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 pIRES2-EGFP-5HRE-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 5HRE 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 pIRES2-EGFP-5HRE-AFPp-NTR is successfully constructed, which may be used for its further investigation in vitro.
Citation：
ZHOU Peihua,SUN Xuejun,WANG Yan,LU Shaoying. Construction of 5 Copies Hypoxia-Responsive Element Enhanced Suicide Gene Vector Targeting for Hepatocellular Carcinoma. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2007, 14(2): 163-167. doi:
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Copyright © the editorial department of CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY of West China Medical Publisher. All rights reserved
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- 1. 禄韶英， 王燕， 孙学军， 等. 缺氧诱导的肝癌靶向性基因治疗载体的构建和检测 [J]. 中国肿瘤生物治疗杂志， 2006； 13（1）∶59.
- 2. Shibata T, Akiyama N, Noda M, et al. Enhancement of gene expression under hypoxic conditions using fragments of the human vascular endothelial growth factor and the erythropoietin genes [J]. Int J Radiat Oncol Biol Phys， 1998; 42(4)∶913.
- 3. 萨姆布鲁克， D.W. 拉塞尔著； 黄培堂,等译. 分子克隆实验指南 [M]. 第3版. 北京： 科学出版社， 2002∶96~97.
- 4. Brown JM, Wilson WR. Exploiting tumour hypoxia in cancer treatment [J]. Nat Rev Cancer， 2004; 4(6)∶437.
- 5. Bussink J, Kaanders JH, van der Kogel AJ. Tumor hypoxia at the micro-regional level: clinical relevance and predictive value of exogenous and endogenous hypoxic cell markers [J]. Radiother Oncol， 2003; 67(1)∶3.
- 6. 李兴睿， 廖晓峰， 易继林. 缺氧对肝癌细胞系HepG2表达VEGF的影响 [J]. 中国普外基础与临床杂志， 2005； 12（5）∶477.
- 7. Goldberg MA, Schneider TJ. Similarities between the oxygen-sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin [J]. J Biol Chem， 1994; 269(6)∶4355.
- 8. Watanabe K, Saito A, Tamaoki T. Cell-specific enhancer activity in a far upstream region of the human alpha-fetoprotein gene [J]. J Biol Chem， 1987; 262(10)∶4812.
- 9. Ido A, Nakata K, Kato Y, et al. Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human alpha-fetoprotein gene promoter [J]. Cancer Res， 1995; 55(14)∶3105.
- 10. 全硕， 潘国宗， 刘东远, 等. 重组人甲胎蛋白基因顺式调控元件功能的研究 [J]. 基础医学与临床， 1999； 19（1）∶38.
- 11. Ido A, Uto H, Moriuchi A, et al. Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human alpha-fetoprotein promoter [J]. Cancer Res， 2001; 61(7)∶3016.
- 12. Helsby NA, Ferry DM, Patterson AV, et al. 2-amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT [J]. Br J Cancer， 2004; 90(5)∶1084.
- 13. Shibata T, Giaccia AJ, Brown JM. Hypoxia-inducible regulation of a prodrug-activating enzyme for tumor-specific gene therapy [J]. Neoplasia， 2002; 4(1)∶40.
- 14. Shibata T, Giaccia AJ, Brown JM. Development of a hypoxia-responsive vector for tumor-specific gene therapy [J]. Gene Ther， 2000; 7(6)∶493.
