NS1基因主要抗原区的可溶性表达及其DotELISA检测方法的建立  

Soluble expression of main antigen region of NS1 gene from H3N2 SIV in Henan and development of Dot-ELISA detection method

作　　者：赵朴[1,2] 苏红辽 刘兴友 赵坤[1,2] 胡建和 王三虎[1,2] 姚四新 郑玉姝[1,2] 

机构地区：[1]河南科技学院动物科学学院,河南新乡453003 [2]动物疫病和残留物防控河南省高校工程技术研究中心,河南新乡453003 [3]滑县动物卫生监督所,河南安阳456400

出　　处：《西北农林科技大学学报（自然科学版）》2015年第6期35-40,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：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)

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250bp的H3N2SIV 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34ku,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H3N2SIV 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。【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,NS1′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/XhoⅠdigestionand 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 ~250bp was amplified by PCR and the recombinant pET32a-NS1′was constructed.SDS-PAGE and Western blotting showed that the expressed protein NS1′was^34ku and soluble and it also reacted specifically with serum from pig infected with SIV.Immune serum prepared with NS1′recognized NS1 expressed in 293 Tcells.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.

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 

分 类 号：S[农业科学]