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Construction of Recombinant Adenovirus Vector Containing Rat Adrenomedullin Gene and Its Effects on Proliferation of Mesenchymal Stem Cells

Author: ChenHua
Tutor: SuJinZi
School: Fujian Medical
Course: Internal Medicine
Keywords: adenovirus vector genetics AM MSCs MTT
CLC: R346
Type: Master's thesis
Year: 2008
Downloads: 97
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Abstract


ObjectiveTo construct the recombinant adenovirus vectors containing Adrenomedulin(AM) gene,and its effects on the proliferation of mesenchymal stem cells (MSCs).MethodsA pair of DNA primers with NotI and HindⅢrestriction Enzyme cutting sites were designed and synthesized. With the primers, AM cDNA was obtained from kidney of the rat by RT-PCR and cloned into pShuttte-CMV in correct direction. The product was pShuttle-CMV-AM. PShuttle-CMV-AM had been identified by double digestion with NotI and HindⅢrestriction emzymes and DNA sequencing.Homologous recombination occurred between pShuttle-CMV-AM and advenoviral backbone plasmid pAdEasy-1 in BJ5183 competent bacteria and the recombinant adenovirus was called rhAdEasy-AM. The recombinant vector was selected by Kanamycin. Identified by digestion with Pac I and PCR, it was amplified broadly in XL10-Gold competent bacteria and was packaged and amplified in AD-293 cell. The infectious titer of the recombinant adenovirus was tested. Western Blot were carried out to monitor the expression of AM protein in mesenchymal stem cells and finally evaluated the effect of AM on the proliferation of MSCs by MTT.ResultsDigestion with NotI and HindⅢgot a fragment size being 579bp. The result of DNA sequencing showed that the sequence of gene combined with pShuttle-CMV was identical to the coding region of AM gene in Genebank, suggesting that AM cDNA had been linked with pShuttle-CMV vector with correct order. Homologous recombination had completed successfully between shuttle vector pShuttle-AM and adenoviral backbone plasmid pAdEasy-1.The recombinant adenoviral plasmid could be digested with PacI to produce a fragment whose size was 3.0kb or 4.5kb.The infectious titer of adenovirus was 1×10~9pfu/ml. The expression of AM was confirmed by western blot and found that AM expressed in the MSCs have positive effect on the proliferation of MSCs.ConclusionThe eukaryotic cell expression vector named rhAdEasy-AM recombinant adenovirus carrying AM cDNA had been constructed successfully and was transfected into mesenchymal stem cells stably at high efficiency. Our data demonstrated that rhAdEasy-AM can expressed AM in the mesenchymal stem cells and promotes MSCs proliferation under hypoxia condition in vitro.

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CLC: > Medicine, health > Basic Medical > Human biochemistry, molecular biology
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