牛传染性鼻气管炎病毒截短gB基因原核表达与间接ELISA诊断方法建立

用DNAstar-Protean分析牛传染性鼻气管炎病毒gB蛋白的亲水性、抗原性和表面展示概率,据IBRVgB全基因序列设计引物,PCR扩增编码385-550位氨基酸的基因序列。将目的片段克隆,酶切鉴定并测序鉴定后,定向连接到pET-28b载体上,转化表达菌DE3并诱导表达。经SDS-PAGE分析,获得大小约为21.4 ku的目的蛋白,与预测值相符。纯化后的蛋白浓度约为2.0 mg/mL,纯度为95.27%。间接ELISA及Western blotting证明,表达的目的蛋白具有抗原性。以该蛋白作为诊断抗原建立的间接ELISA诊断方法,确定的抗原包被量为50μg/mL,血清的最佳稀释倍数为50。与进口IBRV全毒ELISA抗体诊断试剂盒比较,特异性为92.3%、敏感性为93.8%、符合率为93.3%;试验结果表明该诊断方法具有良好的特异性和敏感性。

Prokaryotic Expression of Infectious Bovine Rhinotracheitis Virus Recombinant Truncated Glycoprotein B and Development of Indirect ELISA for Detection of it's Antibody

The hydrophilicity,antigenic index and surface probability of glycoprotein B(gB) of infectious bovine rhinotracheitis virus(IBRV) were analyzed by biosoftware DNAStar-Protean.Amplified the sequence which codes the amino acid residues from 385th to 550th in major antigen determinant region of gB glycoprotein.Then the purified products were cloned into pMD18-T vector and sequenced.The truncated gB gene was inserted into pET-28b vector which was transformed to competent Escherichia coli DE3 for expressing.The ...