5HRE强化的肝癌靶向性自杀基因载体的构建  

Construction of 5 Copies Hypoxia-Responsive Element Enhanced Suicide Gene Vector Targeting for Hepatocellular Carcinoma

作　　者：周培华[1] 孙学军[1] 王燕[2] 禄韶英[1] 

机构地区：[1]西安交通大学医学院第一附属医院普通外科,西安710061 [2]第四军医大学唐都医院疼痛研究所,西安710038

出　　处：《中国普外基础与临床杂志》2007年第2期163-167,共5页Chinese Journal of Bases and Clinics In General Surgery

基　　金：国家自然科学基金资助项目(编号:30400430)~~

摘　　要：目的设计并构建5个拷贝的缺氧反应元件(5HRE)增强子和甲胎蛋白启动子(AFPp)联合调控的自杀基因硝基还原酶(NTR)真核表达载体。方法设计引物作重叠PCR从大肠杆菌中克隆融合有6个组胺酸基因标签(6his-tag)的自杀基因NTR,用合成的HRE寡核苷酸单链退火,多次重组克隆构建5HRE。将NTR基因、5HRE和AFPp与酶切去除了巨细胞病毒即刻早期启动子(Pcmvie)的pIRES2-EGFP载体重组,构建携带5HRE、AFPp和NTR基因的真核表达载体pIRES2-EGFP-5HRE-AFPp-NTR。结果成功克隆了融合6his-tag的NTR基因,NTR基因与Genbank公布的序列一致。成功合成了5HRE,测序与预期相符。将上述片段和AFPp重组到去除了Pcmvie的pIRES2-EGFP载体上,酶切鉴定和DNA序列分析无误。结论成功构建了真核表达载体pIRES2-EGFP-5HRE-AFPp-NTR,为下一步的体外实验奠定了基础。Objective To design and construct the eukaryotic expressed vector of suicide genes, which contained 5 copies of hypoxia-responsive element （5HRE）, promoter of alpha-fetoprotein gene （AFPp） and nitroreductase from Escherichia coli. Methods The constructing processes were as follows： ①The design of primer： Suicide genes of NTR in the Escherichia coli, which contained 6his-tag gene （6his-tag）, were cloned by overlapping PCR. ②The construction of 5HRE： The single strand of synthetized HRE oligonucleotide was annealed, and 5HRE was constructed by multiple recombinant clone. ③The recombination of NTR gene, 5HRE, AFPp and pIRES2-EGFP： pIRES2-EGFP, which had removed the instant early promoter of cytomegalovirus, was recombined with NTR gene, 5HRE, AFPp. In this way, the eukaryotic expressed vector of plRES2-EGFP-SHRE-AFPp-NTR, which carried NTR gene, 5HRE, AFPp was finally constructed. Results NTR gene, which contained the fusion of 684-base pair and 6his-tag gene, was cloned successfully, and its sequence was coincident with the result published by Genbank. A 221-base pair of 5 HRE was also constructed, which was in accordance with the expected sequences. The integrity of the eukaryotic expressed vector was verified by restriction enzyme digestion and DNA sequence analysis, respectively. Conclusion The eukaryotic expressed vector of plRES2-EGFP-5HRE-AFPp-NTR is successfully constructed, which may be used for its further investigation in vitro.

关 键 词：缺氧反应元件 肝癌 自杀基因 

分 类 号：R735.7[医药卫生—肿瘤]