引用本文:
【打印本页】   【下载PDF全文】   查看/发表评论  下载PDF阅读器  关闭
←前一篇|后一篇→ 过刊浏览    高级检索
本文已被:浏览 2193次   下载 1625 本文二维码信息
码上扫一扫!
分享到: 微信 更多
NS1基因主要抗原区的可溶性表达及其Dot-ELISA检测方法的建立
赵 朴1,2, 苏红辽3, 刘兴友,等1,2
1.河南科技学院 动物科学学院;2.动物疫病和残留物防控河南省高校工程技术研究中心;3.滑县动物卫生监督所
摘要:
【目的】可溶性表达猪流感病毒(SIV)NS1基因主要抗原区(NS1′)蛋白,获得具有良好抗原性的NS1′蛋白,并初步建立检测NS1抗体的斑点酶联吸附试验(Dot-ELISA)方法,为鉴别诊断猪流感病毒感染猪与疫苗免疫猪奠定基础。【方法】以质粒pET28a-NS1为模板,PCR扩增NS1基因抗原性较好的区段NS1′,用EcoRⅠ和XhoⅠ 双酶切后插入pET-32a(+),构建pET32a-NS1′,经PCR、EcoRⅠ和XhoⅠ双酶切及DNA测序鉴定后,转化大肠杆菌Rosetta,用IPTG诱导表达,纯化NS1′并免疫家兔制备多克隆血清,对其进行SDS-PAGE、Western blotting 检测和免疫荧光分析,并以纯化的NS1′作为包被抗原初步建立Dot-ELISA检测方法。【结果】PCR扩增获得了长约250 bp的H3N2 SIV NS1′片段;成功构建了重组载体pET32a NS1′;SDS-PAGE 和 Western blotting 检测结果显示,融合蛋白NS1′大小约34 ku,该蛋白可溶,能与感染SIV的猪血清特异性反应,并且用纯化蛋白NS1′制备的多克隆血清能识别293T细胞中表达的NS1,建立的Dot-ELISA检测方法可鉴别猪流感病毒感染猪与疫苗免疫猪。【结论】实现了H3N2 SIV NS1′蛋白的可溶性表达,表达产物具有良好的抗原性,以NS1′作为包被抗原,建立了检测NS1抗体的Dot-ELISA方法。
关键词:  猪流感病毒  非结构蛋白1  主要抗原区域  可溶性表达  斑点酶联免疫吸附试验
DOI:
分类号:
基金项目:河南省基础与前沿技术项目(122300410135);河南省教育厅自然科学研究项目(2011A230008)
Soluble expression of main antigen region of NS1 gene from H3N2 SIV in Henan and development of Dot-ELISA detection method
ZHAO Pu,SU Hong-liao,LIU Xing-you,et al
Abstract:
【Objective】This study aimed to express the soluble main antigen region(NS1′) of NS1 gene,acquire NS1′ with good antigenicity,and develop Dot-ELISA method for detecting NS1 antibody so as to differentiate infected from vaccinated pigs.【Method】Using pET28a-NS1 as template,NS11′ of H3N2 SIV in Henan was amplified by PCR,digested with EcoRⅠ and XhoⅠand cloned into prokaryotic expression vector pET-32a(+).The recombinant pET32a-NS1′ was identified by PCR,EcoRⅠ/XhoⅠdigestion and DNA sequencing.Expression of NS1′ was induced by IPTG in Rosetta and purified,and immune serum was prepared by vaccinating rabbits.The recombinant NS1′ was then analyzed by SDS PAGE,Western blotting and immunofluorescence.Using NS1′ as coating antigen,Dot-ELISA detection method was developed.【Result】NS1′ of H3N2 SIV with length of ~250 bp was amplified by PCR and the recombinant pET32a NS1′ was constructed.SDS-PAGE and Western blotting showed that the expressed protein NS1′ was ~34 ku and soluble and it also reacted specifically with serum from pig infected with SIV.Immune serum prepared with NS1′ recognized NS1 expressed in 293T cells.The established Dot-ELISA method was able to differentiate infected pigs from vaccinated pigs.【Conclusion】NS1′ was successfully expressed in soluble form,expressed NS1′ has good antigenicity and Dot-ELISA method for detecting NS1 antibody was developed.
Key words:  swine influenza virus  nonstructural protein 1  major antigen region  soluble expression  Dot-ELISA