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| Comparison of the Co-expression Level of Ang-1/VEGF165 Mediated by IRES and PolyA-promoter |
| CHEN Yao1, MIAO Jing-cheng1, HAN Ya-li1, SHENG Wei-hua1, BAO Wan-rong1, TIAN Li-na1, ZHANG Feng-juan1, LV Hai-tao2, YANG Ji-cheng1 |
1. Department of Cellular and Molecular Biology, Medical School, Suzhou University, Suzhou 215123, China;
2. Medical Department of Cordis of the Affiliated Children’s Hospital in Soochow University, Suzhou 215006,China |
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Abstract Objective: Construct recombinant adenoviral vector co-expressing angiogenin-1(Ang-1) and vascular endothelial growth factor 165(VEGF165) mediated by IRES and polyA-promoter,then compare the expression of Ang-1/VEGF165 mediated by IRES and polyA-promoter as well as in the downstream and upstream of polyA-promoter/IRES likewise their vascular inducibility,to provid experimental evidence for constructing superior recombinant vector co-expressing double gene or multiple gene.Methods: The VEGF165 and Ang-1 fragments were amplified by PCR using pAdTrack-CMV-Ang-1-IRES-VEGF165 plasmids as templates.The VEGF165 and Ang-1 fragments were subcloned into pAdTrack-CMV-IRES and pAdTrack-CMV-PolyA-promoter transfer vector,and then identified by PCR,double endonuclease digestion,and DNA sequencing.The pTrack-CMV-Ang-1-polyA-promoter-VEGF165、pTrack-CMV-VEGF165-polyA-promoter-Ang-1、pTrack-CMV-VEGF165-IRES-Ang-1 gene recombinated transfer vectors were cotransformed into the bacteria BJ5183 competent cells with pAdEasy-1 backbone vector for homologous recombination,then they were linearized with PacI digestion and transfected into the human embryonic kidney 293 (293A) cells, leading to formation of the recombinant adenoviruses Ad-Ang-1-polyA-promoter-VEGF165、 Ad-VEGF165-polyA-promoter-Ang-1 and Ad-VEGF165-IRES-Ang-1.The transcription of VEGF165 and Ang-1 were identified by RT-PCR,and the expression of VEGF165 and Ang-1 in different adenoviral vector were then identified by enzyme-labeled immunosorbent assay(ELISA),then compared the expression level of Ang-1 and VEGF165 gene in IRES and polyA-promoter mediated vector as well as in the downstream and upstream of the same adenoviral vector.Injected Ad-Ang-1-polyA-promoter-VEGF165,Ad-VEGF165-polyA-promoter-Ang-1,Ad-VEGF165-IRES-Ang-1,Ad-Ang-1-IRES-VEGF165 to the rabbit corneal limbal and detected the new vessels areae, then compared their vascular inducibility.Results: The sequencing of Ang-1 and VEGF165 were correct,three kinds of recombinant adenoviral vector were all successfully constructed and obtained,and their potency were up to 2~5×1010pfu/ml, the transcription of VEGF165 and Ang-1 were also significant by RT-PCR.Moreover VEGF165 and Ang-1 gene expressing in the WI-38 cells was confirmed by ELISA,also found that the IRES-mediated Ang-1 and VEGF165 gene, either upstream or downstream,its expression level was lower than the same location in polyA-promoter-mediated genes,degraded about 60%~70%, moreover either the IRES vector or polyA-promoter vector,the expression of downstream genes volume was significantly lower than the upstream gene volume, degraded about 30%~40%.At the same time the animal experiment of vasiformation in rabbit cornea suggestted that the vascular inducibility of Ad-VEGF165-PolyA-promoter-Ang-1 and Ad-VEGF165-IRES-Ang-1 were better,and the former was stronger than the latter.Conclusion:In the adenoviral expression vector,Ang-1 and VEGF165 gene could both successfully expressed in cell,and both had vascular inducibility.The expression level and vascular inducibility of polyA-promoter-mediated double gene was higher and strongger than the IRES-mediated double gene,at the same time the expression level and vascular inducibility of the gene volume in the downstream of polyA-promoter/IRES were lower than the genes volume in the upstream.
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Received: 21 July 2010
Published: 25 December 2010
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Cite this article:
CHEN Yao, MIAO Jing-cheng, HAN Ya-li, SHENG Wei-hua, BAO Wan-rong, TIAN Li-na, ZHANG Feng-juan, LU Hai-tao, YANG Ji-cheng. Comparison of the Co-expression Level of Ang-1/VEGF165 Mediated by IRES and PolyA-promoter. China Biotechnology, 2010, 30(12): 10-19.
URL:
https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2010/V30/I12/10
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