【摘要】 目的　克隆人胰腺组织激肽原酶基因 ,构建分泌型原核表达载体。方法　使用RT PCR的方法扩增人胰腺组织cDNA ,从中扩增出激肽原酶基因。将扩增的激肽原酶基因用XhoⅠ和EcoRⅠ双酶切后 ,连接到同样双酶切处理的pET 2 0b(+)载体上 ,酶切鉴定后 ,进行核苷酸序列分析和融合蛋白的表达。结果　将IPTG诱导表达的菌体进行SDS PAGE ,在相对分子质量 31 .4× 1 0 3处可见明显的高表达带。蛋白质免疫印迹实验表明 ,重组蛋白具有激肽原酶的抗原性。结论　成功克隆人胰腺组织激肽原酶基因 ,并高效表达融合蛋白 ,为进一步开发基因工程药物打下基础更多还原

【Abstract】 OBJECTIVE To investigate the molecular cloning of the human tissue kininogenase gene and its expression in Escherichia coli .METHODS The kininogenase gene was amplified from human pancreas cDNA synthesized by RT PCR.After digesting by Xho Ⅰ and EcoR Ⅰ,it was then inserted into the pET 20b(+) vector.The clones were respectively identified by enzyme digestion,sequencing and expressed in E. coli .RESULTS The kininogenase fusion protein expressed under the induction of IPTG was detected by SDS PAGE.A significant band with a relative molecular weight of 31.4×10 3 was observed.The western blot indicated that the expressed kininogenase had the antigenicity to human serum kininogenase.CONCLUSION The fusion protein of the cloned kininogenase gene was highly expressed in E.coli and could be used for the development of its biological products.更多还原