發(fā)布時(shí)間：2018-08-01 12:23

【摘要】： 目的和意義:將乙型肝炎病毒DNA整合到細(xì)胞基因組中,建立在體外穩(wěn)定表達(dá)HBV的細(xì)胞模型。為進(jìn)一步研究相關(guān)基因?qū)BV復(fù)制的調(diào)節(jié)作用奠定基礎(chǔ)。方法:pGEM -HBV1.3質(zhì)粒經(jīng)HindIII限制性內(nèi)切酶消化,將HBV1.3全長DNA切下,與同樣經(jīng)HindIII限制性內(nèi)切酶降解過的PU21連接,得到PU21-HBV重組質(zhì)粒。將該重組質(zhì)粒采用電擊轉(zhuǎn)染方法導(dǎo)入HepG2細(xì)胞中,G418篩選陽性克隆并以X-gal染色、PCR、RT-PCR、Southern blot、ELISA等方法驗(yàn)證HBV DNA的插入和表達(dá)。結(jié)果:PU21-HBV重組質(zhì)粒經(jīng)測序證明HBV1.3全長DNA正確與PU21載體連接,該重組質(zhì)粒轉(zhuǎn)染HepG2細(xì)胞后經(jīng)G418篩選,得到一系列陽性克隆, Southern blot證實(shí)HepG2細(xì)胞基因組中含HBV DNA,RT-PCR結(jié)果表明HBV DNA在HepG2細(xì)胞中有功能基因的轉(zhuǎn)錄。結(jié)論:HBV1.3已被整合在HepG2細(xì)胞染色體中并能穩(wěn)定表達(dá)其RNA。穩(wěn)定的HBV感染細(xì)胞模型構(gòu)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R373

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