pEGFP-N1/hIL-1ra重组真核表达载体的构建及其在人牙龈成纤维细胞中的瞬时表达

Construction of the eukaryote expression vector carrying encoding gene of human interleukin-1 receptor antagonist and its transient expression in human gingival fibroblast cells

目的:构建重组人白细胞介素1受体拮抗剂(rhIL-1ra)基因真核表达载体pEGFP-N1/hIL-1ra,瞬时转染人牙龈成纤维细胞并检测其表达,为牙周炎症的基因治疗提供实验基础。方法:TRIzol法提取人乳腺癌组织总RNA进行RT-PCR,将纯化的扩增产物hIL-1ra与克隆载体pMD18-T连接、转化,测序正确后将重组体pMD18-T/hIL-1ra与真核表达质粒pEGFP-N1分别进行双酶切,连接后转化感受态细菌E.coliTOP10。将构建正确的pEGFP-N1/hIL-1ra重组质粒DNA瞬时转染人牙龈成纤维细胞,荧光显微镜观察绿色荧光的表达,RT-PCR方法检测其基因的表达。结果:构建的pEGFP-N1/hIL-1ra真核表达载体经PCR及双酶切鉴定均表明人IL-1ra基因已与pEGFP-N1正确重组。瞬时转染人牙龈成纤维细胞后能观察到绿色荧光,RT-PCR可得到目的片段。结论:成功构建了重组质粒pEGFP-N1/hIL-1ra,瞬时转染人牙龈成纤维细胞后检测到其基因水平的表达,为炎性细胞因子拮抗剂基因用于牙周炎抗炎治疗奠定了实验基础。

Objective：To construct the recombinant pEGFP-N1/hIL-lra and test its transient expression in human gingival fibroblast ceils, this may be useful of the treatment of periodontitis. Methods：Total RNA was extracted from human mammary cancer tissue. RT-PCR was performed to amplify the hIL-1ra encoding gene. Then PCR product was purified and cloned into pMD18-T. After DNA sequencing,both of the recombinant vector pMD18-T/hIL-1ra and plasmid pEGFP-N1 Were digested with Hind III and BamH I respectively. IL-1ra gene fragment was ligated into plasmid pEGFP-N1. The recombinant plasmid DNA was transformed into E.coli competent cells TOP 10 and positive clones were selected and tested. After being transfected by pEGFP-N1/hIL-1ra, the transient expression of IL-1ra gene in human gingival fibroblast （HGF） cells were detected by fluorescence microscope and RT-PCR. Results： hIL-1ra encoding gene fragment was 531 bp and was inserted into the eukaryotic expression vector pEGFP-N1/hIL-1ra correctly. And HGF cells,which were identified by both fluorescence microscope and RT-PCR,had a transient expression of IL-1ra after transfection. Conclusion：The new recombinant expression vector pEGFP-N1/hIL-1ra was constructed successfully and the HGF cells which could express IL-1ra transiently were also successfully made.