引用本文:黄 荣,陈江燕,胡接力,罗英英,黄爱龙.体外培养细胞内乙肝病毒共价闭合环状DNA检测方法的建立[J].重庆医科大学学报,2013,(12):1393~1396
体外培养细胞内乙肝病毒共价闭合环状DNA检测方法的建立
A method for detecting intracellular HBV covalently closed circular DNA in cultured cells in vitro
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中文关键词:  乙型肝炎病毒  共价闭合环状DNA  Southern blot
英文关键词:let-7a  Ewing’s sarcoma  A673 cells  SK-ES-1 cells  lentivirus
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黄 荣,陈江燕,胡接力,罗英英,黄爱龙 重庆医科大学感染性疾病分子生物学教育部重点实验室重庆 400016 
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中文摘要:
      目的:建立一种体外培养细胞内乙肝病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)的检测方法。方法:Southern blot 检测HBV复制中间体,以确认HepAD38、HepG2.2.15和P+P-细胞是否支持HBV复制;用Hirt法分别提取细胞内的去蛋白DNA(protein-free DNA,PF DNA),经加热变性处理,EcoRⅠ酶切,然后用Southern blot检测HBV cccDNA;用同样方法检测不同培养时间的HepAD38细胞内cccDNA,观察培养时间对cccDNA积累量的影响。结果:用Southern blot可在HepAD38、HepG2.2.15和P P-细胞中检测到HBV复制中间体;Hirt法提取物经变性和酶切处理,可有效地将cccDNA与其他形式HBV DNA区分开;HepAD38、HepG2.2.15和P P-细胞内均可检测到HBV cccDNA;HepAD38细胞在铺满后继续培养的时间较长时,其细胞内cccDNA的量较多。结论:成功建立了体外培养细胞内HBV cccDNA检测方法,该方法结合Hirt提取法和地高辛探针Southern blot方法,能可靠、准确地检测HBV cccDNA。
英文摘要:
      Objective:To construct a eukaryotic expression plasmid carrying let-7a and to establish the Ewing’s sarcoma(ES) cell line A673 and SK-ES-1 stably expressing let-7a. Methods:Fragment of attB1-K- eGFP-let-7a-1-attB2 coding let-7a was amplified using Gateway protocols. The let-7a gene was recombined with donor vector pDONR221 to construct an entry plasmid pDONR221-eGFP-let-7a,which forwarded to recombine with pLV.Des2d.P/neo to reconstruct pLV.Des2d.P/neo-EF1A>eGFP/Let-7a. PCR and se-quencing were used to confirm the plasmid. Lenti-let-7a transfected ES cell lines A673/let-7a and SK-ES-1/let-7a which can sta-bly express let-7a were screened out. A673/let-7a and SK-ES-1/let-7a were confirmed by real-time PCR. Western blot analysis was performed to detect the expression of CDK6 in A673/let-7a and SK-ES-1/let-7a cells,which was the target gene of let-7a. Results:PCR results confirmed that the value of target fragment was consistent with the theoretic value. Sequencing analysis showed that let-7a was correctly inserted into the pLV.Des2d.P/neo-EF1A>eGFP/let-7a plasmid. Real-time PCR indicated that the expression of let-7a was increased remarkably in A673/let-7a and SK-ES-1/let-7a compared with that of control and blank cells;expressing values of let-7a were as high as 8.5 folds(P=0.000) and 6.5 folds(P=0.001) in A673/let-7a and SK-ES-1/let-7a respectively. Western blot analysis showed that ectopic expression of let-7a down-regulated the expression of CDK6. Conclusions:ES cell lines A673/let-7a and SK-ES-1/let-7a which can stably express let-7a are successfully established.
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