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  

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

作　　者：李伟[1,2] 孟日增[3] 石建平[3] 何江帅[1] 赵晓[1] 卢强[1] 

机构地区：[1]吉林大学人兽共患病研究所,长春130062 [2]黑龙江生物科技职业学院,哈尔滨150025 [3]吉林出入境检验检疫局,长春130062

出　　处：《中国畜牧兽医》2010年第2期83-86,共4页China Animal Husbandry & Veterinary Medicine

基　　金：国家质检总局科技计划项目(2006IK009)

摘　　要：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。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 385^th to 550^th 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 SDS-PAGE showed that the fusion protein of 21.35 ku as expected.Western blotting and indirect ELISA analysis that the recombinant protein had a specific positive reaction with standard positive blood serum.The concentration of purified protein was 2.0 mg/mL and the purity was 95.27%.The preliminary indirect ELISA method was developed by using the purified recombinant protein of gB as antigen.The results above suggested that the assay was confirmed to be specific,sensitive and quickly,which can be used as antigen for diagnosis.

关 键 词：IBRV gB蛋白 原核表达 间接ELISA 

分 类 号：S854.4[农业科学—临床兽医学]