The Construction And Identification Of The Adenovirus Vector Carring The Extracelluar Fragment Of CD40L

【Object】Recombinant adenoviruses vector carring the extracelluar fragment of CD40L gene of the rhesus was constructed with pAdEasy-1 system by the homologous recombination method in baeteria.【Method】the extracelluar fragment of CD40L gene synthesized using overlapping PCR was connected with the efficient cloning vector pGEM-T, then was converted into E.coli TOP 10 competence baeteria after the test of application of agarose gel electrophoresis and DNA sequencing. the target genes plasmid was extracted after clonal expansion.The pShuttle-CMV-efCD40L was obtained by connecting the pShuttle-CMV in the AdEasy adenovirus vector system ang pGEM-T- efCD40L after double digests。The recombinant adenovirus plasmid was obtained after the transformation of BJ5183 bacteria which was perform with the pAdEasy-1 and linearized pShuttle-CMV-efCD40L.The corresponding gene resistant screeningand PCR analysis certified the successful cloning of the pAd-CMV-efCD40L.The linearized pAd-CMV- efCD40L transfected the AD293 cell by taking advantage of Lipofectamine 2000.After the identification by PCR and RT-PCR,the adenovirus was propagated.【Result】We completed the construction of shuttle plasmid pShuttle-CMV-efCD40L, and further constructed recombined adenovirus vector pAd-CMV-efCD40L by the method of the homologous recombination in bacteria.【Conclusion】 the method of the homologous recombination in bacteria is a conveniently efficient way to construct recombinant adenovirus plasmid with efCD40L.It prepare a condition for the further studies for using adenovirus-mediated efCD40L gene and PDL-1 contransfect immature dendritic cells(iDCs) to induce immunologic tolerance in liver transplantation.