基于ORF3蛋白的PCV2间接ELISA诊断方法的建立

发布时间：2018-06-26 18:23

  本文选题：猪圆环病毒型 + ORF　； 参考：《中国兽医科学》2017年04期

【摘要】：ORF3蛋白是猪圆环病毒2型(porcine circovirus type,2PCV2)的一个重要的非结构蛋白。根据GenBank数据库中PCV2的ORF3基因序列设计引物,以PCV2基因组为模板进行PCR扩增,并构建重组表达质粒。将重组质粒转染至BL21感受态细胞,进行最佳诱导条件的选择,采用Ni-NTA亲和层析柱纯化表达产物。以纯化后的重组ORF3蛋白作为酶标包被抗原,优化反应条件,建立PCV2间接ELISA(indirect ELISA,iELISA)诊断方法。结果显示,重组质粒经PCR鉴定、酶切鉴定和测序鉴定构建成功。重组蛋白表达的最佳诱导条件为37℃、8 h、1 mmol/LIPTG。Western-blot结果表明,该重组蛋白具有较好的免疫原性。Ni-NTA亲和层析纯化结果表明,280 mmol/L咪唑为最佳洗脱浓度。iELISA的最佳包被抗原质量浓度为0.03 mg/m L,待检血清最佳稀释度为1∶100,酶标抗体最佳工作浓度为1∶4 000。批内和批间试验结果表明,该方法具有较好的重复性。此iELISA与PCR检测方法具有良好的一致性。结果表明,基于ORF3蛋白建立的PCV2间接ELISA诊断方法,可以为临床上rCap蛋白基因工程疫苗免疫猪场进行鉴别诊断。
[Abstract]:ORF3 protein is an important nonstructural protein of porcine circovirus type 2 (porcine circovirus type 2 PCV2. According to the sequence of ORF3 gene of PCV2 in GenBank database, primers were designed and PCR amplification was carried out using PCV2 genome as template, and the recombinant expression plasmid was constructed. The recombinant plasmid was transfected into BL21 cells and the optimal induction conditions were selected. The expression product was purified by Ni-NTA affinity chromatography. The purified recombinant ORF3 protein was used as the coated antigen, and the reaction conditions were optimized. An indirect Elisa method for the diagnosis of PCV2 was established. The results showed that the recombinant plasmid was successfully constructed by PCR, enzyme digestion and sequencing. The optimal inducing conditions for the expression of recombinant protein were as follows: 1 mmol / L LIPT G. Western-blot at 37 鈩，

本文编号：2070992