| 摘要: |
| ［目的］ 通过分别克隆白细胞介素-23（IL-23）基因 的双亚基p19和p40，构建pcDNA3-IL-23真核双表达载体，为通过IL-23基因转染树突状细胞 （dendritic cell， DC）增强其介导的免疫抗肿瘤作用提供基础。［方法］ 一步法从小鼠脾脏及胸腺中提取p19和p40总RNA ，RT-PCR法扩增p19和p40的cDNA，扩增产物与pMD18-T vector连接并热转化至大肠杆菌JM10 9, PCR法筛选阳性克隆，提取质粒并进行DNA的测序鉴定，将pMD18-p19和pMD18-p40分别 限制性酶切，在对pcDNA3表达载体限制性酶切后将切胶回收的p19和p40片段分别克隆至pcDNA3.0表达载体， 对阳性质粒进行XhoI/KpnI双酶切检测，琼脂糖凝胶电泳分析。［结果］ 脾脏及胸腺中提取p19和p40总RNA电泳鉴定是完整的，克隆的p19和p40 cDNA经测序鉴定与Genebank中序列完全一致。构建的pcDNA3.0-p19和pcDNA3.0-p40质粒分别限制性酶切后得到了593 bp和1028 bp的基因片段；构建的pcDNA3.0-IL-23表达载体限制性酶切后得到了1.62 kbp的p19+p40片段。［结论］ 成功克隆了小鼠IL-23基因，并分别构建了pcDNA3-p19、pcDNA3-p40和pcDNA3-IL-23真核表达载体，为IL-23基因转染DC来增强其免疫活性创造了条件。 |
| 关键词: IL-23基因 克隆 真核表达载体 |
| DOI：10.11724/jdmu.2006.04.02 |
| 分类号:R73 |
| 基金项目: |
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| Construction of dual-expression vector pcDNA3-IL-23 after cloning active cytokine Interleukin 23 |
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TAN Guang1, WANG Xiao-gang2, CHENG Lei1, LUO Hai-feng1, WANG Zhong-yu1, YIN Shuo1
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1.Department of General Surgery, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, China;2.Deparement of General Surgery, Pucheng Country Hospital, Xi'an 715500,China
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| Abstract: |
| ［Objective］ To clone two subunits p19 and p40 of the Interleukin 23 gene, to construct the dual-expression vector pcDNA3-IL- 23 in order to study the anti-tumor therapy inducing by the dendritic cells genetically transfect by Interleukin 23.［Methods］ Total RNA of p19 and p40 were extracted from thymus and spleen of mice respectively by one step way, and cDNA was amplified by RT-PCR, the PCR product was linked with pMD18-T vector and E.Coli JM109 was transformed by pMD18-p19 and pMD18-p40, positive plasmid was selected by PCR method. p19 and p40 gene were digested from pMD18-p19 and pMD18-p40, the product was inserted in
to pcDNA3.0 expression vector, selected positive plasmid was tested by Xho I /Kpn I restrict enzyme. ［Results］ Total RNA of p19 and p40 from thymus and spleen respectively was complete. The sequence of cloned gene is consistent with the reported sequence of the genebank. The constructed pcDNA3-IL-23 eukaryotic dual-expression vector was proved to have p19 and p40 subunits of 1.62 kbp by restriction endonuclease analysis. ［Conclusion］ Successfully constructed eukaryotic dual-expression vector pcDNA3-IL-23.It is the basic work for the further study of immunotherapy against pancreatic carcinoma leading by the dendritic cells genetically modified by interleukin 23. |
| Key words: gene clone interleukin-23 eukaryotic expression vector |
