人肝癌靶向性CD80基因表达重组腺病毒载体的构建及鉴定

Construction of Human Hepatoma-targeting Recombinant Adenovirus Vector of CD80 and Its Identification

目的:构建人肝癌靶向性CD80基因重组腺病毒载体。方法:首先,从人基因组DNA中克隆AFP增强子、启动子,利用腺病毒穿梭质粒pShuttle和pShutte-CMV,采用AFP增强子替换CMV启动子,构建含有AFP启动子和增强子的穿梭质粒,命名为pShuttle-AFP。将人CD80基因克隆到pShuttle-AFP的AFP增强子和启动子之后,构建pShuttle-AFP-CD80质粒。再将其与腺病毒骨架质粒pAdEasy-1共转化E.coliBJ5183,利用同源重组构建腺病毒质粒pAd-AFP-CD80。以获得的重组子转染AD293细胞后制备重组腺病毒,并对重组的病毒进行鉴定和病毒滴度测定。结果:测序结果与Genebank中公布的AFP启动子、核心增强子和CD80基因序列相符。双酶切结果与预期结果相符。PacI酶切结果与目的结果一致。穿梭质粒pShutte-AFP-CD80和腺病毒质粒pAd-AFP-CD80经酶切和PCR鉴定均获得期望大小的目的片段。结论:成功的构建了人肝癌靶向性pAd-AFP-CD80腺病毒载体,为CD80基因在肝癌靶向性治疗的研究奠定了基础。

Objective： To construct the hepatoma targeting CD80 gene recombinant adenovirus vector. Methods： The enhancer and promoter of AFP were cloned from the human genomic DNA, and pShuttle-AFP plasmid was generated by replacing the CMV promoter of pShuttle-CMV. CD80 gene was inserted into the downstream of AFP enhancer and promoter, to construct the pShuttle-AFP-CD80 plasmid. After digestion with Pme I, pShuttle-AFP-CD80 was co-transformed into E. coli BJ5183 with adenovirus backbone plasmid, and pAd-AFP-CD80 was constructed by homologous recombination. The recombinant adenovirus DNA was transfected into AD293 cells to prepare adenovirus. After identification and titer of the prepare adenovirus. Results： the cloned AFP enhancer promoterand CD80 gene were identical to those reported in GeneBank. Double-digestion results in line with the expected results. Pac I digestion results are consistent with the purpose of. the pShuttle-AFP-CD80 and pAd-AFP-CD80 were successfully constructed after identification by digestion and PCR. Conclusion： We successfully constructed the human hepatoma targeting CD80 expression adenovirus vector, which laid the foundation for the hepatoma targeting immunotherapy with CD80 molecular.