| Autor: |
Tao LI, Wen-yan YU, Xiao-li OU, Ai-hua LIU, Zhu-zhong MEI, Yong JIANG |
| Jazyk: |
čínština |
| Rok vydání: |
2011 |
| Předmět: |
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| Zdroj: |
Medical Journal of Chinese People's Liberation Army, Vol 36, Iss 4, Pp 352-355 (2011) |
| ISSN: |
0577-7402 |
| Popis: |
Objective To construct a eukaryotic expression vector for HuR and analyze its expression and biological function in NIH3T3 cells.Methods The total RNA was extracted from NIH3T3 cells and reverse transcribed to cDNAs.The coding region sequence of mouse HuR was then amplified by PCR and subcloned into the pcDNA3.1-FLAG plasmid.The recombinant plasmid pcDNA3.1-HuR-FLAG was verified by PCR and restriction endonuclease analysis,confirmed by DNA sequence analysis,and then transiently transfected into NIH3T3 cells with Lipofectamine LTX.The expression of HuR protein was determined by Western blotting,and the mRNA level of HuR and DUSP1 were analyzed by using real-time PCR.Result The recombinant plasmid pcDNA3.1-HuR-FLAG was correctly constructed.Twenty-four hours after transfection of the recombinant plasmid into NIH3T3 cells,the fusion protein was found to have highly expressed in the cells as revealed by Western blotting.Real-time PCR results detected that the over-expression of HuR could up-regulate the expression of DUSP1.Conclusion The eukaryotic expression vector for HuR-FLAG fusion protein has been successfully constructed and transiently expressed in NIH3T3 cells.It can be used in further analysis of the posttranscriptional regulation of DUSP1 by HuR in cancer cells. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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