摘要
以重组克隆质粒p0390-MF-NASHOR1为模板,通过聚合酶链式反应(polymerase chain reaction,PCR)扩增出烟碱酰胺合成酶基因(nas)片段并将其克隆到原核表达载体pGEM-KG中,获得重组表达质粒pKG-NAS。将此重组表达载体质粒转化到大肠杆菌DH5α中,经异丙基-β-D硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)诱导表达。聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-poly acrylamide gel electrophoresis,SDS-PAGE)结果显示,烟碱酰胺合成酶基因在大肠杆菌中成功表达,表达的烟碱酰胺合成酶融合蛋白分子量大约为61 kDa。将初步的纯化产物作为免疫原去免疫新西兰大白兔制备兔抗烟碱酰胺合成酶血清,用蛋白免疫印迹法(western blot-ting)对制备的兔抗血清中的多克隆抗体进行了鉴定,显示该抗体具有较好的特异性。烟碱酰胺合成酶基因多克隆抗体的成功制备为检测该基因在草坪草中的表达提供了可靠的手段和技术平台,为进一步阐明该基因的作用机理奠定了基础。
Using the recombination cloning plasmid p0390-MF-NASHOR1 as template, the nicotianamine synthase （nas） gene was cloned into the prokaryotic expression vector pGEM-KG to construct the recombinant plasmid pKG-NAS. Recipient cells of Escherichia coli DH5α were transformed with pKG-NAS. NAS was expressed under the induction of IPTG and was identified by SDS-PAGE. The molecular mass of expressed NAS-GST was about 61 kD. An emulsion was prepared with the purified NAS and iniected into rabbits. Polyclonal antibodies in the rabbit serum with specific responses to the NAS protein were identified by Western blotting . The successful preparation of a polyclonal antibody to the nas gene provided a reliable method and technical platform for detecting its expression in turfgrass. Moreover, it laid a foundation for investigating the mechanisms of action of the nas gene.
出处
《草业学报》
CSCD
2008年第3期65-70,共6页
Acta Prataculturae Sinica
基金
湖南省自然科学基金项目“抗旱、抗除草剂目的基因转化草坪草获得转基因新材料的研究”(05JJ30036)
湖南省教育厅科学研究重点项目“转抗旱、抗除草剂双价基因冷季型草坪草新品系的培育”(05A022)资助
关键词
烟碱酰胺合成酶
原核表达
多克隆抗体
nicotianamine synthase （NAS）
prokaryotic expression
polyclonal antibody
