中文摘要：

目的 构建特异性过表达大鼠IL-6基因的重组逆转录病毒载体，并在大鼠嗜铬细胞瘤PC12细胞和人胚肾HEK293细胞中检测IL-6的表达。方法以大鼠骨髓间充质干细胞mRNA为模板，经PCR获得目的基因IL-6，将其定向克隆到逆转录病毒载体pSEB-3H中，构建重组逆转录病毒质粒pSEB—IL-6，经脂质体分别转染到PC12细胞和HEK293细胞中，应用Real—timePCR和ELISA的方法在mRNA和蛋白质水平检测IL-6的表达变化。继而用HEK293细胞中包装获得的含有pSEB—IL-6的病毒颗粒进一步感染PC12细胞，Real—timePCR检测，IL-6mRNA的表达水平变化。结果PCR电泳及酶切鉴定证实目的基因正确克隆至逆转录病毒载体中，其基因序列与Genbank报道一致；Real—timePCR和ELISA结果均显示，逆转录病毒质粒pSEB—IL-6转染PC12细胞和HEK293细胞后，IL-6的表达水平较对照组显著上调；经pSEB—IL-6逆转录病毒颗粒感染的PC12细胞中，IL-6mRNA表达水平较对照组提高4倍。结论成功构建了特异性表达大鼠儿-6基因的重组逆转录病毒载体pSEB—IL-6，并获得了具有感染能力的逆转录病毒颗粒，感染真核细胞后可高表达IL-6，为进一步研究IL-6的功能及其在多种疾病中的免疫调节机制提供重要的分子手段。

英文摘要：

Objective To construct recombinant retroviral vector containing rat interleukin-6 （IL-6） gene, and identify IL-6 expression level in PC12 cells and HEK293 cells. Methods The full-length of target IL-6 gene was amplified by PCR, and directional cloned into the pSEB-3H tag retroviral vector. The recombinant pSEB-IL-6 retroviral vector was transfected in HEK293 cells by using lipofectamine 2000. PC12 cells were infected by the pSEB-IL-6 retrovirus particles through packaging in HEK293 cells. The levels of IL-6 mRNA and protein expression were detected with real-time PCR and ELISA. Results Construction of IL-6 target gene in the recombinant pSEB-IL-6 retroviral vector was verified by PCR and sequencing analysis. Compared with the control pSEB-3H plasmid, pSEB-IL-6 retroviral vector expressed significantly high level of IL-6 in both PC12 cells and HEK293 cells. Conclusions The pSEB-IL-6 recombinant retroviral vector was well constructed. pSEB-IL-6 retroviral vector showed efficient ability of infection and specifically expressed rat IL-6 protein in eukaryotic cells, suggesting its further applicability in study of IL-6 function and its immune regulatory mechanisms in different diseases.