【摘要】 根据已筛选到的手掌参(Gymnadnia conopsea)中赤霉酸诱导的富含半胱氨酸蛋白部分cDNA序列,设计带有EcoRⅠ和HindⅢ酶切位点的引物,对编码区全长(gcgasa)及信号肽缺失的片段(Δgcgasa)进行扩增,获得大小约319和238bp的片段。克隆到表达载体pET-32a,转化大肠杆菌(Escherichia coli)BL21(DE3),经1mmol/LIPTG诱导后,在分子量约26.0和25.2kD出现融合蛋白条带。SDS-PAGE和细胞透射电镜结果表明:含有信号肽的外源基因在大肠杆菌中以包含体形式存在于周质空间,而信号肽缺失的片段主要以可溶形式表达,通过Ni2+-NTA亲和层析和凝胶过滤进一步纯化了目的蛋白。更多还原

【Abstract】 According to the known partial cDNA sequence of gibberellin-induced cysteine-rich protein from Gymnadnig conopsea,the primers bearing restriction enzyme site of EcoR I and HindⅢ were designed for amplifying the full-length of ORF and the signal peptide-truncated fragment of gcgasa gene. Two fragments with the length of 319 and 238 bp were obtained and then cloned into the plasmid pET-32(a). Following the transformation into Escherichia coli BL21(DE3) , the fusion proteins were observed at approximately 26.0 and 25.2 kD with the induction of 1 mmol/L IPTG. The results of SDS-PAGE and transmission electron micrograph of ultrathin section revealed that the presence of signal peptide gave rise to the formation of inclusion body, which was located in periplasmic space, however, the absence of signal peptide greatly enhanced the solubility of the target protein, and the expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods.更多还原