人白细胞介素24真核表达载体的构建及其在HepG2细胞中的表达  

Construction of eukaryotic expression vector of human interleukin-24 and its expression in HepG2 cells

作　　者：于培霞[1] 冀永进[2] 王桂琴[1] 常江[1] 景刚[1] 王艳红[1] 

机构地区：[1]山西医科大学微生物学与免疫学教研室,太原030001 [2]山西医科大学第二附属医院耳鼻喉科,太原030001

出　　处：《国际生物制品学杂志》2009年第6期281-284,共4页International Journal of Biologicals

摘　　要：目的构建人白细胞介素24（humaninterleukin-24，hIL-24）真核表达载体，并在HepG2细胞中稳定表达，为进一步研究其抗肿瘤作用奠定基础。方法采用逆转录聚合酶链反应（RT-PCR），从植物血凝素活化的人外周血单个核细胞中克隆得到hIL-24基因目的片段。应用DNA重组技术将IL-24PCR产物双酶切后定向克隆至真核表达载体peDNA3．1（＋），转化大肠杆菌DH5a获得重组载体，进行PCR、酶切及测序鉴定。应用脂质体将鉴定正确的重组质粒转染至HepG2细胞，用G418筛选稳定转染细胞株。采用RT—PCR检测稳定转染细胞HepG2中IL-24mRNA的表达。结果通过RT—PCR获得与预期大小一致约600bp的IL-24基因片段；重组载体pcDNA3．1（＋）-IL-24经PCR、双酶切及测序证实，IL-24cDNA片段已正确插入真核表达载体中；在稳定转染的HepG2细胞株中可见到IL-24mRNA表达。结论成功构建了hIL-24真核表达载体peDNA3．1（＋）-IL-24，并获得了稳定转染该重组质粒的HepG2细胞株。Objective To construct a eukaryotic expression vector of human interleukin-24 （hIL-24） and to observe its expression in HepG2 cells. Methods The hIL-24 gene was cloned from phytohemaggluti- nin-treated human peripheral blood mononuclear cells with RT-PCR. The eukaryotic expression vector pcDNA3.1（ ＋ ）-IL-24 was constructed by subcloning IL-24 cDNA into the eukaryotic expressing vector pcDNA3.1 （ ＋ ） with DNA recombination technique and was identified by PCR, restriction enzyme digestion and DNA sequencing. The pcDNA3. 1 （ ＋ ）-IL-24 was stably transfected into HepG2 cells by liposome transfection with using G418 to screen positive transformants. RT-PCR was performed to determine hIL-24 mRNA expression in HepG2 cells. Results The target gene with expected molecular mass was obtained with RT-PCR. The results of PCR, restriction enzyme digestion and DNA sequencing showed that the IL-24 gene had cloned into the eukaryotic vector pcDNA3.1 （ ＋ ） correctly. The mRNA of IL-24 was detected in the stably transfected HepG2 cells. Conclusions The recombinant eukaryotic expression vector pcDNA3. 1 （ ＋ ）-IL-24 is constructed successfully and the HepG2 cell strains stably transfected with the recombinant plasmid are obtained.

关 键 词：白细胞介素类 基因表达 载体 HEPG2细胞 

分 类 号：Q78[生物学—分子生物学]