[1]张红飞),李军锋),户义),等.重组表达乙肝病毒x蛋白的腺病毒构建及在HepG2细胞的表达*[J].郑州大学学报(医学版),2012,(04):450.
 ZHANG Hongfei,LI Junfeng,HU Yi,et al.Construction and expression of recombinant HBxexpressed adenovirus and HBx expression in HepG2[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2012,(04):450.
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重组表达乙肝病毒x蛋白的腺病毒构建及在HepG2细胞的表达*
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2012年04期
页码:
450
栏目:
论著
出版日期:
2012-07-20

文章信息/Info

Title:
Construction and expression of recombinant HBxexpressed adenovirus and HBx expression in HepG2
作者:
张红飞12)李军锋2)户义2)张慧娜1)鲍春旸2)王凯娟1)#周育森2)
1)郑州大学公共卫生学院流行病学教研室 郑州 450001 2)军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室 北京 100071
Author(s):
ZHANG Hongfei12)LI Junfeng2)HU Yi2)ZHANG Huina1)BAO Chunyang2)WANG Kaijuan1) ZHOU Yusen2)
1)Department of Epidemiology,College of Public Health, Zhengzhou University, Zhengzhou 450001 2)State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071
关键词:
腺病毒载体HBx基因肝癌细胞
Keywords:
adenovirus vectorHBx genehepatoma cell
分类号:
R575.1
摘要:
目的:构建重组表达HBx的腺病毒,并通过感染细胞建立HBx高表达的细胞模型。方法:首先将HBx基因插入腺病毒穿梭质粒pAdTrack 中,限制性酶切鉴定正确后将其用PmeⅠ酶切线性化,转到BJ5183感受态细菌进行同源重组,重组后的腺病毒载体pAdHBx经PacⅠ酶切线性化后,转染到QBI293A细胞中,根据EGFP的表达判断重组腺病毒的包装情况。RTPCR鉴定重组腺病毒的HBx基因表达。根据GFP的表达检测病毒滴度和感染效率。将病毒感染人肝癌HepG2细胞,48 h后收集裂解细胞,Western blot检测HBx蛋白的表达。结果:重组腺病毒载体pAdHBx经酶切鉴定确认构建成功。将pAdHBx转染到QBI293A细胞,荧光检测提示病毒获得成功包装,RTPCR检测到细胞中HBx基因的表达。重组病毒感染人肝癌HepG2细胞,Western blot检测到HBx蛋白表达。结论:成功构建了携带HBx基因的重组腺病毒,感染肝癌HepG2细胞后检测到了HBx蛋白的表达,提示建立了HBx高表达的细胞模型,为后续的HBx的功能研究奠定了基础。
Abstract:
Aim:To construct recombinant HBxexpressed adenovirus, and establish a cell culture model with highlevel HBx expression by infection.Methods:HBx gene was cloned into the shuttle vector pAdTrack. After confirmed by restriction digestion, the resultant plasmid was linearized by digestion with PmeⅠ and was subsequently transformed into competent E.coli cells BJ5183 for homologous recombination. The recombination vector pAdHBx was linearized with PacⅠ and transfected into QBI293A cells. The packing of adenovirus was determined by the expression of EGFP. The expression of HBx gene in recombinant adenovirus was identified by RTPCR. Viral titer and infection efficiency of adenovirus were detected according to the expression of GFP. HepG2 cells were infected by recombinant adenovirus. Fortyeight hours later, cells were collected and lysed to detect the expression of HBx protein by Western blot.Results:Restriction digestion showed that the recombinant adenovirus vector pAdHBx was successfully constructed.After pAdHBx transfected into QBI293A cells, fluorescencetection indicated that the adenovirus was successfully packed. The expression of HBx gene was detected by PCR. After infection to the hepatoma HepG2 cells, the expression of HBx protein was detected by Western blot.Conclusion: The recombinant adenovirus expressing HBx gene has been successfully constructed. After infecting HepG2 cell,the HBx protein expression is detected,suggesting cell culture model with highlevel expression of HBx is established.

参考文献/References:

[1]Hong A, Han DD, Wright CJ, et al. The interaction between hepatitis B virus X protein and AIB1 oncogene is required for the activation of NFκB signal transduction[J]. Biochem Biophys Res Commun, 2012 May 22[Epub ahead of print]
[2]MartinVilchez S, LaraPezzi E, TraperoMarugán M, et al. The molecular and pathophysiological implications of hepatitis B X antigen in chronic hepatitis B virus infection[J]. Rev Med Virol,2011 Jul 14.doi:10.1002/rmv.699[Epub ahead of print]
[3]卢宏柱,周建华.乙型肝炎病毒X蛋白通过激活细胞外蛋白调节激酶转导机制上调大鼠系膜细胞肿瘤坏死因子α的表达[J]. 实用儿科临床杂志,2009,24(10):738
[4]Srisuttee R, Koh SS, Kim SJ, et al. Hepatitis B virus X (HBX) protein upregulates βcatenin in a human hepatic cell line by sequestering SIRT1 deacetylase[J]. Oncol Rep,2012,28(1):276
[5]刘凯歌,高红艳,赵慧,等. HBx和VEGF与乙肝相关性肝癌组织血管生成及转移的关系[J].吉林大学学报:医学版, 2010, 36(2): 372
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备注/Memo

备注/Memo:
*国家自然科学基金青年科学基金资助项目30900753 #通讯作者,女,1964年10月生,博士,教授,研究方向:肿瘤流行病学,Email: kjwang@163.com
更新日期/Last Update: 2012-08-14