人白细胞介素7重组蛋白原核表达载体在大肠杆菌中的表达和鉴定  

Expression and identification of recombinant human interleukin 7 product in Escherichia coli

作　　者：耿梅[1,2] 陈清[1,2] 徐丽慧[3] 张延亭[1,2] 何贤辉[1,2] 

机构地区：[1]暨南大学生命科学技术学院组织移植与免疫实验中心,广东广州510632 [2]暨南大学再生医学教育部重点实验室,广东广州510632 [3]暨南大学生命科学技术学院生物工程研究所,广东广州510632

出　　处：《暨南大学学报（自然科学与医学版）》2010年第4期331-335,共5页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：国家自然科学基金项目(30572199;30371651)

摘　　要：目的:构建重组人白细胞介素7(rhIL-7)的原核表达载体,在大肠杆菌中表达并鉴定。方法:以RT-PCR方法扩增编码IL-7的cDNA片段,插入至pET-3d载体中,构建rhIL-7的原核表达载体,阳性克隆经测序鉴定,转化至大肠杆菌BL21(DE3),优化rhIL-7表达条件,利用免疫印迹鉴定重组蛋白。结果:重组载体经DNA测序显示包含正确的rhIL-7编码序列,重组载体转入大肠杆菌后,经IPTG诱导后外源蛋白表达,相对分子质量为17 400,与IL-7理论分子质量一致;免疫印迹显示,外源蛋白可以被人IL-7特异性抗体识别,表明在原核表达的外源蛋白是rhIL-7。rhIL-7主要表达于包涵体中,包涵体表达优化条件为:温度为37℃、IPTG浓度为0.4 mmol/L、诱导时间为4h。结论:成功的构建了rhIL-7的原核表达载体。Aim：To construct a prokaryotic expression vector for human interleukin7（IL-7）,identify its expression product and optimize its expression condition. Methods： The cDNA fragment encoding human IL-7 was amplified by RT-PCR from the total RNA of human PBMC,and then the IL-7 gene was cloned into pET-3d plasmid to construct a prokaryotic expression vector.The positive clone was analyzed by DNA sequencing.The recombinant protein was expressed in Escherichia coli（E.coli） BL21（DE3） strain and identified with Western blotting.Results： The prokaryotic expression vector forIL-7 was constructed and confirmed by sequencing.The expression vector was transformed into E.coli and was expressed in after induction with IPTG.The relative molecular weight of the protein was 17 400,which is consistent with theoretical value.Western blotting showed that the recombinant protein was mainly presented in inclusion bodies.The optimized expression condition was 4 h induction at 37 ℃ with 0.4 mmol/L IPTG.Conclusion：The prokaryotic expression vector for human IL-7 has been constructed successfully and the expression condition has been optimized,which has paved the way for further study on human IL-7.

关 键 词：白细胞介素7 原核表达载体 包涵体表达 免疫印迹 

分 类 号：Q786[生物学—分子生物学]