摘要
[目的]在基因工程菌中实现猪繁殖与呼吸综合征病毒(PRRSV)ORF7基因的高效表达,对表达的重组蛋白进行纯化,并探讨其免疫学活性及应用价值。[方法]将已构建好的重组表达载体pET-ORF7转化大肠杆菌BL21(DE3),用IPTG在最适条件下诱导表达,其表达产物用SDS-PAGE和Western Blot进行检测。应用Ni-NTAHis.Bind Resin层析柱在变性条件下纯化表达产物,通过梯度透析进行复性。采用Western Blot及间接ELISA法检测复性后重组蛋白的免疫学活性。[结果]重组质粒pET-ORF7在大肠杆菌中以融合形式成功表达,融合表达产物主要以包涵体形式存在。SDS-PAGE检测所表达的重组蛋白分子量为33 kD左右,与预期大小相符。Band-scan软件对表达蛋白分析发现,表达产物约占菌体蛋白的50%。Western Blot及间接ELISA法显示复性后的重组蛋白与PRRSV阳性血清有特异性结合反应,表明其具有良好的免疫学活性。[结论]该试验将为进一步研制PRRSV单克隆抗体及诊断试剂盒奠定基础。
[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus（PRRSV） ORF7 gene in genetic engineering bacteria and analyze the immun ological activity of the recombinant protein after purification.[Method]The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia coli BL21（DE3） and induced by IPTG under the optimal condition.After analysis by SDS-PAGE and Western Blot,the expression products were purified by Ni-NTA His·Bind Resin chromatographic column under denaturing condition and renatured by gradient dialysis.Subsequently,the immun ological activity of the renatured recombinant protein was detected by Western Blot and indirect ELISA.[Result] The recombinant plasmid pET-ORF7 expressed in E.coli successfully,and the fusion protein was in the form of inclusion body.By SDS-PAGE detection,the molecular weight of the expression protein was approximate 33 kD,according with the expectation.Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21（DE3）.Western Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically,indicating its good immun ological activity.[Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.
出处
《安徽农业科学》
CAS
北大核心
2009年第10期4439-4442,4445,共5页
Journal of Anhui Agricultural Sciences
基金
北京市农林科学院青年基金项目(2007020122)
北京市科委猪高致病性蓝耳病防控技术子课题
关键词
猪繁殖与呼吸综合征病毒
ORF7基因
表达
纯化
免疫学活性
Porcine reproductive and respiratory syndrome virus
ORF7 gene
Expression
Purification
Immun ological activity
