细菌内同源重组法构建pAd-EGFP-hSDF-1α腺病毒质粒

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

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。

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 polymersse chain reaction （PCR） ,and subcloned into pGEM - T Easy vector to construct pGEM - T - hSDF - 1α. The fragment of 279 bp hSDF - 1α cDNA recovered from EcoR Ⅴ/Xho Ⅰ - digested pGEM -T- hSDF -1α was subcloned imo 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 idemification of recombinant adenoviral 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. Conciusion 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 hone marrow -derived stem cells into injured tissue.