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

Abstract

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.

CLC Number:

R-33

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.

References

[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]	YUAN Qiang, LIU Da-lie, YUAN Ji-long, SHAN Lei Department of Plastic and Reconstructive Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. Establishment of rabbit orbital hypertelorism model and evaluation of effects of surgical correction [J]. Journal of Southern Medical University, 2006, 26(10): 1476-.
[2]	CUI Yan-zhi1, HAN Ya-guang1, LUO Rong-cheng1, YANG Hai-qing2, JIANG Da3 1Department of Oncology, Nanfang Hospital, 3Department of Statistics, Southern Medical University, Guangzhou 510515, China; 2Department of Oncology, Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China. Establishment of an rat model of tumor-associated depression [J]. Journal of Southern Medical University, 2006, 26(10): 1513-.
[3]	. [J]. Journal of Southern Medical University, 2006, 26(08): 1209-1210.
[4]	LI Hao, PENG Han-wei, ZENG Zong-yuan, GUO Zhu-ming State Key Laboratory of Oncology in Southern China, Department of Head and Neck, Oncology Center, Sun Yat-sen University, Guangzhou 510060, China. Research of modified rat laryngeal transplantation model [J]. Journal of Southern Medical University, 2006, 26(07): 994-.
[5]	. [J]. Journal of Southern Medical University, 2006, 26(07): 1069-1070.
[6]	CHEN Xiao-lin,HE Xiao-hong,ZHANG Yan,QIAN Yue-tao,LIANG Lu-guang,FANG Dian-qiu,ZHAN Cheng-yang,ZHENG Zhen-sheng,MA Hong Department of Cardiology,First Affiliated Hospital,Sun Yat-sen University,Guangzhou 510089,China. Establishment of a pig model of chronic ally enhanced external counterpulsation [J]. Journal of Southern Medical University, 2006, 26(05): 613-614.
[7]	DONG Gao-hong1,ZHANG Zhao-da2,HU Wei-ming2,TANG Yong2,LI Jian-shui2 1Center of Organ Transplantation,First Affiliated Hospital of Jinan University,Guangzhou 510630,China;2West China Hospital of Sichuan University,Chengdu 610041,China. Establishment of whole pancreaticoduodenal allotransplantation model with portal venous drainage and enteric drainage in pigs [J]. Journal of Southern Medical University, 2006, 26(05): 626-628.
[8]	LI qi1,HUANG De-liang2 1Institute of Otolaryngology,2Department of Otolaryngology-Head and Neck Surgery,General Hospital of PLA,Beijing 100853,China. Expression of aquaporin-1 in a guinea pig model of labyrinth destruction [J]. Journal of Southern Medical University, 2006, 26(05): 664-666.
[9]	. [J]. Journal of Southern Medical University, 2006, 26(05): 707-708.
[10]	LIN Xiao-jing, ZOU Fei, LI Ya-jie, WANG Bin, HE Wen-jing, ZHAO Zhi-rong, ZHU Shun-fang Nanfang Hospital, Institute of Public Hygiene and Tropical Medicine, Southern Medical University, Guangzhou 510515, China. Establishment of a rat model of severe heatstroke complicated with endotoxemia [J]. Journal of Southern Medical University, 2006, 26(01): 86-89.
[11]	XIONG Bing¹, CHEN Hua-de¹, LAI Wen¹, XIONG Shi-qiu², ZHEN Shao-yi¹, GAO Hui¹, BIAN Hui-ning¹, LIU Zu-an¹. Establishing a parabiosis animal model of two-way paradigm [J]. Journal of Southern Medical University, 2005, 25(12): 1511-1513,1516.
[12]	HU Jing¹, YU Shou-yi¹, KAN Biao², LIU Zhi-hua³. Construction and characterization of mutant Enteroaggregative E. coli O42 strain with high-pathogenicity island deletion [J]. Journal of Southern Medical University, 2005, 25(11): 1352-1356.
[13]	DENG Hong, ZOU Fei, LUO Hai-ji. Differentiation of neonatal rat striatal neural stem cells induced by all-trans retinoic acid [J]. Journal of Southern Medical University, 2005, 25(11): 1357-1360,1374.
[14]	QIU Xiao-zhong¹, YU Lei¹, LIAO Hua¹, ZHANG Li-sheng², QIN Jian-qiang¹, YANG Jun¹, OUYANG Jun¹. Oxidative stress-induced differentiation of L6 myoblasts [J]. Journal of Southern Medical University, 2005, 25(11): 1384-1386.
[15]	WANG Ping¹, WU Xiao-hua¹, CHEN Wen-xue¹, SHAN Bao-en², GUO Qing³. Expression of lysophosphatidic acid receptor in human ovarian cancer cell lines 3AO,SKOV3,OVCAR3 and its significance [J]. Journal of Southern Medical University, 2005, 25(11): 1422-1424,1431.