【摘要】：目的探討表達(dá)載脂蛋白E-增強(qiáng)型綠色熒光蛋白(apolipoprotein E-enhanced green fluorescence protein,apoEEGFP)的細(xì)胞系是否支持感染性丙型肝炎病毒(HCV)顆粒的組裝。方法利用shRNA基因沉默技術(shù)建立apoE穩(wěn)定下調(diào)的Huh7.5.1細(xì)胞系,然后通過(guò)基因工程技術(shù)在該細(xì)胞系中建立穩(wěn)定表達(dá)apoE-EGFP融合蛋白的細(xì)胞系。將HCV RNA轉(zhuǎn)染進(jìn)入野生型細(xì)胞(Huh7.5.1細(xì)胞)、對(duì)照組sh-NT細(xì)胞(轉(zhuǎn)導(dǎo)非靶向野生型細(xì)胞基因的shRNA質(zhì)粒的細(xì)胞)、apoE下調(diào)的Huh7.5.1細(xì)胞(sh-apoE細(xì)胞)以及表達(dá)apoE-EGFP融合蛋白的sh-apoE細(xì)胞(apoE-EGFP細(xì)胞)中,收集病毒液;通過(guò)半數(shù)組織培養(yǎng)感染劑量(TCID50)方法檢測(cè)釋放到Huh7.5.1、sh-NT、sh-apoE和apoE-EGFP細(xì)胞培養(yǎng)上清液中的HCV的滴度。利用免疫熒光技術(shù)檢測(cè)apoE與HCV的結(jié)構(gòu)蛋白E2的相互作用,利用蛋白質(zhì)印跡法檢測(cè)用具有特異親和性FLAG-gel純化的HCV顆粒表面的apoE-EGFP的表達(dá)。結(jié)果 apoE-EGFP融合蛋白在apoE-EGFP細(xì)胞系中高效表達(dá);apoE-EGFP細(xì)胞來(lái)源的HCV的感染性與Huh7.5.1、sh-NT細(xì)胞相比差異無(wú)統(tǒng)計(jì)意義;在apoE-EGFP細(xì)胞系中,apoE-EGFP融合蛋白與HCV結(jié)構(gòu)蛋白E2存在共定位,并且可以在HCV顆粒表面上檢測(cè)到apoE-EGFP融合蛋白。結(jié)論 apoE-EGFP融合蛋白是HCV顆粒的組分,apoE-EGFP細(xì)胞系支持感染性HCV顆粒的組裝。
[Abstract]:Objective to investigate whether the cell lines expressing apolipoprotein E-enhanced green fluorescent protein (apolipoprotein E-enhanced green fluorescence protein,apoEEGFP) support the assembly of infectious hepatitis C virus (HCV) particles. Methods shRNA gene silencing technique was used to establish a stable down-regulated Huh7.5.1 cell line of apoE, and then a cell line stably expressing apoE-EGFP fusion protein was established by genetic engineering technique. HCV RNA was transfected into wild-type cells (Huh7.5.1 cells), control sh-NT cells (shRNA plasmids transducted non-target wild-type cells) Huh7.5.1 cells (sh-apoE cells) and sh-apoE cells expressing apoE-EGFP fusion protein (apoE-EGFP cells). The viral solution was collected and the titer of HCV released into the supernatant of Huh7.5.1,sh-NT,sh-apoE and apoE-EGFP cells was detected by half of the tissue culture infection dose (TCID50) method. The interaction between apoE and HCV structural protein E2 was detected by immunofluorescence technique, and the expression of apoE-EGFP on the surface of HCV particles purified by specific affinity FLAG-gel was detected by Western blot. Results there was no statistical significance between the infection of HCV derived from apoE-EGFP fusion protein and that of Huh7.5.1,sh-NT cells in apoE-EGFP cell line, and the co-localization of HCV fusion protein and HCV structural protein E2 in apoE-EGFP cell line. ApoE-EGFP fusion protein can be detected on the surface of HCV particles. Conclusion apoE-EGFP fusion protein is a component of HCV particles, which supports the assembly of infective HCV particles in apoE-EGFP cell line.
【作者單位】： 上海大學(xué)生命科學(xué)學(xué)院;中國(guó)科學(xué)院上海巴斯德研究所;
【基金】：國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展規(guī)劃項(xiàng)目(2015CB554301)~~
【分類(lèi)號(hào)】：R512.63

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