人肿瘤坏死因子-α基因真核表达载体的构建  

Construction of a Recombinant Eukaryotic Vector Expressing Human Tumor Necrosis Factor alpha

作　　者：冯凤芝[1] 张卫光[2] 向阳[1] 刘芝华[3] 杨秀玉[1] 

机构地区：[1]中国医学科学院中国协和医科大学北京协和医院妇产科,北京100730 [2]北京大学医学部解剖学与组织胚胎学系,北京100083 [3]中国医学科学院肿瘤研究所分子肿瘤学国家重点实验室,北京100021

出　　处：《肿瘤防治杂志》2003年第2期121-124,共4页China Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金资助课题 ( 30 0 0 0 179)

摘　　要：目的 :构建可在真核细胞内表达人肿瘤坏死因子alpha(hTNF α)基因的真核表达载体。方法 :应用RT PCR的方法从足月妊高征患者的胎盘组织中 ,扩增出hTNF αcDNA ,连接至载体pMD 18 Tvec tor中 ,经酶切鉴定与序列测定证实后 ,以亚克隆法构建于真核表达载体pcDNA3.1 myc his(- )A ,B ,C的相应酶切位点 ,通过RT PCR方法检测其在体外转导绒癌耐药细胞后hTNF α基因的表达。结果 :自胎盘中克隆的hTNF αcDNA经序列测定证实 ,与Genbank的序列完全一致 ;用EcoRⅠ和HindⅢ双酶切鉴定证实 ,克隆基因正确插入载体pcDNA3 1 myc his(- )A ,B ,C。经脂质体介导转导绒癌耐药细胞后 ,检测到其在转导的细胞中稳定表达。结论 :通过DNA重组技术成功地构建了可在体外稳定表达hTNFObjective To construct a eukaryotic expression vector expressing human tumor necrosis factor alpha (hTNF-α) for the study on hTNF-α gene therapy.Methods The full length gene of hTNF-α cDNA was amplified by using RT-PCR from placenta tissue of a pregnant woman with pregnancy-induced hypertension, and inserted into a pMD 18-T vector.After gene sequence of hTNF-α was confirmed by cleavage of restriction enzymes and DNA sequence analysis,the fragment was inserted into corresponding enzymes cleavage sites in an eukaryotic expression vector pcDNA3.1-myc-his(-)A,B,C,and expression of hTNF-α mRNA of hTNF-α-transduced drug-resistant choriocarcinoma cell line was investigated using RT-PCR.Results The sequence of hTNF-α cDNA cloned from placenta tissue was completely correct,compared with the sequence in Genbank.Digestion with EcoR Ⅰand Hind Ⅲ confirmed that hTNF-α cDNA was inserted correctly into pcDNA3.1-myc-his(-)A,B,C eukaryotic expression vector.The drug resistant cell line of choriocarcinoma was transduced with an eukaryotic expression plasmid containing hTNF-α gene by lipofectin,and then expressed steadily hTNF-α cDNA.Conclusion The pcDNA3.1-myc-his(-)A,B,C/hTNF-α,a eukaryotic expression plasmid for the full length gene of hTNF-αcDNA,was constructed successfully by DNA recombinant technique and expressed steadily in vitro.

关 键 词：肿瘤坏死因子 DNA 基因疗法 聚合酶链反应 妊娠高血压综合症 

分 类 号：R73-36[医药卫生—肿瘤]