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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2015, Vol. 35 Issue (4): 30-41    DOI: 10.13523/j.cb.20150406
    
Construction of HPD Promoter Reporter Vector and Analysis of Its Transcriptional Activity
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Abstract  

 HPD is a kind of tyrosine metabolizing enzyme with specific expression in liver. The current research mainly focuses on the relationship between HPD and tyrosinemia diseases, while there is less reporter about the transcription regulational mechanism of HPD, and the mechanism of HPDliver specific expression is not yet known. The aim was to construct HPD gene promoter luciferase reporter gene vector, analyze its transcriptional activity in the human hepatocellular cells, and begin to clarify the regulational mechanisms of its specific expression in liver. Methods: The 5′flanking sequence from －2 000 to +39 of the human HPD gene was analyzed by the UCSC software for the promoter character. This sequence were cloned from the human hepatocellular carcinoma cell HepG2 genomic DNA by PCR and inserted into the pGL3Basic vector.Through the design of different primers, a series of 5′deleted constructs were made to product seven luciferase reporter gene vectors in different lengths. Then the seven reconstructors were transfected into HepG2,L02 and HEK293 cells respectively to determine the transcriptional activity of HPD gene promoter by the dualluciferase analysis.Results: The dualluciferase analysis results showed that the fragment from －600 to －400 had a stronger transcriptional activity in HepG2and L02 than in HEK293,while the fragment from －400 to +39 had the transcriptional activity in all of the three cells. Bioinformatics analysis results showed that there are many liver specific transcription factor binding sites in －600 ~－400 area of HPD gene promoter sequence. Conclusion: The luciferase reporter gene vectors with the HPD gene promoters are constructed successfully. In the fragment from －600 to －400,there would have the key regulatory elements for the HPD liverspecific expression,which provides an important basis and lays a theoretical foundation for further revealing the transcriptional regulational mechanism of HPDliver specific expression.

Received: 19 January 2015      Published: 25 April 2015
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Cite this article:

. Construction of HPD Promoter Reporter Vector and Analysis of Its Transcriptional Activity. China Biotechnology, 2015, 35(4): 30-41.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20150406     OR     https://manu60.magtech.com.cn/biotech/Y2015/V35/I4/30

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