人canstatin腺病毒载体的构建及在卵巢癌细胞的表达  

Construction of recombinant adenovirus containing human canstatin gene and its expression in ovarian cancer cells.

作　　者：朱宝菊[1] 乔玉环[2] 陈琛[3] 李印[4] 

机构地区：[1]郑州大学第二附属医院妇产科,郑州450014 [2]郑州大学第一附属医院妇产科 [3]郑州大学第二附属医院病理科,郑州450014 [4]河南省肿瘤医院胸外科

出　　处：《现代妇产科进展》2008年第11期805-808,共4页Progress in Obstetrics and Gynecology

基　　金：河南省卫生厅科技攻关项目(No:200703064);河南省科技厅重点攻关项目(No:豫财社[2005]158号)

摘　　要：目的:构建并鉴定人canstatin基因腺病毒表达载体Ad-can及研究在卵巢癌的表达。方法:(1)提取人卵巢癌组织总RNA,设计带有特殊酶切位点的引物,用RT-PCR方法扩增canstatin片段,克隆入载体pMD-18T,并转化至大肠杆菌DH5α,大量扩增目的基因(pMD-can);(2)双酶切pMD-can和pAdtrack质粒,酶切产物在T4DNA连接酶作用下连接,获得pAdtrack-canstatin(pAdtr-can)穿梭载体;(3)PmeⅠ酶切pAdtr-can,使其线性化,并与腺病毒骨架质粒pAdeasy共同转化大肠杆菌BJ5183,使其同源重组,获得pADeasy-canstatin(pAd-can)载体;(4)内切酶PacⅠ再次将重组子线性化,脂质体转染细胞株HEK293,借助GFP表达观察包装病毒Ad-can。重组腺病毒载体感染HEK293细胞扩增病毒,经4次扩增、纯化后,用空斑形成实验法测病毒滴度;(5)用包装的病毒转染卵巢癌细胞HO8910PM,借助GFP的荧光表达观察卵巢癌细胞转染情况;(6)RT-PCR检测转染后卵巢癌细胞中目的基因的表达。结果:各中间载体经酶切鉴定,DNA测序证实正确。转染的HEK293细胞2～3天产生病毒,7～10天出现病毒斑。最终包装的病毒中携带有目的基因canstatin,并能够在包装细胞中表达。用空斑形成实验法测定病毒滴度约为1.6×1011pfu/ml。将包装的病毒转染卵巢癌细胞,荧光显微镜下可观察到大部分卵巢癌细胞发出绿色荧光,经RT-PCR检测可发现canstatin基因扩增片段。结论:大肠杆菌内同源重组法能有效和较方便的构建canstatin基因腺病毒载体Ad-can。重组子Ad-can能转染卵巢癌细胞,并在卵巢癌细胞中表达,为进一步研究canstatin基因治疗卵巢肿瘤提供了良好的转导载体。Objective：To construct recombinant adenovirus containing human canstatin gene and to investigate its expression in ovarian cancer cells. Methods： （ 1 ） Total RNA was extracted from ovarian cancer tissues and the fragment of canstatin was obtained by RT-PCR. Then the gene was cloned into pMD-18T vector and sequenced;（2）After double digests with restriction enzymes, canstatin cDNA fragment was subcloned into the shuttle vector pAdtrack ; （3） The newly constructed vector was linearized by Pine I following co-transformation with the backbone vector pAdEasy-1 to BJ5183. The correct recombinant pAd-can was selected by endonucleases and Kana resistance;（4）Linearize pAd-can with Pac I and transfect to HEK293 cell by means of Lipofectamine. The recombinant adenovirus was confirmed by GFP expression in HEK 293 cell. Then it was amplificated four times by infecting HEK 293 cells. The titer of virus was measured by plaque assay after primary purification; （5） The recombinant adenovirus was transfected to ovarian cancer HO8910PM and confirmed by GFP expression; （6） After transfected, the ovarian cancer cells were collected and canstatin gene was checked by RT-PCR. Results ：The result of identifica- tion of intermediary plasmids during the adenovirus vector construction were conformed by restricted enzyme analysis and sequencing. The adenoviral vector constructed by homologous recom- bination could be examined 2 -3days after transfection ,plaque formation at about 7 - 10days. The final virus had the targeted gene of canstatin and could be expressed in package cell line. The ti- ter of the recombinant adenovirus vector measured by plaque assay was 1.6 × 10^11 pfu/ml which fulfilled the requirement of gene therapy in vitro. Premodinantly ovarian cancer transfected by Ad- can erupt green fluorescence. The targeted gene of canstatin could be expressed in the transfected ovarian cancer cells by RT-PCR. Conclusion：The adenovirus vector containing canstatin gene is constructed successfully. Homogenous

关 键 词：CANSTATIN 基因治疗 腺病毒 载体构建 卵巢肿瘤 

分 类 号：R737.31[医药卫生—肿瘤]