[Construction and expression analysis of recombinant vector pcDNA3.1+-HIF-1alpha].
OBJECTIVE
To construct the eukaryotic expression vector for human hypoxia-inducible factor-1alpha (HIF-1alpha) gene and examine its expression.


METHODS
Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on the total RNA extracted from HT29 cells to obtain the cDNA of HIF-1alpha, which was inserted into T vector pUC18. DNA sequencing was performed before the amplified products were cloned into the eukaryotic expression vector pcDNA3.1+ identified by endonuclease digestion. This recombinant vector was transfected into HEK293 cells by means of liposome and its expression examined.


RESULTS
The amplified products were confirmed as the cDNA of HIF-1alpha by DNA sequencing, and pcDNA3.1+ -HIF-1alpha obtained was verified by endonuclease digestion, being capable of expression in HEK293 cells.


CONCLUSIONS
We have successfully constructed the eukaryotic expression vector for HIF-1alpha, which can be expressed in HEK293 cells.
Fu
Rui-bin
RB
Department of Cardiology, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China. furb7@sohu.com
Wu
Ping-sheng
PS
Dai
Tie-ying
TY
Lai
Wen-yan
WY
Qiu
Jian
J
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics*
D018076:DNA, Complementary; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D014157:Transcription Factors