Ksp-Gpx1-klk1载体的构建及鉴定

Construction and identification of Ksp-cadherin-Gpx1-Klk1 expression vector

目的构建含有肾组织特异性启动子(Ksp-cadherin)的Gpx1与klk1载体质粒,为下一步构建转基因动物、研究在动物模型体内表达和基因治疗提供基础。方法以KLK1cDNA、GPX1cDNA、Ksp-cadherinBAC为模板,PCR扩增获得人源Gpx1、KLK1、Ksp-cadherincDNA。PCR产物大小与预期结果一致,序列分析完全正确。选择多个相应酶切位点,分步将Ksp-cadherin、Gpx1、KLK1插入至pIRES-EGFP质粒中,构建pKSP-GPX1-IRES-KLK1重组质粒。应用酶切鉴定和序列分析方法验证所构建载体的正确性。结果成功构建了含有Ksp-cadherin的Gpx1与klk1载体质粒。结论该重组载体的构建为下一步构建转基因动物,研究其在哺乳动物肾组织内过表达及在移植肾缺血再灌注损伤的基因治疗中的作用奠定了基础。

Objective To construct a Gpx1 and klk1 recombinant vector containing the kidney-specific promoter Ksp-cadherin. Methods Human Gpx1, Klk1 and Ksp-cadherin cDNAs were amplified with PCR and inserted in a stepwise manner into the expressive vector pIRES-EGFP to construct the recombinant vector Ksp-cadherin-Gpx1-Klk1. The constructed vector was verified with restriction enzyme digestion and sequence analysis. Results and Conclusion The recombinant expression vector Ksp-cadherin-Gpx1-Klk1 was constructed and identified successfully, which provides a potent tool for preparing transgenic animals to investigate gene therapy for ischemia-reperfusion injury in kidney transplantation.