丁型肝炎病毒ORF5基因片段的原核表达及在ELISA中的初步应用  

Prokaryotic Expression of Hepatitis Delta Virus ORF5 Gene and Its Application in ELISA

作　　者：王云龙[1,2] 王娟[1] 李玉林 董彩文 孙新城 张怡青[1] 王国强 李恒思 刘旺根 

机构地区：[1]河南师范大学,河南新乡453007 [2]郑州职业技术学院,河南郑州450001 [3]河南省生物工程技术研究中心,河南郑州450001

出　　处：《动物医学进展》2012年第5期31-34,共4页Progress In Veterinary Medicine

基　　金：河南省科技创新团队项目;郑州市科技创新团队项目

摘　　要：为获得HDAg抗原,以含有ORF5编码区基因的pETHD质粒为模板进行PCR扩增得到500bp大小片段,经过BamHⅠ、HindⅢ双酶切,与载体pET-30a连接,构建重组表达质粒pET-30a/HDAg,再转入细胞BL21(DE3),经过IPTG诱导表达和SDS-PAGE分析,表达蛋白分子质量约27ku,经过Ni-NTA亲和层析纯化后,纯度达95%以上。将纯化的蛋白经HRP标记,初步建立ELISA捕获法,通过对24份标本检测和交叉反应试验,结果表明表达的抗原具有很好的反应原性和特异性,为建立丁型肝炎早期检测试剂盒提供材料。To obtain recombinant hepatitis delta virus antigen,the fragments of HDAg amplified by PCR from pETHD was inserted into expression vector pET-30a.The recombinant plasmid was transformed into BL21（DE3）,where hepatitis delta virus antigen was induced to express by IPTG.Analyzed by SDS-PAGE,the product was 27 ku.Purified by Ni-NTA metal-affinity chromatography,protein＇s purity was 95%.The HRP-labeled protein was used to detecte 24 samples by ELISA and cross-reactivity experiments,results showed that the antigen has specific reactogenicity,which provide material source for the establishment of HDV diagnostic kit.

关 键 词：丁型肝炎病毒 丁肝抗原 原核表达 ELISA 

分 类 号：S852.659.1[农业科学—基础兽医学]