论文部分内容阅读
目的 开发激肽释放酶基因工程产品,为开展基因治疗高血压奠定基础。方法 采用RT-PCR的方法合成人胰腺 cDNA,从中扩增出Kallikrein(KK)基因。经 XhoI和EcoR I双酶切后,连接到pET-28b(+)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电泳,与蛋白标准品比较,在相对分子质量31800处可见明显的高表达带。免疫印迹实验表明重组蛋白具有KK的抗原性。结论 已成功克隆并表达了人胰腺组织激肽释放酶基因,为进一步开发基因工程产品及进行基因治疗高血压研究打下了基础。
Objective To develop kallikrein gene engineering products and lay the foundation for the gene therapy of hypertension. Methods The human pancreatic cDNA was synthesized by RT-PCR and the Kallikrein (KK) gene was amplified. After digested with XhoI and EcoR I, the recombinant plasmid was ligated into pET-28b (+) vector. After restriction analysis, nucleotide sequence analysis and fusion protein expression were carried out. Results The IPTG induced SDS - PAGE electrophoresis showed that the expression of IPTG was significantly higher than that of the protein standard at the relative molecular mass of 31800. Western blotting experiments showed that the recombinant protein has the antigenicity of KK. Conclusion The human pancreatic tissue kallikrein gene has been successfully cloned and expressed, which laid the foundation for the further development of genetically engineered products and gene therapy of hypertension.