Construction of recombinant adenoviral vector Ad—CMV—hTGFβ1 for reversion of intervertebral disc degeneration by gene transfer-中国期刊网

摘要 Objective:ToprovideahighlyefficientadenoviralvectorAd-CMV-hTGFβ1forthestudyofgenetherapyforreversionoftheintervertebraldiscdegeneration.Methods:Anewlydevelopedrecombinantadenoviralvectorconstructionsystemwasusedinthestudy.ThecDNAofhTGFβ1wasfirstsubclonedintoashuttleplasmidpShuttle-CMV.TheresultantplasmidwaslinearizedbydigestingwithrestrictionendonucleasePmeI,andsubsequentlytransformedintoE.coll.BJ5183cellswithanadenoviralbackboneplasmidpAdEasy-1.Recombinantswereselectedbykanamycinresistanceandconfirmedbyrestrictionendonucleaseanalysis.Finally,therecombinantplasmidlinearizedbyPmeIwastransfectedinto293cells.Recombinantadenovirusesweregeneratedwithin2weeks.Results:TherecombinantadenoviralplasmidswerecutbyBamHIandPacIrespectively,andthediagnosticfragmentsappearedin0.8%agaroseelectrophoresis.Theinfected293cellsshowedevidentcytopathlceffect(CPE).TheproductionsofPCRconfirmedthepresenceofrecombinantadenovirus.TheexpressionofhTGFβ1wasverifiedbyimmunohistochemicalstaining.Conclusions:ThesuccessfulgenerationoftheadenoviralvectorAd-CMV-hTGFβ1andtheconfirmationoftheinterestgeneexpressionmakeitpossiblefortheexperimentalstudyofthereversionoftheintervertebraldiscdegenerationbygenetherapy.

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Construction of recombinant adenoviral vector Ad—CMV—hTGFβ1 for reversion of intervertebral disc degeneration by gene transfer

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Construction of recombinant adenoviral vector Ad—CMV—hTGFβ1 for reversion of intervertebral disc degeneration by gene transfer

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