【摘要】 目的构建热休克蛋白70(HSP70)与黑色素瘤抗原-4(MAGE-4)抗原表位基因的原核表达载体,并对表达产物进行鉴定。方法在热应激条件下,用PCR法从结肠腺癌细胞获取HSP70基因。在线用蛋白质二级结构预测软件分析MAGE-4抗原表位,PCR获取MAGE-4抗原特征表位基因。将二者分别克隆入pGEM-Teasy载体,酶切鉴定后,将HSP70基因亚克隆入pET28a原核表达载体,再将MAGE-4基因克隆入HSP70基因的下游,构建重组质粒pET28a-HSP70-MAGE-4。转化大肠杆菌,IPTG诱导表达并鉴定。结果所构建的HSP70与MAGE-4抗原表位基因表达载体pET28a-HSP70-MAGE-4,经PCR及DNA测序结果表明构建正确。SDS-PAGE及Western blot鉴定均在预期位置出现阳性条带。结论已成功构建了人HSP70与MAGE-4抗原表位基因的原核表达载体,为疫苗研究提供了依据。更多还原

【Abstract】 Objective To construct a prokaryotic expression vector for human heat shock protein 70(HSP70) and melanoma antigen-4(MAGE-4) epitope genes and identify the expressed product.Methods Under a condition of heat stress,amplify HSP70 gene from colon cancer cells by PCR.Analyze MAGE-4 antigen epitope by online predict software of protein secondary structure and amplify the epitope gene by PCR.Clone the amplified HSP70 and MAGE-4 epitope genes into pGEM-T easy vector and,after identification of the constructed recombinant plasmid by restriction analysis,subclone HSP70 gene into prokaryotic expression vector pET28a,and MAGE-4 gene downstream to HSP70.Transformed the constructed recombinant plasmid pET28a-HSP70-MAGE-4 to E.coli for expression under induction of IPTG.Identify the expressed product by SDS-PAGE and Western blot.Results Both PCR and DNA sequencing proved that recombinant plasmid pET28a-HSP70-MAGE-4 was correctly constructed.Both SDS-PAGE and Western blot showed positive bands consistent with those expected.Conclusion The prokaryotic expression vector for HSP70 and MAGE-4 epitope genes was successfully constructed,which provided a basis for the development of vaccine.更多还原