【摘要】 目的原核表达华支睾吸虫鳞状上皮癌相关肿瘤基因(SCCRO)全长编码区序列,获得纯化的重组蛋白,为进一步功能研究做好准备。方法用生物信息学方法从华支睾吸虫成虫全长cDNA文库中识别出与人鳞状上皮癌相关基因的同源序列及其全长编码区,将其编码区序列克隆到原核表达载体PET-30a(+),测序鉴定重组质粒,在大肠杆菌BL-21/DE3中用IPTG诱导表达,重组产物用His-镍蛋白纯化柱纯化。结果华支睾吸虫鳞状上皮癌相关基因长度为1 218bp,其全长编码序列长度为780bp,编码259个氨基酸,1-22位为分泌信号肽,在羧基端有一个高度保守的区域。该基因在大肠杆菌中得到了高效的可溶性表达,重组蛋白达总蛋白的39%,纯化后的重组蛋白纯度可达98%。结论鳞状上皮癌相关基因的PET-30a(+)原核重组质粒在大肠杆菌BL-21/DE3中可以稳定高效地表达可溶性蛋白。更多还原

【Abstract】 The homologous sequence of adult Clonorchis sinensis with the squamous cell carcinoma-related oncogene(SCCRO) and its fulllength coding region were obtained from the full-length cDNA library of C.sinensis by PCR and sequenced.Meanwhile,the encoding sequence was cloned into the prokaryotic expression vector pET-30a(+),and then expressed in E.coli BL21/DE3 after IPTG induction.The recombinant product was purified according to the protocol NTA agarose(QINGEN,Germany) and analyzed by SDS-PAGE.It was demonstrated that the whole length of SCCRO was 1218 bps,while the full-length coding sequence was 780 bps,encoding 259 amino acids with the secretion signal peptide at 1-22 residues and a highly conserved domain at its carboxyl terminal.This gene could be efficiently expressed in E.coli BL-21/DE3 with the recombinant protein accounting to 39% of the total bacterial proteins.The purity of the purified recombinant protein was 98%.It is concluded that the prokaryotic expression plasmid pET-30a(+)carried with squamous carcinoma cell-related oncogene can stably and highly express the soluble proteins in E.coli BL-21/DE3 cells.更多还原