利用基因捕获技术建立稳定表达HBV的细胞模型

中国生物工程杂志

研究报告

利用基因捕获技术建立稳定表达HBV的细胞模型

何云燕谭畅李毅黄爱龙汤华

重庆医科大学重庆医科大学重庆医科大学

Construction of a human liver carcinoma cell line that stable expression of HBV with gene trap vector

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摘要： pGEM-HBV1.3质粒经HindIII限制性内切酶消化，将HBV1.3全长DNA切下，与同样经HindIII限制性内切酶降解过的PU21连接，得到PU21-HBV重组质粒。将该重组质粒采用电击转染方法导入HepG2细胞中，G418筛选阳性克隆并以X-gal染色，RT-PCR、Southern blot等方法验证HBV DNA的插入和表达。 PU21-HBV重组质粒经测序证明HBV1.3全长DNA正确与PU21载体连接，该重组质粒转染HepG2细胞后经G418筛选，得到一系列阳性克隆， Southern blot证实HepG2细胞基因组中含HBV DNA，RT-PCR结果表明HBV DNA在HepG2细胞中有功能基因的转录。HBV1.3已被整合在HepG2细胞染色体中并能稳定表达其RNA。稳定的HBV表达细胞模型构建成功。HBV表达细胞模型的建立，为进一步研究相关基因对HBV的转录、复制、转录后调节以及HBV各种蛋白的表达机理研究提供实验材料。

Abstract: Objective To establish a cell model in vitro that stable expressing HBV by integrating HBA1.3 DNA into cell chromosome. Methods The HBV1.3 full-length DNA was obtained by digested pGEM-HBV1.3 plasmid with HindIII and then was linked with PU-21 vector digested by HindIII. This was resulted in generation of a recombined plasmid named PU21-HBV plasmid. The recombined plasmid was introduced into HepG2 cells by electroporation. The transfected cells were screened with G418. The insertion and expression of HBV were identified by X-gal staining, RT-PCR and Southern blot. Results The result of PU21-HBV plasmid sequence demonstrated that HBV1.3 DNA was linked correctly with PU-21 vector. A series of positive cell colonies were obtained with G418 screening followed transfecting PU21-HBV plasmid into HepG2 cells. The results of Southern blot and RT-PCR exhibit that HBV1.3 DNA had successfully integrated into the chromosomes of HepG2 cells and had functional HBV gene transcription. Conclusion HBV1.3 DNA was inserted into HepG2 genome and could stable transcript HBV RNA. The stable HBV expression cell line was constructed successfully.

收稿日期: 2006-10-12 出版日期: 2007-02-25

通讯作者: 汤华

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引用本文:

何云燕,谭畅,李毅,黄爱龙,汤华. 利用基因捕获技术建立稳定表达HBV的细胞模型[J]. 中国生物工程杂志, .

. Construction of a human liver carcinoma cell line that stable expression of HBV with gene trap vector. China Biotechnology, .

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2007/V27/I2/24

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