重组腺病毒载体pAd-NY-ESO-1-EGFP的构建及鉴定

Construction and Identification of Recombinant Adenovirus Vector Containing NY-ESO-1 Gene

目的:利用细菌内同源重组法构建含人肿瘤特异性抗原的腺病毒质粒。方法:设计NY-ESO-1 cDNA扩增引物,从pET15b-NY-ESO-1质粒中扩增NY-ESO-1的DNA序列,与pShuttle-IRES-hrGFP-2进行连接,PCR和EcoRⅤ酶切鉴定重组质粒pShuttle-NY-ESO-1-EGFP,经Pm eⅠ酶切线性化后,转化含pAdeasy-1的超感受态B J5183大肠杆菌,细菌内同源重组法构建腺病毒质粒pAd-NY-ESO-1-EGFP;质粒经PacⅠ酶切鉴定,DNA测序。结果:线性化的pShuttle-NY-ESO-1-EGFP转化含pAdeasy-1的超感受态B J5183大肠杆菌,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340 bp的片段。结论:用细菌内同源重组法成功地构建了含NY-ESO-1的重组腺病毒质粒,为进行表达NY-ESO-1的重组腺病毒的制备及肿瘤特异性抗原NY-ESO-1的核酸疫苗在肿瘤方面的研究奠定了基础。

Objective To construct recombinant advenovirus plasmid containing human tumor-specific antigen NY-ESO-1 gene by using the method of homologous recombination in bacteria. Methods The NY-ESO-1 cDNA primers were designed, the DNA sequence of NY-ESO-1 was amplified from recombinant vector pET15b-NY-ESO-1 and ligated into pShuttle-IRES- hrGFP-2. The recombinant plasmid was named after pShuttle-NY-ESO-1-EGFP and was identified with PCR and EcoR V digestion, pShuttle-NY-ESO-1-EGFP was linealized with Pine I and transformed into ultracompletent BJ5183 containing pAdeasy-1, then recombinant advenovirus pAd-NY-ESO-1-EGFP was constructed by homologous recombination in bacteria. The recombinant adenoviral plasmid pAd-NY-ESO-1-EGFP was identified by Pac I digestion and DNA sequencing. Resuits There were two bands ,4.5kb and larger than 23 kb when pAd-NY-ESO-1-EGFP was digested with Pac I . A 340bp NY-ESO-1cDNA fragment was amplified by PCR. Conclusion The recombinant adenoviral plasmid containing NY-ESO-1 was successfully constructed with homologous recombination in bacteria. This study provides a basis for the preparation of recombinant adenovirus expressing NY-ESO-1 and tumor-specific antigen NY-ESO-1 DNA vaccine for research in cancer therapy.