利用基因捕获技术建立稳定的HBV感染细胞模型

发布时间：2018-08-01 12:23

【摘要】： 目的和意义:将乙型肝炎病毒DNA整合到细胞基因组中,建立在体外稳定表达HBV的细胞模型。为进一步研究相关基因对HBV复制的调节作用奠定基础。方法: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、PCR、RT-PCR、Southern blot、ELISA等方法验证HBV DNA的插入和表达。结果: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。结论: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感染细胞模型构建成功。
[Abstract]:Aim and significance: to integrate hepatitis B virus DNA into cell genome and establish a cell model of stable expression of HBV in vitro. It lays a foundation for further study on the regulation of HBV replication by related genes. Methods plasmid 1. 3 was digested by HindIII restriction endonuclease, then the full-length DNA of HBV1.3 was digested and ligated with PU21 which was also degraded by HindIII restriction endonuclease. The recombinant plasmid of PU21-HBV was obtained. The recombinant plasmid was transfected into HepG2 cells by electroporation. The positive clones were screened by G418 and the insertion and expression of HBV DNA were verified by X-gal staining. Results the HBV1.3 full-length DNA was correctly ligated with the PU21 vector by sequencing. The recombinant plasmid was transfected into HepG2 cells and screened by G418. A series of positive, Southern blot clones were obtained to confirm that HepG2 cells contain HBV DNA by RT-PCR. The results showed that HBV DNA had functional gene transcription in HepG2 cells. Conclusion: 1. 3 was integrated into the chromosome of HepG2 cells and expressed stably. A stable cell model of HBV infection was successfully constructed.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373

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