摘要
将单拷贝人α心钠素基因3′端用Ban Ⅱ酶解除去包括终止密码在内的36个碱基对,代之以人工合成的含Glu-Lys-Phe-Glu连接片段与另一单拷贝人α心钠素基因的5′端串连成编码60肽的双拷贝心钠素基因,克隆于大肠杆菌分泌型表达载体pIN-Ⅲ-OmpA_2质粒中,表达生成60肽的双拷贝人α型心钠素衍生物,在信号肽的作用下分泌至胞膜间质并自动切割为60肽的外源基因产物。分子量约8K的表达产物用分子筛或超滤膜分离后再经HPLC纯化,表达产物具有明显的心钠素放免活性和舒张血管活性。
A synthetic gene for a-hANF was reconstructed as a double joined gene,linked in tandem.The two copies in the gene were seperated by codons specifying a Glu-Lys-Phe-Glu linker with Glu residues flanking the authentic N and C termini of the 28-a.a.hormone.This linker is provided for enzymatic cleavage of expressed product by endoproteinaese Glu-c or Lys-c.The double joined gene fragment was cloned and expressed in) secretive expression vector PIN-*******III-OmpA2 in E.coli.The product was located in periplasmic space of the bacteria and purified by gel filtration and HPLC.The expressed protein after gently oxidized exhibited Vaso-dilatation activity similar to natural *********a-ANF.
关键词
人心钠素
重组DNA
分泌型表达
Human artrial natriuretic factor (ANF)
Recombinant DNA
Tandem gene
Secretive expression vector
