Construction of Recombinant Adenovirus Plasmid Containing hSDF-1α cDNA By Homologous Recombination in Bacteria

Objective To construct recombinant adenovirus plasmid containing hSDF-1α cDNA by using a novel and high efficient method of homologous recombination in bacteria.Methods hSDF-1α cDNA primers were synthesized with forward primer and reverse primer containing EcoR V and Xho I,respectively.hSDF-1α cDNA was amplified by polymerase chain reaction(PCR),and subcloned into pGEM-T Easy vector to construct pGEMT-hSDF-1α.The fragment of 279 bp hSDF-1α cDNA recovered from EcoR V/Xho I-digested pGEM-T-hSDF-1α was subcloned into pShuttle-IRES-hrGFP-2.The recombinant plasmid was named after pShuttle-EGFP-hSDF-1α and identified with EcoR V and Xho I digestion.Adenovirus genomic DNA plasmid of pAdeasy-1 was transformed into BJ5183 bacteria and ultracompletent BJ5183 containing pAdeasy-1 was prepared,pShuttle-EGFP-hSDF-1α was linealized with Pme I and transformed into ultracompletent BJ5183 containing pAdeasy-1,the identification of recombinant adenovira1 plasmid pAd-EGFP-hSDF-1α was performed with digestion with Pac I and PCR.Results There were two bands,4.5kb and larger than 23 kb when pAd-EGFP-hSDF-1α was digested with Pac I and electrophoresis.There appeared 279 bp hSDF-1α cDNA fragment when PCR was performed with pAd-EGFP-hSDF-1α as template.Conclusion The recombinant adenoviral plasmid carrying hSDF-1α cDNA was successfully constructed with homologous recombination in bacteria.This study provides a basis for the preparation of recombinant adenovirus expressing hSDF-1α and for exploring the migration mechanism of bone marrow-derived stem cells into injured tissue.