细菌内同源重组法构建pAd-EGFP-hSDF-1α腺病毒质粒

目的:利用细菌内同源重组法构建和制备含人基质细胞源衍生因子-1α(hSDF-1α)重组腺病毒质粒。方法:设计5'端分别具有EcoR V/Xho I酶切位点的扩增hSDF-1αcDNA片段上下游引物,聚合酶链反应法(PCR)从pORF-hSDF-1α质粒中扩增hSDF-1α的cDNA,插入pGEM-T Easy载体中,构建pGEM-T-hSDF-1α质粒,EcoR V/Xho I双酶切后回收279 bp片段,与经过相同酶切的8.8 kb pShuttle-IRES-hrGFP-2进行连接,连接产物转化感受态DH5α,挑选克隆,重组质粒pShuttle-EGFP-hSDF-1α用EcoR V/Xho I酶切鉴定。pShuttle-EGFP-hSDF-1α经Pm e I酶切线性化后,转化含pAdeasy-1的超感受态B J5183,采用细菌内同源重组法构建腺病毒质粒pAd-EGFP-hSDF-1α;pAd-EGFP-hSDF-1α质粒经PacⅠ酶切鉴定和PCR鉴定。结果:线性化的pShuttle-EGFP-hSDF-1α转化含pAdeasy-1的超感受态B J5183,重组质粒经酶切获得一大于23 kb的大片段和4.5 kb的片段,PCR反应扩增出了279 bp的片段。结论:用细菌内同源重组成功地构建了含hSDF-1αcDNA的重组腺病毒质粒,为进行表达hSDF-1α的重组腺病毒的制备及hSDF-1α在骨髓源干细胞动员迁移中的作用研究奠定了基础。

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.