Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (9): 1848-1856.doi: 10.3864/j.issn.0578-1752.2012.09.021

• VETERINARY SCIENCE • Previous Articles     Next Articles

Constructions of Arkadia and UCH37 Expression Vectors and Effects on TGF-β1/Smad7 Signal Pathway

 LIAO  Fang-Fang, YUAN  Si-Chun, ZHANG  Zhong-Wen, WU  Guo-Juan   

  1. 北京农学院动物科技学院，北京 102206
  • Received:2011-11-15 Online:2012-05-01 Published:2012-02-13

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Abstract: 【Objective】The objective of the study is to construct expression vectors of ubiquitination starting enzyme Arkadia and ubiquitin COOH-terminal hydmlase-L5 (also known as UCH37), and to investigate the effect of Smad7 modified by ubiquitination on TGF-β1/Smad7 signal pathway in aristolochic acid nephropathy (AAN). 【Method】The transfection condition was optimized by observing expressions of green fluorescences and undertaking real-time PCR reactions. After transferring the siRNA of Arkadia and UCH37, mRNA inhibition ratios were detected by real-time PCR. Then the expression vectors pGPU6/GFP/Neo of Arkadia and UCH37 were constructed, and transferred into cells after evaluations, and the expression changes of Arkadia, UCH37 and Smad7 were detected by real-time PCR and western blotting. 【Result】 Results showed that 30 pmol siRNA and 1.5 μL LipofectamineTM 2000 were the best transfection conditions. The interferences of Arkadia-1490 and UCH37-721 were most obvious. The inserted sequences of vectors were found to be correct, and pGPU6/GFP/Neo-Arkadia could increase Smad7 expression in AAN, while pGPU6/GFP/Neo-UCH37 was the opposite.【Conclusion】Expression vectors pGPU6/GFP/Neo-Arkadia and pGPU6/GFP/ Neo-UCH37 were constructed successfully, and it was found that Arkadia and UCH37 could separately enlarge, and inhibit TGF-β1/ Smad7 signal pathway.

Key words: Arkadia gene, UCH37 gene, transfection, expression vector, TGF-β1/Smad7 signal pathway

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