HBx和mIL-12双基因重组腺病毒的构建

Construction of Recombinant Adenovirus Carrying HBx and mIL-12

目的构建携带双基因HBx和mIL-12的重组腺病毒Ad-HBx-mIL-12。方法采用酶切、连接的方法分别将基因HBx和mIL-12连接到穿梭质粒载体pAdenoVator-CMV5。将构建正确的穿梭质粒pAdV-HBx-mIL-12经EcoRⅠ酶切线性化后,转化于含腺病毒骨架质粒pAdenoVatorΔE1/E3的BJ5183感受态细菌,实现细菌内同源重组。将鉴定正确的重组腺病毒质粒pAd-HBx-mIL-12经PacⅠ酶切线性化后,以脂质体法转染至293细胞,包装并扩增获得携带双基因HBx和mIL-12的重组腺病毒Ad-HBx-mIL-12。结果经鉴定携带HBx和mIL-12双基因的重组腺病毒构建成功,并可在293细胞中有效表达。结论成功构建了携带双基因HBx和mIL-12的重组腺病毒Ad-HBx-mIL-12,为探讨其联合抗肿瘤机制及后续基因治疗奠定了基础。

Objective To construct a recombinant adenovirus Ad-HBx-mIL-12 carrying HBx and mIL-12. Methods HBx and mIL-12 were cloned into the shuttle plasmid pAdenoVator-CMV5 and confirmed by means of enzymatic manipulation. After linearization by EcoR Ⅰ digestion, the recombinant shuttle plasmid pAdV-HBx-mIL- 12 was transformed into competent BJ5183 germs with the adenoviral backbone plasmid pAdenoVator AE1/E3 and then homologically recombined to obtain the recombinant adenovirus plasmid. After confirmation, the recombinant adenovirus plasmid pAd-HBx-mIL-12 was linearized with Pac Ⅰ digestion and transfected into 293 cells via liposome, and then adenovirus package and amplification were performed. Results It was confirmed that the recombinant adenovirus Ad-HBx-mIL-12 had been successfully constructed and both HBx and raiL-12 were expressed in 293 cells. Conclusion The recombinant adenovirus carrying HBx and mIL-12 has been successfully constructed, which lays a foundation for the further study of antitumor mechanism and gene therapy.