摘要
为了进一步揭示菠菜非共生血红蛋白(SoHb)的功能,通过RT-PCR的方法从菠菜根中克隆了SoHb基因编码区全长序列,并将其克隆到原核表达载体pET32a上,构建原核表达载体p ET32a-SoHb,并转化大肠杆菌BL21 star(DE3)获得原核表达工程菌株。通过IPTG法诱导该重组质粒在大肠杆菌中得到高效表达,融合蛋白分子质量约为38 kDa,且在上清液和包涵体中均有表达,可溶性部分经Ni2+NTA亲和柱纯化,获得纯化的融合蛋白。与空载体相比,重组菌对硝化胁迫的抗性增加。以纯化的融合蛋白为抗原免疫昆明小鼠,制备多克隆抗体,并对其进行了Western blot检测。实验结果为进一步研究菠菜SoHb基因功能奠定了基础。
To further investigate the function of non symbiotic hemoglobin of spinach （SoHb）, the full length cDNA encoding SoHb was amplified by RT-PCR from spinach root and cloned into inducible expression vector pET32a. The recombinant prokaryotic expression plasmid was transformed into E.coli BL21 star （DE3） strain for genetic engineering strain. The transformed strain was induced with isopropyl-beta- -D-thiogalactoside （IPTG） for expressing fusion protein. SDS-PAGE analysis showed that the recombinant protein with a molecular mass about 38 kDa was highly expressed in E.coli and presented both in the supernatant and the pellet part of E.coli lysates. The pET32a-SoHb strain was more resistant to nitrosative stress than the pET32a empty vector strain. The supernatant was further purified by Ni2＋ NTA affinity chromatography and immunized white mouses as antigen. The polyclonal antibody was obtained and analyzed by Western blot. It laid foundation for investigating the function of SoHb.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2015年第4期54-59,共6页
China Biotechnology
基金
国家自然科学基金(31101557
31460526)
云南省应用基础研究面上项目(2010ZC053)
云南省教育厅科学研究基金(2011Z109)资助项目
关键词
非共生血红蛋白
大肠杆菌表达
硝化胁迫
Non-symbiotic hemoglobin
Escherichia coli expression
Nitrosative stress
