突变低氧诱导因子1α基因真核表达载体的构建和表达

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (11): 1348-1351.

突变低氧诱导因子1α基因真核表达载体的构建和表达

傅锐斌¹, 吴平生², 宋云峰¹, 邱建¹, 戴铁英², 李建华², 修建成²

1. 广州军区广州总医院心内科, 广东, 广州, 510010;
2. 南方医科大学南方医院心内科, 广东, 广州, 510515

出版日期:2005-11-20 发布日期:2005-11-20
基金资助:
收稿日期:2005-6-7。
基金项目:国家自然科学基金项目(30370587);广东省科技计划项目(C31206)
作者简介:傅锐斌(1974-),男,2003年毕业于第一军医大学,硕士,主治医师,电话:020-36653366,E-mail:furb7@sohu.com

Construction of eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 and their expressions in human microvascular endothelial cells

FU Rui-bin¹, WU Ping-sheng², SONG Yun-feng¹, QIU Jian¹, DAI Tie-ying², LI Jian-hua², XIU Jian-cheng²

1. 广州军区广州总医院心内科, 广东, 广州, 510010;
2. 南方医科大学南方医院心内科, 广东, 广州, 510515

Online:2005-11-20 Published:2005-11-20

摘要/Abstract

摘要： 目的构建人低氧诱导因子-1α(HIF-1α)真核表达载体pcDNA3.1⁺-HIF-1α的两种突变体pcDNA3.1⁺-HIF-1α-564Ala和pcDNA3.1⁺-HIF-1α-564Ala-803Ala,并检测它们在人肺微血管内皮细胞(HMVECs)中的表达。方法用定点突变的方法将pcDNA3.1⁺-HIF-1α的HIF-1α第564位脯氨酸(Pro)密码子CCC突变为丙氨酸(Ala)密码子GCC,构建成单个突变HIF-1α真核表达载体pcDNA3.1⁺-HIF-1α-564Ala。在第一次突变的基础上,仍然用定点突变的方法将pcDNA3.1⁺-HIF-1α-564Ala的HIF-1α-564Ala第803位天冬酰胺(Asn)密码子ATT突变为丙氨酸(Ala)密码子GCT,构建成双个点突变HIF-1α真核表达载体pcDNA3.1⁺-HIF-1α-564Ala-803Ala。将3种HIF-1α表达载体用脂质体法分别转入HMVECs,以RT-PCR、免疫荧光法和Westernblotting法分别对转染细胞进行表达分析。结果测序证实,定点突变成功,构建成重组真核表达载体pcDNA3.1⁺-HIF-1α-564Ala和pcDNA3.1⁺-HIF-1α-564Ala-803Ala;3种载体均能表达相应的蛋白和mRNA,但转化突变HIF-1α表达载体的细胞蛋白量比空白细胞和转化无突变HIF-1α载体的细胞显著增加。结论成功构建能够在HMVECs中表达的单个和双个点突变的HIF-1α基因的真核表达载体。

Abstract: Objective To construct eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 (HIF-1α) and study their expressions in human microvascular endothelial cells (HMVECs). Methods Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Pro564 (ccc) in HIF-1α into gcc (Ala) in pcDNA3.1⁺-HIF-1α to obtain single-site-mutated vector pcDNA3.1⁺-HIF-1α-564Ala, which was then subjected to a second site-directed mutagenesis to convert the codons for Asn803 into that of Ala (gct) to acquire double-site-mutated pcDNA3.1⁺-HIF-1α-564Ala-803Ala. After lipofectin-mediated transient transformation of HMVECs with the 3 recombinant plasmids including the two plasmids containing the mutations and the one without mutation, respectively, the expression levels of HIF-1α mRNA and protein were determined using RT-PCR, immunofluorescent staining and Western blotting. Results DNA sequence analysis demonstrated success of the two-step mutagenesis and the two plasmids of pcDNA3.1⁺-HIF-1α-564Ala and pcDNA3.1⁺-HIF-1α-564Ala- 803Ala were obtained, both of which could produce HIF-1α protein resistant to oxidation degradation in HMVECs as compared with the non-mutated one. Conclusion The recombinant plasmids pcDNA3.1⁺-HIF-1α-564Ala and pcDNA3.1⁺-HIF-1α- 564Ala-803Ala have been successfully constructed with efficient expressions in HMVECs.

中图分类号:

R-33

傅锐斌¹, 吴平生², 宋云峰¹, 邱建¹, 戴铁英², 李建华², 修建成². 突变低氧诱导因子1α基因真核表达载体的构建和表达[J]. 南方医科大学学报, 2005, 25(11): 1348-1351.

FU Rui-bin¹, WU Ping-sheng², SONG Yun-feng¹, QIU Jian¹, DAI Tie-ying², LI Jian-hua², XIU Jian-cheng². Construction of eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 and their expressions in human microvascular endothelial cells[J]. Journal of Southern Medical University, 2005, 25(11): 1348-1351.

