pcDNA3肿瘤坏死因子α真核表达载体的构建及其在真核细胞中的表达  

Construction of pcDNA3 tumor necrosis factor-α(TNFα) vector and its expression in eukaryotic cells

作　　者：齐力[1] 季雪莲[2] 王秀梅[2] 张智清[3] 

机构地区：[1]武警内蒙古总队医院外一科,呼和浩特010019 [2]内蒙古自治区医院神经内科,呼和浩特010017 [3]中国预防医学科学院病毒所基因室,北京100015

出　　处：《武警医学》2003年第2期76-78,共3页Medical Journal of the Chinese People's Armed Police Force

摘　　要：目的 构建pcDNA3肿瘤坏死因子α(Tumor necrosis factor-alpha,TNFα)真核表达质粒并了解其在真核细胞内的表达。方法 用双酶切方法由pBluescript/TNFa克隆载体上切下702hp的INFa基因片段,将其克隆至pcDNA3真核表达载体上,经酶切鉴定及测序分析并以脂质体介导法转染真核细胞,了解其在真核细胞内的表达及其表达蛋白的生物学活性。结果 pcDNA3 TNFa重组体经酶切后出现699 bp片段,测序分析与文献报告结果完全一致,表明重组pcDNA3 TNFa表达质粒克隆成功。可见pcDNA3 TNFa质粒在真核动物细胞中得到表达,TNFa蛋白能够杀伤L_(929)细胞,表明重组质粒能在真核动物细胞中表达出具有生物活性的TNFa蛋白。结论 以脂质体介导pcDNA3 TNFa质粒转染真核细胞为基因治疗奠定了一定基础。Objective To construct pcDNAS TNFa recombinant eukaryotic expression plasmid and investigate its expression in eukaryotic cells. Methods The TNFa gene fragment was obtained from pBluescript cloning vector by restricting enzyme digesting and cloning into pcDNA 3 eukaryotic expression vector. There combinant pcDNA3 TNFa plasmid was transfected into eukaryotic cells mediated by using lipofectin method. Analysis by restricting enzyme digesting and DNA sequencing was carried out to demonstrate the sequence of the plasmid . TNFa protein and its activity were then determined using pcDNA3 TNFa plasmid transfected eukaryotic cells. Results The regained TNFa gene fragment was 702 bp.DNA sequence of pcDNA3 TNFa recombinant showed 699 bp segment. DNA sequenceing revealed that TNFα cloning was successful. The recombinant plasmid could express active TNFa protein in eukaryotic cells. Conclusions The transfection of pcDNA3 TNFa plasmid into eukaryotic cells mediated by lipofectin provides a basis for gene therapy.

关 键 词：人脑胶质细胞瘤 基因疗法 真核细胞 转染 肿瘤坏死因子Α PCDNA3 TNFΑ 基因表达 

分 类 号：R730.51[医药卫生—肿瘤]