基于PCV3-Cap蛋白间接ELISA检测方法的建立及临床应用

Establishment and Clinical Application of an Indirect ELISA for Detection of Antibodies against PCV3 Based on Recombinant ORF2 Protein

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 a part 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-30a vector.The recombinant plasmid was transfected into E.coli BL21 competent cells,and the best induction conditions were determined.The expression protein was purified by Ni-NTA 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-30a,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 1 μg·mL^-1,1:20 and 1:5000,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.