Construction of eukaryotic expression vector encoding the human transforming growth factor-β1 and its expression in COS-7 cells

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Author:
YU Hong(First Affiliated Hospital,Chongqing Medical University)
ZHOU Jing(First Affiliated Hospital,Chongqing Medical University)
SUN Wei(First Affiliated Hospital,Chongqing Medical University)
ZHOU Zhou(First Affiliated Hospital,Chongqing Medical University)
WU Ying(First Affiliated Hospital,Chongqing Medical University)
JIANG Zhen(First Affiliated Hospital,Chongqing Medical University)
WANG Pei-Long(First Affiliated Hospital,Chongqing Medical University)
Journal Title:
JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
Issue:
Volume 30, Issue 24, 2009
DOI:
Key Word:
hepatic fibrosis; hepatin stellate cell; transforming growth factor-β_1 ; gene therapy; transfection

Abstract: AIM:To construct the recombinant human transforming growth factor-β_1 eukaryotic expression vector and to detect its expression in COS-7, in order to lay the foundation for elucidating pathogenesis of hepatic fibrosis and constructing the correspondence genetherapy with RNA interference. METHODS:Total mRNA was extracted from human hepatic tissue and the TGFβ_1 cDNA was obtained by RT-PCR and cloned into pEGFP-N1 vector, and then the pEGFP-NI-TGF-β_1 vector was constructed,identificated and transfected into COS-7 cells. Its expression was detected by fluorescent microscopy, RT-PCR and Western Blot.RESULTS:Enzyme digestion analysis and sequencing showed that the target genes was found to be 1173 bp, and had been inserted into recombinant vector, as compared with gene reported by GenBank, O. 08% of the gene mutation(531 th:T→C)and no amino acid residues change in hTGF-β_1. RT-PCR and Western Blot demonstrated that the recombinant vector pEGFP-N1-TGF-β_1 was expressed in COS-7 ceils, and the relatived molecular mass of the GFP-TGF-β_1 fusion protein was about 52×10~3, while the relatived molecular mass of the GFP protein expressed by pBud-GFP was about 27×10~3. The Western Blot analysis indicated that the GFP-TGF-β_1 fusion protein could be recognized by anti-GFP antibody, suggesting that this fusion protein had good biological activity.CONCLUSION:The eukaryotie expression plasmid pEGFP-N1TGF-β_1 has been constructed and expressed successfully in COS-7 cells, which would lay a foundation for further study on the applications of TGF-β_1 in gene therapy of fibrosis.

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