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携带人白细胞介素10和肝细胞生长因子双基因的重组腺病毒载体构建与鉴定
引用本文:邱红,朱月蓉,邢继成,江淑芳,苏长青,曹祥荣,方琳.携带人白细胞介素10和肝细胞生长因子双基因的重组腺病毒载体构建与鉴定[J].江苏大学学报(医学版),2010,20(1):42-45,60.
作者姓名:邱红  朱月蓉  邢继成  江淑芳  苏长青  曹祥荣  方琳
作者单位:1. 南京八一医院生化科,江苏,南京,210002
2. 上海第二军医大学东方肝胆外科医院病毒及基因治疗实验室,上海,200438
3. 南京师范大学生命科学学院,江苏,南京,210097
基金项目:南京军区医学科技创新经费资助项目 
摘    要:目的:构建携带人白细胞介素10(hIL-10)和人肝细胞生长因子(hHGF)双基因的重组腺病毒载体。方法:分别以pDC316-hIL10-EGFP和pCDNA3-hHGF重组质粒为模板,PCR扩增获得hIL-10和hHGF基因片段,与pIRES连接成为hIL10-IRES—hHGF,测序正确后酶切,与pDC315连接,获得穿梭质粒pDC315-hIL10-IRES—hHGF,与腺病毒包装系统转染293细胞,包装产生重组腺病毒hIL10—hHGF,PCR鉴定后,扩增病毒,并测定病毒滴度。用重组腺病毒转染BEL-7402和WRL-68细胞,ELISA法检测上清液中hIL—10和hHGF的含量。结果:成功构建了重组腺病毒hIL10—hHGF,扩增纯化后测得重组腺病毒滴度为1×10^9pfu/ml,转染BEL-7402和WRL-68细胞72h后,表达hIL-10的量分别为(6271.7±5.2)pg/ml和(350.6±12.4)pg/ml,表达hHGF的量分别为(20827.1±345.5)pg/ml和(201.6±10.1)pg/ml。结论:本实验为进一步研究Ad—hIL10—hHGF在动物体内抗肝炎、肝纤维化作用奠定了基础。

关 键 词:白细胞介素10  肝细胞生长因子  腺病毒载体

Construction and identification of recombinant adenovirus expression vector of hIL-10 and hHGF double genes
QIU Hong,ZHU Yue-rong,XING Ji-cheng,JIANG Su-fang,SU Chang-qing,CAO Xiang-rong,FANG Lin.Construction and identification of recombinant adenovirus expression vector of hIL-10 and hHGF double genes[J].Journal of Jiangsu University Medicine Edition,2010,20(1):42-45,60.
Authors:QIU Hong  ZHU Yue-rong  XING Ji-cheng  JIANG Su-fang  SU Chang-qing  CAO Xiang-rong  FANG Lin
Institution:QIU Hong1,ZHU Yue-rong1,XING Ji-cheng1,JIANG Su-fang1,SU Chang-qing2,CAO Xiang-rong3,FANG Lin3(1.Department of Biochemistry,PLA 81 Hospital,Nanjing Jiangsu 210002,2.Virus & Genetherapy Lab,Eastern Heptobilaiary Hospital,the Second Military Medical University of Chinese PLA,Shanghai 200438,3.College of Life Science,Nanjing Normal University,Nanjing Jiangsu 210097,China)
Abstract:Objective:To construct the recombinant adenovirus vector carrying human interlerkin-10(hIL-10) and human hepatocyte growth factor(hHGF).Methods:We amplified hIL-10 and hHGF genes by PCR with plasmids pDC316-hIL10-EGFP and pCDNA3-hHGF,respectively,and subcloned into pIRES to generate hIL10-IRES-hHGF.Confirmed by sequence analysis,the hIL-10-IRES-hHGF genes were inserted into the vector pDC315,then the shuttle plasmid pDC315-hIL10-IRES-hHGF was constructed,which was cotransfected with the adenovirus skeleton ...
Keywords:interlukin-10  heptocyte growth factor  adenovirus vector  
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