人IL-24基因的克隆及在COS-7细胞中的表达  

Cloning & expression of human IL-24 in COS-7 cells

作　　者：陈雄艳[1] 盛伟华[1] 谢宇锋[1] 缪竞诚[1] 白艳艳[1] 杨吉成[1] 

机构地区：[1]苏州大学医学院细胞和分子生物学教研室,苏州215007

出　　处：《中国免疫学杂志》2004年第10期672-676,共5页Chinese Journal of Immunology

基　　金：江苏省高校自然科学研究项目 (0 1KJB180 0 0 1)

摘　　要：目的 :克隆人IL 2 4基因 ,构建真核表达载体 ,在COS 7细胞中进行表达 ,并检测重组表达蛋白hIL 2 4的抗肿瘤活性。方法 :分离人外周血单个核细胞 ,提取细胞总RNA ,采用RT PCR技术克隆人IL 2 4基因 ,将其重组于pUC19,双脱氧末端终止法测定其核苷酸序列 ,构建真核重组表达载体pcDNA3 hIL 2 4 ,进行双酶切和PCR鉴定 ,以质粒pcDNA3 hIL 2 4转染COS 7细胞进行瞬时表达 ,用RT PCR检测mRNA表达 ,并用MTT法、TUNEL法和流式细胞术检测其诱导肿瘤细胞凋亡的活性。结果 :成功获得 6 2 1bp的人IL 2 4基因 ,测序正确 ,经双酶切和PCR鉴定真核表达质粒构建正确 ,以脂质体转染COS 7细胞后 ,用RT PCR法、MTT法、TUNEL法和流式细胞术检测表明COS 7细胞可表达hIL 2 4 ,且所表达的人IL 2 4具有较强的诱导A5 4 9肺腺癌细胞凋亡的作用。结论 :人IL 2 4基因的瞬时表达和凋亡效应的初步研究已获成功 ,为研究人IL 2Objective:To clone human IL-24 gene,and construct its eukaryotic expression vector,and express it in COS-7 cells & detect its anti-tumor activities of recombinant hIL-24 protein.Methods:The total RNA was extracted from peripheral blood mononuclear cells.And the functional fragment of human IL-24 gene was amplified by RT-PCR.It was cloned into pUC19 & sequenced and was subcloned into pcDNA3 vector.Constructed pcDNA3-hIL-24 was identified by endonucleases digestion & PCR.The recombinant expression plasmids was transfected into COS-7 cells,human hIL-24 expressed in COS-7 cells was deteced by RT-PCR.The apoptosis-inducing activities of recombinant protein hIL-24 was tested by MTT assey,TUNEL assey & FCM assey.Results:Obtained full encoding sequence of hIL-24 was identical with that included in Genbank,& the eukaryotic expression vector pcDNA3-hIL-24 was constructed correctly.Expression of human IL-24 in COS-7 cells was identified by RT-PCR.The apoptosis of A549 cells induced by hIL-24 was proved by TUNEL & FCM assay.Conclusion:The successful cloning,transient expression & preliminary study of apoptosis effect of human IL-24 protein provide experimental basis for the further studying of molecular mechanism and clinical application of hIL-24 on anti-tumors.

关 键 词：人IL-24 克隆 表达 COS-7细胞 凋亡 

分 类 号：R392.13[医药卫生—免疫学]