本文關(guān)鍵詞：慢病毒-HBx表達(dá)載體的構(gòu)建及其在人正常肝細(xì)胞系Chang liver的穩(wěn)定表達(dá)　出處：《世界華人消化雜志》2015年28期 　論文類型：期刊論文

  更多相關(guān)文章： 乙型肝炎病毒X蛋白 Chang liver細(xì)胞 慢病毒載體

【摘要】：目的:構(gòu)建乙型肝炎病毒X基因(hepatitis B virus x,HBx)慢病毒表達(dá)載體,建立穩(wěn)定表達(dá)HBx蛋白的人正常肝細(xì)胞系Chang liverHBx.方法:應(yīng)用聚合酶鏈?zhǔn)椒磻?yīng)(polymerase chain reaction,PCR)方法從質(zhì)粒中擴(kuò)增HBx基因,并克隆到慢病毒p EB-3xflag-GP-Puro載體上,經(jīng)PCR、酶切和測(cè)序鑒定正確后經(jīng)慢病毒包裝感染人肝細(xì)胞Chang liver,再用嘌呤霉素篩選出穩(wěn)定表達(dá)HBx蛋白的細(xì)胞株,最后用免疫熒光和Western blot技術(shù)檢測(cè)HBx蛋白的表達(dá).結(jié)果:酶切鑒定和基因測(cè)序證實(shí)HBx基因成功克隆到慢病毒表達(dá)載體上,重組慢病毒經(jīng)包裝純化后獲得滴度為1×108 TU/m L,用包裝好的重組慢病毒感染人肝細(xì)胞Chang liver,經(jīng)嘌呤霉素篩選獲得單克隆細(xì)胞株Chang liver-HBx,利用免疫熒光和Western blot技術(shù)檢測(cè)發(fā)現(xiàn)細(xì)胞株Chang liver-HBx可穩(wěn)定表達(dá)HBx蛋白.結(jié)論:成功構(gòu)建了HBx的重組慢病毒表達(dá)載體,獲得了穩(wěn)定表達(dá)HBx的Chang liver細(xì)胞系Chang liver-HBx,為進(jìn)一步研究HBx誘導(dǎo)正常肝細(xì)胞惡性轉(zhuǎn)化提供細(xì)胞模型.
[Abstract]:Objective: to construct the lentivirus expression vector of hepatitis B virus X gene, hepatitis B virus and HBX. A human normal liver cell line Chang liver HBx. stably expressing HBx protein was established. Methods: polymerase chain reaction (PCR) was used. Polymerase chain reaction. HBx gene was amplified from plasmid and cloned into lentivirus p EB-3xflag-GP-Puro vector by PCR. The Chang livers of human hepatocytes were infected by lentivirus packaging and confirmed by restriction endonuclease digestion and sequencing. Then the cell lines expressing HBx protein stably were screened by purine mycin. Finally, the expression of HBx protein was detected by immunofluorescence and Western blot. Results: the HBx gene was successfully cloned into lentivirus expression vector by restriction endonuclease digestion and gene sequencing. The titer of recombinant lentivirus was 1 脳 10 ~ 8 TU/m / L, and the recombinant lentivirus was used to infect Chang liver of human hepatocytes. A monoclonal cell line Chang liver-HBx was obtained by purine mycin screening. Immunofluorescence and Western blot techniques were used to detect the stable expression of HBx protein in cell line Chang liver-HBx. The recombinant lentivirus expression vector of HBx was successfully constructed. Chang liver cell line Chang liver-HBx, which stably expressed HBx, was obtained, which provides a cell model for further study on the malignant transformation of normal hepatocytes induced by HBx.
【作者單位】： 海南醫(yī)學(xué)院分子生物學(xué)重點(diǎn)實(shí)驗(yàn)室
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目,Nos.81560450,81360307,81260306,81160261,31060164 海南省社會(huì)發(fā)展專項(xiàng)基金資助項(xiàng)目,No.2015SF03 海南省重點(diǎn)科技基金資助項(xiàng)目,No.DZXM20110038 海南醫(yī)學(xué)院培育基金資助項(xiàng)目,No.HY2013-19 海南醫(yī)學(xué)院大學(xué)生創(chuàng)新計(jì)劃基金資助項(xiàng)目,N o.HYCX2014031~~
【分類號(hào)】：R373.21;Q78
【正文快照】： 核心提示:乙型肝炎病毒X(hepatitis B virus x,HBx)是誘導(dǎo)肝細(xì)胞惡性轉(zhuǎn)化的關(guān)鍵分子.研究發(fā)現(xiàn)HBx能激活生長(zhǎng)信號(hào),或抑制P53的轉(zhuǎn)錄調(diào)節(jié)功能,促進(jìn)肝細(xì)胞的惡性轉(zhuǎn)化.本研究成功構(gòu)建p EB-GFP-HBx載體,并成功轉(zhuǎn)染Chang Liver細(xì)胞,為研究HBx誘導(dǎo)肝細(xì)胞惡性轉(zhuǎn)化建立了細(xì)胞模型.魯琰,，

本文編號(hào)：1392767