26-30.
[17]NAWAGITGUL P,HARMS P A,MOROZOV I,et al.Modified indirect porcine circovirus(PCV)type2-based and recombinant capsid protein(ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV[J].Clin Diagn Lab Immunol,2002,9(1):33-40.
[2]TISCHER I,RASCH R,TOCHTERMANN G.Characterization of papovavirus-and picornavirus-like particles in permanent pig kidney cell lines[J].Zentralbl Bakteriol Orig A,1974,226(2):153-167.
[3]KIUPEL M,STEVENSON G W,MITTAL S K,et al.Circovirus-like viral associated disease in weaned pigs in Indiana[J].Vet Pathol,1998,35(4):303-307.
[4]ALLAN G M,NEILLY F M,MEEHAN B M,et al.Isolation andcharacterisation of circoviruses from pigs with wasting syndromes in Spain,Denmark and Northern Ireland[J].Vet Microbiol,1999,66(2):115-123.
[5]PALINSKI R,PIEYRO P,SHANG P C,et al.Anovel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure[J].JVirol,2017,91(1):e01879-16.
[6]湛洋,王东亮,王乃东,等.猪圆环病毒3型检测及其Cap结构序列和抗原性预测分析[J].畜牧兽医学报,2017,48(6):1076-1084.ZHAN Y,WANG D L,WANG N D,et al.Survey on detection and analyses of Cap antigenicity prediction of porcine circovirus type 3isolated from partial provinces of southern China[J].Acta Veterinaria et Zootechnica Sinica,2017,48(6):1076-1084.(in Chinese)
[7]NAWAGITGUL P,MOROZOV I,BOLIN S R,et al.Open reading frame 2of porcine circovirus type 2encodes a major capsid protein[J].J Gen Virol,2000,81(9):2281-2287.
[8]WEN S,SUN W,LI Z,et al.The detection of porcine circovirus 3in Guangxi,China[J].Transbound Emerg Dis,2018,65(1):27-31.
[9]ZHENG S,WU X,ZHANG L,et al.The occurrence of porcine circovirus 3without clinical infection signs in Shandong Province[J].Transbound Emerg Dis,2017,64(5):1337-1341.
[10]DENG J H,LI X D,ZHENG D D,et al.Establishment and application of an indirect ELISA for porcine circovirus 3[J].Arch Virol,2018,163(2):479-482.
[11]STADEJEK T,WO Z′NIAK A,MILEK D,et al.First detection of porcine circovirus type 3on commercial pig farms in Poland[J].Transbound Emerg Dis,2017,64(5):1350-1353.
[12]KWON T,YOO S J,PARK C K,et al.Prevalence of novel porcinecircovirus 3in Korean pig populations[J].Vet Microbiol,2017,207:178-180.
[13]KU X,CHEN F,LI P,et al.Identification and genetic characterization of porcinecircovirus type 3in China[J].Transbound Emerg Dis,2017,64(3):703-708.
[14]WANG J C,ZHANG Y N,WANG J F,et al.Development of a TaqMan-based real-time PCR assay for the specific detection of porcine circovirus 3[J].JVirol Methods,2017,248:177-180.
[15]KIM H R,PARK Y R,LIM D R,et al.Multiplex real-time polymerase chain reaction for the differential detection of porcinecircovirus 2and 3[J].J Virol Methods,2017,250:11-16.
[16]PARK Y R,KIM H R,KIM S H,et al.Loop-mediated isothermal amplification assay for the rapid and visual detection of novel porcine circovirus 3[J].JVirol Methods,2018,253:26-30.
[17]NAWAGITGUL P,HARMS P A,MOROZOV I,et al.Modified indirect porcine circovirus(PCV)type2-based and recombinant capsid protein(ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV[J].Clin Diagn Lab Immunol,2002,9(1):33-40.