首页 | 本学科首页   官方微博 | 高级检索  
     

存活蛋白重组腺病毒载体的构建及其在树突状细胞的表达
引用本文:朱雄鹏,陈志哲,林旭,胡建达,李纯团,杨婷,许贞书,吕璐璐,陈彩平,张浪辉. 存活蛋白重组腺病毒载体的构建及其在树突状细胞的表达[J]. 中国实验血液学杂志, 2006, 14(4): 791-794
作者姓名:朱雄鹏  陈志哲  林旭  胡建达  李纯团  杨婷  许贞书  吕璐璐  陈彩平  张浪辉
作者单位:1. 福建医科大学附属协和医院,福建省血液病研究所,福州,350001;福建泉州市第一医院血液科,泉州,362000
2. 福建医科大学附属协和医院,福建省血液病研究所,福州,350001
3. 福建医科大学分子医学研究中心,福州,350004
4. 福建泉州市第一医院血液科,泉州 362000
摘    要:本研究旨在构建含有人存活蛋白(survivin)基因的重组腺病毒载体,并探讨其在转染树突状细胞中的表达。以质粒pcDNA3.0-survivin为模板,通过PCR扩增获得survivin基因全长序列。PCR产物回收后经酶切,定向插入腺病毒穿梭质粒,获得重组质粒pShuttle-CMV-survivin。通过双酶切、PCR及插入片段测序鉴定后,将正确重组体pShuttle-CMV-survivin转化E.coliBJ5183菌(含腺病毒骨架质粒)并进行同源重组,然后筛选阳性克隆,提取质粒,将此重组腺病毒质粒分别进行酶切、线性化、纯化,用脂质体LipofectamineTM2000介导转染293细胞。制备病毒上清并测定其滴度,将病毒上清转染树突状细胞,应用Westernblot方法分析survivin的表达。结果表明成功构建了含有人survivin基因的重组腺病毒,病毒滴度为2.65×109pfu/ml。Westernblot鉴定显示,经重组腺病毒转染的DC可有效表达survivin。结论含survivin基因的重组腺病毒载体的构建成功,为下一步开展抗白血病免疫研究奠定实验基础。

关 键 词:腺病毒载体  存活蛋白基因  树突状细胞
文章编号:1009-2137(2006)04-0791-04
收稿时间:2006-05-16
修稿时间:2006-07-13

Construction of Recombinant Adenovirus Vector Containing Human Survivin Gene and Its Expression in Dentritic Cells
ZHU Xiong-Peng,CHEN Zhi-Zhe,LIN Xu,HU Jian-Da,LI Chun-Tuan,YANG Ting,XU Zheng-Shu,L Lu-Lu,CHEN Cai-Ping,ZHANG Lang-Hui. Construction of Recombinant Adenovirus Vector Containing Human Survivin Gene and Its Expression in Dentritic Cells[J]. Journal of experimental hematology, 2006, 14(4): 791-794
Authors:ZHU Xiong-Peng  CHEN Zhi-Zhe  LIN Xu  HU Jian-Da  LI Chun-Tuan  YANG Ting  XU Zheng-Shu  L Lu-Lu  CHEN Cai-Ping  ZHANG Lang-Hui
Affiliation:Fujian Institute of Hematology, Union Hospital, Fujian Medical University, Fuzhou 350001, China. xiongpengzhu@163.com
Abstract:The study was aimed to construct the recombinant adenovirus vectors containing human survivin gene, and to investigate their expression in transfected dendritic cells. Full length cDNA encoding survivin was obtained by PCR amplification from plasmid pcDNA3.0-survivin. The PCR product was restricted, and then inserted into pShuttle-CMV. The plasmids of pShuttle-CMV-survivin were linearized with PmeI, and the fragment containing survivin was ligated with pShuttle-CMV and transfected into E. coli BJ5183. After homologous recombination in bacteria, the extracted plasmid from the positive bacteria were linearized with PacI, transfected into HEK293 cells with liposome Lipofectamine 2000. Then, the harvested adenovirus supernatants were transfected into dendritic cells. The results showed that the recombinant adenovirus-survivin was constructed successfully and its titer was about 2.65 x 10(9) pfu/ml. The expression of survivin in transfected dendritic cells was confirmed by Western blot analysis. It is concluded that the recombinant adenovirus vector containing human survivin was constructed successfully, which may provide preliminary laboratory evidence for anti-leukemia immunotherapy.
Keywords:adenovirus vector   survivin gene   dendritic cell
本文献已被 CNKI 万方数据 等数据库收录!
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号