表達載脂蛋白E-增強型綠色熒光蛋白的細胞系支持感染性丙型肝炎病毒的組裝
發(fā)布時間：2018-10-05 11:08
【摘要】：目的探討表達載脂蛋白E-增強型綠色熒光蛋白(apolipoprotein E-enhanced green fluorescence protein,apoEEGFP)的細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基因沉默技術建立apoE穩(wěn)定下調(diào)的Huh7.5.1細胞系,然后通過基因工程技術在該細胞系中建立穩(wěn)定表達apoE-EGFP融合蛋白的細胞系。將HCV RNA轉(zhuǎn)染進入野生型細胞(Huh7.5.1細胞)、對照組sh-NT細胞(轉(zhuǎn)導非靶向野生型細胞基因的shRNA質(zhì)粒的細胞)、apoE下調(diào)的Huh7.5.1細胞(sh-apoE細胞)以及表達apoE-EGFP融合蛋白的sh-apoE細胞(apoE-EGFP細胞)中,收集病毒液;通過半數(shù)組織培養(yǎng)感染劑量(TCID50)方法檢測釋放到Huh7.5.1、sh-NT、sh-apoE和apoE-EGFP細胞培養(yǎng)上清液中的HCV的滴度。利用免疫熒光技術檢測apoE與HCV的結(jié)構蛋白E2的相互作用,利用蛋白質(zhì)印跡法檢測用具有特異親和性FLAG-gel純化的HCV顆粒表面的apoE-EGFP的表達。結(jié)果 apoE-EGFP融合蛋白在apoE-EGFP細胞系中高效表達;apoE-EGFP細胞來源的HCV的感染性與Huh7.5.1、sh-NT細胞相比差異無統(tǒng)計意義;在apoE-EGFP細胞系中,apoE-EGFP融合蛋白與HCV結(jié)構蛋白E2存在共定位,并且可以在HCV顆粒表面上檢測到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】： 上海大學生命科學學院;中國科學院上海巴斯德研究所;
【基金】：國家重點基礎研究發(fā)展規(guī)劃項目(2015CB554301)~~
【分類號】：R512.63
本文編號：2253151
[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】： 上海大學生命科學學院;中國科學院上海巴斯德研究所;
【基金】：國家重點基礎研究發(fā)展規(guī)劃項目(2015CB554301)~~
【分類號】：R512.63
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