重组腺病毒rAd-mIL-15的构建及表达  

Construction and expression of recombinant adenovirus rAd-mIL-15

作　　者：谭榜云[1] 李莉[1] 刘志武[1] 

机构地区：[1]兰州大学第一医院检验科,甘肃兰州730000

出　　处：《检验医学与临床》2017年第22期3300-3302,共3页Laboratory Medicine and Clinic

基　　金：甘肃省青年科技基金计划资助项目(1606RJYA266)

摘　　要：目的构建并制备鼠白细胞介素-15(mIL-15)重组腺病毒(rAd-mIL-15),感染HEK293细胞,为肝细胞肝癌的免疫治疗奠定基础。方法采用聚合酶链反应(PCR)扩增mIL-15,将扩增产物连接到穿梭载体pDC316上,构建重组穿梭质粒pDC316-mIL-15。在Lipofectamine2000脂质体介导下将腺病毒骨架载体pBHGlox△E1,3Cre和穿梭质粒pDC316-mIL-15共转染入HEK293细胞,进行同源重组,得到腺病毒重组质粒pAd-mIL-15。随后在HEK293细胞中包装扩增病毒并测定病毒滴度。采用PCR对重组腺病毒进行鉴定。结果重组腺病毒质粒经PCR鉴定,证实含有mIL-15基因,重组腺病毒载体构建成功,病毒滴度达1.25×1010 PFU/mL。结论采用细胞内同源重组方法可成功构建含mIL-15基因的重组腺病毒,并可获得高滴度病毒,其能高效感染HEK293细胞,能为进一步研究奠定基础。ObjectiveTo construct and prepare the recombinant adenovirus carrying mouse interleukin 15（mIL 15）,and to transfect it into HEK293 cells on order to lay the foundation for immunotherapy of hepatocellular carcinoma（HCC）.MethodsThe PCR was adopted to amplify mIL 15,the amplified products were connected into shuttle vector DC316,the recombinant shuttle plasmid pDC316 mIL 15 was constructed.Under mediation of lipofectamine2000,recombinant adenovirus backbone vector pBHGlox△E1,3 Cre and shuttle plasmid pDC316 mIL 15 were co transfected into HEK293 cells for conducting the homologous recombination.The recombinant adenovirus plasmid rAd- mIL- 15 was obtained.Then the amplified virus was packed in HEK293 cells and the viral titer was measured.The recombinant adenovirus was identified by PCR.ResultsThe recombinant adenovirus plasmid was verified to contain mIL 15 gene by PCR identification.The recombinant adenovirus vector was successfully constructed,the viral titer reached 1.25 ×10^10 PFU /mL.ConclusionRecombinant adenovirus vector containing mIL 15 can be successfully constructed by adopting the intracellular homologous recombination method,moreover which can obtain high titer virus,can efficiently infect HEK293 cells and lay a foundation for further study.

关 键 词：白细胞介素-15 重组腺病毒载体 包装 

分 类 号：R734.2[医药卫生—肿瘤]