重组真核表达载体pEGFP-N1/hIL-1ra的构建及其在NIH3T3细胞中的稳定表达

Construction of the eukaryotic expression vector carrying encoding gene of human interleukin-1 receptor antagonist and Its stable expression in NIH3T3 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,稳定转染小鼠成纤维细胞(NIH3T3)并检测其表达,为使用细胞因子拮抗剂基因治疗牙周炎奠定实验基础。方法Trizol法提取人乳腺癌组织总RNA,RT-PCR方法扩增目的基因。胶回收、纯化后产物与克隆载体pMD18-T连接、转化,测序鉴定正确后将重组克隆载体与真核表达质粒pEGFP-N1分别用HindⅢ和BamHⅠ双酶切,体外连接。重组DNA转化感受态细菌E.coliTOP10,筛选阳性克隆并鉴定。将构建正确的pEGFP-N1/hIL-1ra重组质粒DNA由脂质体介导转染NIH3T3细胞,G418加压筛选20d,RT-PCR检测基因的表达,ELISA检测蛋白的表达。结果重组质粒pEGFP-N1/hIL-1ra经PCR及双酶切鉴定均证实人IL-1ra基因已与pEGFP-N1正确重组。稳定转染NIH3T3细胞后,RT-PCR得到目的基因片段,ELISA检测到目的蛋白的表达。结论本实验成功构建了编码人IL-1ra基因的重组质粒pEGFP-N1/hIL-1ra,并获得了稳定表达目的蛋白人IL-1ra的NIH3T3细胞系,为牙周炎基因治疗的后续研究奠定了实验基础。

Objective To construct the recombinant eukaryotic expression vector pEGFP - NI/hIL - Ira carrying encoding gene of human interleukin - 1 receptor antagonist and to test its stable expression in NIH3T3 cells. Methods Total RNA was extracted from human mammary cancer tissue. RT - PCR was performed to amplify the hIL - Ira encoding gene. Then PCR product was purified and cloned into pMD18 -T. After DNA sequencing, both of the recombinant vector pMDI8 - T/hIL - Ira and plasmid pEGFP - N1 were digested with Hind Ⅲ, BamH I respectively, IL - Ira gene fragment was ligated into plasmid pEGFP - N1. The recombinant plasmid DNA was transformed into E. coli competent ceils TOP 10 and positive clones were selected by G418 and tested. After being transfected by pEGFP - N1/hlL - Ira, the expression of IL -lra gene in NIH3T3 cells was detected by RT - PCR at mRNA level and by ELISA at protein level. Results hlL -lra encoding gene fragment was 531bp and was inserted into the eukaryotie expression vector pEGFP - N1 correctly. And NIH3T3, which was identified by both RT - PCR and ELISA, had a stable expression of IL - Ira after being transfected by recombinant plasmid DNA. Conclusion The IL - Ira eukaryotie expression plasmid was constructed successfully and the NIH3T3 ceils could express IL - Ira stably and successfully.