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-Tvector连接并热转化至大肠杆菌JM109,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pcD-NA3.0-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。
[ Objective] To clone two subunits p19 and p40 of the Interleukin 23 gene, to construct the dual- expression vector pcD- NA3- 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 into 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 dendritio cells genetically modified by interleukin 23.
出处
《大连医科大学学报》
CAS
2006年第4期276-279,共4页
Journal of Dalian Medical University
基金
辽宁省科技厅自然基金资助(90042138)
关键词
IL-23基因
克隆
真核表达载体
gene clone
interleukin - 23
eukaryotic expression vector
