[1]陈竞红,常志伟,贾永旭,等.AZIN1慢病毒的制备及EC109稳定感染细胞株的建立*[J].郑州大学学报(医学版),2016,(05):561-564.
 CHEN Jinghong,CHANG Zhiwei,JIA Yongxu,et al.Preparation of AZIN1 lentivirus and establishment of stable infected EC109 cell strain[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2016,(05):561-564.
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AZIN1慢病毒的制备及EC109稳定感染细胞株的建立*()
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2016年05期
页码:
561-564
栏目:
食管癌研究
出版日期:
2016-08-20

文章信息/Info

Title:
Preparation of AZIN1 lentivirus and establishment of stable infected EC109 cell strain
作者:
陈竞红常志伟贾永旭秦艳茹#
郑州大学第一附属医院肿瘤科 郑州 450052
Author(s):
CHEN JinghongCHANG ZhiweiJIA YongxuQIN Yanru
Department of Oncology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052
关键词:
AZIN1基因 RNA编辑 慢病毒 EC109细胞
Keywords:
AZIN1 RNA editing lentivirus EC109 cell
分类号:
R735.1
摘要:
目的:制备野生型和编辑型AZIN1的慢病毒并建立稳定感染的食管癌EC109细胞株。方法:用RT-PCR法从人食管鳞状细胞癌组织中扩增出AZIN1基因的cDNA序列,选取测序结果中目标位点的A峰与G峰均高且与NCBI公布的基因编码序列一致的PCR产物,克隆至慢病毒表达载体pLenti6/V5-D-TOPO,构建野生型和编辑型AZIN1的重组慢病毒表达载体,经酶切和测序鉴定后,经293FT细胞包装,制备相应的慢病毒,然后感染EC109细胞株,行RT-PCR、测序和Western blot鉴定稳定感染的EC109细胞株。结果:双酶切重组慢病毒表达载体后产生了1 347 bp和6 915 bp的片段,经测序证实1 347 bp片段为正确的AZIN1基因编码序列; RT-PCR、测序和Western blot结果证实成功建立了稳定感染的EC109细胞株。结论:成功制备了野生型和编辑型AZIN1的慢病毒并建立了稳定感染的EC109细胞株。
Abstract:
Aim: To prepare the lentivirus of the wild type and the edited type of AZIN1, and then establish stable infected EC109 cell strain.Methods: The cDNA came from human esophageal squamous cell carcinoma tissue was amplified by RT-PCR. The PCR products with high peak A and peak G in the target site and the gene coding sequence matching the NCBI published were chosen and inserted into lentiviral vector pLenti6/V5-D-TOPO to construct the recombinant lentiviral vectors of the wild type and the edited type of AZIN1.The recombinant lentiviral vectors were verified with restriction enzyme digestion and DNA sequencing. Then the lentivirus were packed in 293FT cells and collected to infect EC109 cells. The stable infected EC109 cell strain was identified by RT-PCR,sequencing and Western blot.Results: By restriction enzyme digestion, the recombinant lentiviral vectors were digested into fragments of 1 347 bp and 6 915 bp. DNA sequencing results showed that the 1 347 fragments were the right gene coding sequence of AZIN1.The results of RT-PCR,sequencing and Western blot showed that the stable infected EC109 cell strain was established successfully.Conclusion: The lentivirus of the wild type and the edited type of AZIN1 and the stable infected EC109 cell strain have been constructed successfully.

参考文献/References:

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备注/Memo

备注/Memo:
*国家自然科学基金资助项目 81472605
#通信作者,女,1964年1月生,博士,主任医师,教授,研究方向:消化道肿瘤基础与临床,E-mail:yanruqin@163.com
更新日期/Last Update: 2016-08-20