参考文献

[1] Drake C J, Little CD. Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization[J]. Proc Natl Acad Sci, 1995, 92 (17): 7657-61.
[2] Epstein SE, Komowski R, Fuchs S, et al. Angiogenesis therapy:amidst the hype, the neglected potential for serious side effects[J].Circulation, 2001, 104(1): 115-9.
[3] Palmer LA, Semenza GL, Stoler MH, et al. Hypoxiainducestype Ⅱ NOS gene expression in pulmonary and systemic vascular cells[J]. Am J Physiol, 1998, 274(2 Pt 1): L212-9.
[4] Shweiki D, Itin A, Soffer D, et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis[J].Nature, 1992, 359(6398): 843-5.
[5] Firth JD, Ebert BL, Pugh CW, et al. Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: similarities with the erythropoietin 3’ enhancer[J]. Proc Natl Acad Sci, 1994, 91(14): 6496-500.
[6] Semenza GL, Roth PH, Fang HM, et al. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1[J]. J Biol Chem, 1994, 269(38): 23757-63.
[7] Wang GL, Jiang BH, Rue EA, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2tension[J]. Proc Natl Acad Sci, 1995, 92(6): 5510-4.
[8] Bruick RK, McKnight SL. A conserved family of rolyl-4-hydroxylases that modify HIF[J]. Science, 2001, 294(5545): 1337-40.
[9] Lando D, Peet DJ, Whelan DA, et al. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch[J]. Science, 2002,295(5556): 858-61.
[10] 傅锐斌,吴平生,戴铁英,等.pcDNA3.1⁺-HIF-1α载体构建和初步表达鉴定[J].第一军医大学学报,2003,23(11):1134-36.Fu RB, Wu PS, Dai TY, et al. Construction and expression analysis of recombinant vector pcDNA3.1⁺-HIF-1α[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2003, 23(11): 1134-36.
[11] Elson DA, Thurston G, Huang LE, et al. Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor-l alpha[J]. Genes Dev, 2001, 5(19):2520-32.

[1]	袁强；柳大烈；袁继龙；单磊；. 兔眶距增宽手术模型的建立与手术效果观察[J]. 南方医科大学学报, 2006, 26(10): 1476-.
[2]	崔彦芝；韩亚光；罗荣城；杨海青；姜达；. 肿瘤相关性抑郁动物模型的建立[J]. 南方医科大学学报, 2006, 26(10): 1513-.
[3]	白遵光；陈志强；王树声；王昭辉；欧润妹；. 一种梗阻性肾病动物模型的建立[J]. 南方医科大学学报, 2006, 26(08): 1209-1210.
[4]	李浩；彭汉伟；曾宗渊；郭朱明；. 大鼠喉移植改进模型的研究[J]. 南方医科大学学报, 2006, 26(07): 994-.
[5]	潘明新；李爱辉；高毅；谢金敏；张翌；张新建；张鸿飞；郭跃虎；张会迎；. 中国小型猪原位肝移植的实验研究[J]. 南方医科大学学报, 2006, 26(07): 1069-1070.
[6]	陈小林；何小洪；张焰；钱月桃；梁陆光；方典秋；詹澄扬；郑振声；马虹；. 慢性增强体外反搏猪模型的建立[J]. 南方医科大学学报, 2006, 26(05): 613-614.
[7]	董高宏；张肇达；胡伟明；唐勇；李建水；. 胰-十二指肠移植肠内引流的新型大动物模型[J]. 南方医科大学学报, 2006, 26(05): 626-628.
[8]	李琦；黄德亮；. 膜迷路破坏动物模型中水通道蛋白-1的表达及其意义[J]. 南方医科大学学报, 2006, 26(05): 664-666.
[9]	朱宝义；郭勇；王永强；. 糖尿病性白内障动物模型的建立与评价[J]. 南方医科大学学报, 2006, 26(05): 707-708.
[10]	林晓静;邹飞;李亚洁;王斌;贺文静;赵志荣;朱顺芳; . 重度热射病合并内毒素血症大鼠模型的建立[J]. 南方医科大学学报, 2006, 26(01): 86-89.
[11]	熊兵¹, 陈华德¹, 赖文¹, 熊仕秋², 郑少逸¹, 高辉¹, 卞徽宁¹, 刘族安¹. 双向排斥反应联体动物模型的建立[J]. 南方医科大学学报, 2005, 25(12): 1511-1513,1516.
[12]	胡静¹, 俞守义¹, 阚飙², 刘志华³. 缺失HPI毒力岛的EAggEC O42突变菌株的构建及鉴定[J]. 南方医科大学学报, 2005, 25(11): 1352-1356.
[13]	邓红, 邹飞, 罗海吉. 全反式视黄酸对新生大鼠纹状体神经干细胞向神经元样细胞分化的作用[J]. 南方医科大学学报, 2005, 25(11): 1357-1360,1374.
[14]	邱小忠¹, 余磊¹, 廖华¹, 张黎声², 秦建强¹, 杨俊¹, 欧阳钧¹. 活性氧促使L6成肌细胞的分化[J]. 南方医科大学学报, 2005, 25(11): 1384-1386.
[15]	汪萍¹, 吴小华¹, 陈文雪¹, 单保恩², 郭清³. 溶血磷脂酸受体在人卵巢癌细胞系3AO·SKOV3·OVCAR3中的表达及其意义[J]. 南方医科大学学报, 2005, 25(11): 1422-1424,1431.