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.
出处
《动物医学进展》
CSCD
北大核心
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
关键词
丁型肝炎病毒
丁肝抗原
原核表达
ELISA
Hepatitis delta virus
Hepatitis delta antigen
prokaryotic expression
ELISA
