[Construction and expression analysis of recombinant vector pcDNA3.1+-HIF-1alpha]

Rui-bin Fu, Ping-sheng Wu, Tie-ying Dai, Wen-yan Lai, Jian Qiu
Di 1 Jun Yi da Xue Xue Bao, Academic Journal of the First Medical College of PLA 2003, 23 (11): 1134-6

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.

CONCLUSION: We have successfully constructed the eukaryotic expression vector for HIF-1alpha, which can be expressed in HEK293 cells.

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