摘要
利用RT-PCR方法扩增旋毛虫HSP70基因,将其扩增产物克隆到pEASY-Blunt Simple载体中,经限制性酶切后与pET-28a(+)载体连接,进一步转化至感受态细胞Transetta(DE3)中,用IPTG诱导表达重组旋毛虫HSP70。以纯化的重组HSP70制备多克隆抗体,并对HSP70进行免疫印迹分析及免疫组织化学染色。结果表明,重组质粒的鉴定和DNA测序均正确;SDS-PAGE和Western-blot分析结果显示,在70ku处获得一目的条带;免疫组织化学染色结果显示,该HSP70具有很好的免疫原性。证实,成功构建了重组表达质粒pET28a-HSP70,为深入研究旋毛虫HSP70的功能奠定了基础。
HSP70 gene of Trichinella spiralis was amplified by RT-PCR and then cloned into pEASY-Blunt Simple vector.The recombinant was digested and linked with pET-28a（＋） vector.The recombinant plasmid was confirmed by PCR and DNA sequencing.The positive plasmid was transformed into competent Escherichia coli Transetta（DE3） and induced by IPTG for expression.The recombinant Trichinella HSP70 was purified to prepare polyclonal antibody.Trichinella HSP70 was analysed by Western-blot and stained by immunohistochemistry.The recombinant protein was confirmed to be about 70 ku in size.The positive signals were localized in the Trichinella by immunohistochemistry.The results showed that the recombinant expression plasmid pET28a-HSP70 was successfully constructed and laid the foundation for further studies on the function of HSP70.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2012年第3期275-279,共5页
Chinese Veterinary Science
基金
"十一五"国家科技支撑计划重大项目(2010BAD04B01)
国家自然科学基金资助项目(31172312)
家畜疫病病原生物学国家重点实验室开放课题(SKLVEB2009KFKT005)
