【摘要】 目的:克隆烟草富含甘氨酸RNA结合蛋白基因(glycine-rich RNA-binding protein,GRRBPs)并进行原核表达,为制备抗体和研究烟草抗逆性分子机理打下基础。方法:从总RNA中反转录扩增并克隆烟草富含甘氨酸RNA结合蛋白NtRGP-1a和NtRGP-3全长cDNA,将cDNA序列克隆到表达载体pGEX4T-1上,构建了重组表达质粒pGEX4T-1/NtRGP-1a、pGEX4T-1/NtRGP-3,转化大肠杆菌rosetta,IPTG诱导表达,GSTrap 4B亲和层析柱对重组蛋白进行纯化。结果:重组蛋白在大肠杆菌中可以高效表达,SDS-PAGE显示其相对分子质量与预计大小一致,蛋白纯度占总蛋白的95%以上。结论:成功克隆和表达了烟草富含甘氨酸RNA结合蛋白NtRGP-1a和NtRGP-3基因序列,为制备抗体和烟草抗逆性分子机理等进一步的研究奠定了基础。更多还原

【Abstract】 Objective:To clone,express the Glycine-rich RNA-binding protein of tobacco. Methods:Amplified the full length NtRGP-1a and NtRGP-3 gene of tobacco,then ligated the gene into pGEX4T-1.Expression vectors pGEX4T-1/NtRGP-1a,pGEX4T-1/ NtRGP-3 were constructed and transformed into E.coli rosetta for expression induced by IPTG. Recombinant proteins were purified through GSTrap 4B affinity chromatography. Results: The recombinant gene can be overexpressed in E.coli. SDS-PAGE showed that the molecular weight of the expressed product were the same as expected. The purity of the protein is greater than 95%. Conclusion:The NtRGP-1a and NtRGP-3 protein of tobacco has been successful cloned and expressed,which could be useful for developing monoclonal antibody and the research of stress resistance.更多还原