携带人6Ckine/IFNγ融合基因腺病毒载体的构建和鉴定  

Construction and Expression of Recombinant Adenovirus Carrying Human 6Ckine/IFNγ Fusion Gene

作　　者：薛刚[1] 程莹[2] 曹永宽[1] 刘然义[3] 田伏洲[1] 黄文林[3] 

机构地区：[1]成都军区总医院全军普通外科中心,成都610083 [2]成都军区总医院内分泌科,成都610083 [3]中山大学肿瘤防治中心肿瘤学国家重点实验室,广州510060

出　　处：《中国生物制品学杂志》2008年第4期261-264,共4页Chinese Journal of Biologicals

基　　金：国家基础研究计划(973计划;2004CB518801);广东省科委重大攻关项目(2003A10902)

摘　　要：目的构建携带人次级淋巴组织趋化因子(6Ckine)和γ-干扰素(IFNγ)融合基因的重组腺病毒载体Ad-6Ckine/IFNγ,并在真核细胞中表达。方法RT-PCR法分别从人淋巴结和外周血单个核细胞中扩增人6Ckine cDNA和IFNγ cDNA,分两步先后将6Ckine cDNA和IFNγ cDNA连接到腺病毒穿梭质粒的多克隆位点上,二者之间以XbaⅠ酶切位点相连,应用AdMax腺病毒包装系统在HEK293细胞内同源重组,构建重组腺病毒载体Ad-6Ckine/IFNγ,经扩增和纯化后感染真核细胞HepG2,并检测6Ckine/IFNγ融合蛋白的表达。结果所构建的重组腺病毒Ad-6Ckine/IFNγ经扩增和纯化后,滴度为1.26×1010IFU/ml。PCR法鉴定无野生型腺病毒的污染,所携带的6Ckine/IFNγ融合基因能在真核细胞中得到表达,表达形式为分泌型。结论已成功构建重组腺病毒载体Ad-6Ckine/IFNγ,并在真核细胞中表达,为进一步研究肿瘤的基因和免疫治疗奠定了基础。Objective To construct the recombinant adenovirus carrying human secondary lymphoid organ chemokine （6Ckine）/IFNT fusion gene and express in eukaryotie cells. Methods Amplify human 6Ckine eDNA and IFNy eDNA from human lymph node and peripheral blood mononuelear cells by RT-PCR respectively and insert into adenoviras shuttle plasmid pShuttle sequentially. Amplify 6Ckine/IFNγ fusion gene from the construeted recombinant plasmid pShuttle-6Ckine/IFNγ by PCR and clone to plasmid pDC316. Co-transfect HEK293 cells with large adenoverus helper plasmid pBHGlox△E1 and the constructod shuttle plasmid pDC316-6Ckine/IFNy to prepare recombinant adenoviras Ad-6Ckine/IFNγ. The obtained recombinant adenevirtts Ad-6Ckine/IFNγ was amplified, purified and used for infection of HepG2 cells, and the expressed fusion protein was identified by immunofluorescenee method and Western blot. Results The constructed recombinant adenovirus Ad6Ckine/IFNy reaehod a titer of 1.26 × 10^10 IFU/ml after amplification and purification and showed no contamination with wild type adenovirus as proved by PCR. The 6Ckine/IFNγ was expressed in a secretory form in HepG2 cells. Conetusion Recombinant adenovirus Ad-6Ckine/ IFNT was sueeessfully constructed and expressed in eukaryotie ceils, which laid a foundation of further study on gene therapy and immunotherapy of tumors.

关 键 词：次级淋巴组织趋化因子 Γ-干扰素 融合基因 腺病毒载体 

分 类 号：R373.1[医药卫生—病原生物学]