水稻HL-CMS中orf216蛋白的原核表达和多克隆抗体制备

Protokaryotic Expression and Polyclonal Antibody Preparation of orf216 Protein in Honglian Cytoplasmic Male Sterile Rice Line

以常规基因重组技术,将orf216基因克隆入含有内含肽标签的原核表达载体pTYB1中,得到重组质粒pTYB1-orf216,经鉴定正确后,转化至大肠杆菌ER2566感受态细胞,15℃下0.8 mmol/LIPTG诱导15 h后,SDS-PAGE检测到大小约85 ku的特异性蛋白条带,融合蛋白以可溶组分形式存在。融合蛋白经亲和纯化后制备多克隆抗体,Western blot分析表明多克隆抗体具有较高的特异性,可与免疫原特异结合。

The recombinant prokaryotic expression vector pTYB1-orf216 with the intein tag was constructed and transformed into E.coli ER2566.The optimized protein expression induction condition in E.coli was 0.8 mmol/L IPTG（isopropy-β-D-thiogalacto side） at 15℃ for 15 hours.A band with 85 ku of molecular weight was observed on SDS-PAGE,and the protein was mainly existing in the soluble composition.This protein was isolated and purified through affinity chromatography,and then polyclonal antibody was prepared.The results of Western blot indicated that the prepared polyclonal antibody was with high specificity.