小鼠IL-33基因cDNA克隆与真核质粒的构建与表达_临床医学论文.docVIP

小鼠IL-33基因cDNA克隆与真核质粒的构建与表达_临床医学论文 小鼠IL-33基因cDNA克隆与真核质粒的构建与表达_临床医学论文 作者：张林，程晓刚，练栩辉，李鑫，刘林娜，赵柏程，任飞，李晗郡，刘平 【摘要】 　　目的 克隆小鼠IL-33基因(mIL-33)cDNA构建其真核表达质粒，并转染EL-4细胞检测其表达。方法 提取小鼠脑与肺组织总RNA，经逆转录－聚合酶链反应（RT-PCR）扩增小鼠IL-33基因cDNA，酶切后插入pcDNA-3.1构建其真核表达载体pcDNA-mIL-33，重组质粒转染EL-4细胞，RT-PCR与ELISA法检测目的基因表达。结果 pcDNA-mIL-33中插入的DNA序列测定结果与mIL-33 cDNA序列一致，重组质粒转染EL-4细胞后检测到相应基因表达。结论 成功克隆小鼠IL-33基因cDNA，并构建其真核表达质粒。 【关键词】 IL-33；基因克隆；真核表达 　　Abstract: Objective To clone and construct the eukaryotic expression plasmids containing mouse IL-33 cDNA and investigate their expression. Methods Total RNA was extracted from mouse lung and brain tissues to amplify mouse interleukin-33 (mIL-33) cDNA by reverse transcription-polymerase chain reaction (RT-PCR). The eukaryotic expression plasmids pcDNA-mIL-33 was constructed with the insertion of enzyme-digested pcDNA-3.1(+) vector to construct eukaryotic expression vector pcDNA-mIL-33. The recombinant plasmids were transfected into EL-4 cells and the expression of target gene was detected by RT-PCR and ELISA. Results The inserted DNA sequence in pcDNA-mIL-33 was identical to the mIL-33 cDNA, which was verified correctly by sequencing. The corresponding gene expression was detected in the recombinant plasmid-transfected EL-4 cells.Conclusion The mouse IL-33 cDNA was successfully cloned and its eukaryotic expression plasmids were constructed. 　　Key words: IL-33; gene cloning; eukaryotic expression 　　白细胞介素33（IL-33）是近几年发现的细胞因子,为IL-1类细胞因子超家族成员［1］，已发现其在小鼠及人各组织内皮与上皮细胞中均有广泛表达。其相应受体为由IL-1受体1（interleukin-1 receptor-like 1，IL1RL1）和IL-1受体辅助蛋白（IL-1R accessory protein，IL-1RAcP）2个亚基组成的异源二聚体，其中IL1RL1又称蛋白ST2［2］，上述两亚基均为IL-1受体超家族成员。研究表明IL-33和其配体结合后可促进肥大细胞与Th2细胞分泌IL-4、IL-5、IL-13等细胞因子并促进炎症反应［3-4］，在过敏性哮喘、过敏性休克、肺纤维化、风湿性关节炎等自身免疫性疾病及脓毒血症、败血性休克、某些恶性肿瘤与心肌炎等发生发展中起重要作用［5-6］。本研究旨在克隆小鼠IL-33基因的cDNA，构建其真核表达质粒pcDNA-mIL-33并实现在EL-4细胞中表达，为研究IL-33蛋白的生物学功能并为实现其原核表达奠定基础。 　　1 材料和方法 　　1.1 动物、质粒和菌株 　　BALB/c小鼠来自本校科研中心动物室，真核表达质粒pcDNA3.1(+)（5.4 kb) 、大肠杆菌E.coli DH5α菌株、小鼠淋巴瘤细胞株EL-4为本校免疫学教研室保存。 　　1.2 主要试剂