[Construction of eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 and their expressions in human microvascular endothelial cells].
OBJECTIVE
To construct eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 (HIF-1alpha) 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-1alpha into gcc (Ala) in pcDNA3.1(+)-HIF-1alphato obtain single-site-mutated vector pcDNA3.1(+)-HIF-1alpha-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-1alpha-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-1alpha 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-1alpha-564Ala and pcDNA3.1(+)-HIF-1alpha-564Ala- 803Ala were obtained, both of which could produce HIF-1alpha protein resistant to oxidation degradation in HMVECs as compared with the non-mutated one.


CONCLUSIONS
The recombinant plasmids pcDNA3.1(+)-HIF-1alpha-564Ala and pcDNA3.1(+)-HIF-1alpha- 564Ala-803Ala have been successfully constructed with efficient expressions in HMVECs.
Fu
Rui-bin
RB
Department of Cardiology, Guangzhou General Hospital of Guangzhou Command, Guangzhou 510010, China. furb7@sohu.com
Wu
Ping-sheng
PS
Song
Yun-feng
YF
Qiu
Jian
J
Dai
Tie-ying
TY
Li
Jian-hua
JH
Xiu
Jian-cheng
JC
D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D018919:Neovascularization, Physiologic; D017354:Point Mutation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger