【摘要】 目的构建X盒结合蛋白1-u(XBP1-u)基因原核表达质粒,表达并纯化XBP1-u蛋白。方法利用RT-PCR技术从肝癌细胞HepG2中扩增XBP1-u基因,先插入中间载体pGEM-Teasy,再将其克隆至原核表达载体pET32a,构建重组原核表达质粒pET32a-XBP1-u,转化E.coliBL21(DE3),IPTG诱导表达。表达产物经Ni-NTA树脂柱亲和层析纯化后,进行Western blot鉴定。结果测序分析证实,克隆入pET32a的XBP1-u序列与GenBank中登录的XBP1-ucDNA序列一致。IPTG的最佳诱导浓度为0.5mmol/L,最佳诱导时间为6h。目的蛋白以包涵体形式表达,相对分子质量约为33000。纯化的蛋白经SDS-PAGE分析显示单一条带,且具有良好的反应原性。结论已成功构建了XBP1-u基因原核表达质粒,表达并纯化了XBP1-u蛋白,为XBP1-u在肿瘤发病中的作用机制及在应激性疾病临床治疗中的研究奠定了基础。更多还原

【Abstract】 Objective To construct a prokaryotic expression vector for X-box binding protein 1-u(XBP1-u)and purify the expressed protein. Methods XBP1-u gene was amplified from liver cancer HepG2 cells by RT-PCR and inserted into pGEM-T easy vector,then cloned into prokaryotic expression vector pET32a. The constructed recombinant plasmid pET32a-XBP1-u was transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed product was purified by Ni2+-NTA affinity chromatography and identified by Western blot. Results The sequence of XBP1-u cloned into pET32a was identical to that of XBP1-u cDNA reported in GenBank. The optimal IPTG concentration and time for induction were 0. 5 mmol / L and 6 h respectively. The target product with a relative molecular mass of about 33 000 was expressed in a form of inclusion body. The purified target protein showed a single band on SDS-PAGE profile,with good reactogenicity. Conclusion The prokaryotic expression vector for XBP1-u was successfully constructed,and XBP1-u protein was expressed and purified,which laid a foundation of study on the role of XBP1-u in onset of tumor and in clinical treatment of irritable diseases.更多还原