【摘要】：旨在構建編碼大鼠鐵蛋白重鏈多肽1(ferritin heavy chain 1,Fth1)的慢病毒,并檢測其在大鼠骨髓間充質(zhì)干細胞(RMSC)中的表達。PCR擴增大鼠Fth1基因,克隆至p Lenti-GFP-C慢病毒表達載體,包裝慢病毒,感染RMSC細胞。熒光顯微鏡觀察細胞內(nèi)GFP熒光情況,Western blot檢測Fth1的表達情況,WST-1試劑檢測Fth1慢病毒感染組(RMSC-Fth1)和空載體組(RMSC-GFP)的細胞增殖活性。DNA測序結果表明,p Lenti-GFP-C-Fth1慢病毒載體構建成功。包裝慢病毒感染RMSC細胞后,熒光顯微鏡下可見明顯綠色熒光,表明感染成功。Western blot結果顯示,實驗組細胞裂解液中可檢測到特異的Fth1表達條帶。細胞增殖實驗顯示,與空載體組相比,過表達Fth1并不影響RMSC細胞生長。成功構建并包裝攜帶大鼠Fth1基因的慢病毒,其能夠成功感染RMSC細胞并表達Fth1蛋白,且對細胞增殖無明顯影響。
[Abstract]:The aim of this study was to construct a lentivirus encoding rat ferritin heavy chain polypeptide 1 (ferritin heavy chain 1 and detect its expression in rat bone marrow mesenchymal stem cells (RMSC). Rat Fth1 gene was amplified by PCR. Cloning into p Lenti-GFP-C lentivirus expression vector, packaging lentivirus, infected RMSC cells. GFP fluorescence was observed by fluorescence microscope, Fth1 expression was detected by, Western blot, proliferation activity of Fth1 lentivirus infected group (RMSC-Fth1) and empty vector group (RMSC-GFP) was detected by WST-1 reagent. P Lenti-GFP-C-Fth1 lentivirus vector was successfully constructed. After packaging lentivirus was infected with RMSC cells, green fluorescence could be seen under fluorescence microscope. The results showed that the specific Fth1 expression bands could be detected in the cell lysate of the experimental group. Cell proliferation assay showed that overexpression of Fth1 did not affect the growth of RMSC cells compared with empty vector group. The lentivirus carrying rat Fth1 gene was successfully constructed and packaged. It can infect RMSC cells and express Fth1 protein successfully, and has no obvious effect on cell proliferation.
【作者單位】： 浙江省農(nóng)業(yè)科學院植物保護與微生物研究所;浙江大學醫(yī)學院附屬第二醫(yī)院放射科;
【基金】：浙江省自然科學基金項目(LY15H180004) 浙江省公益項目(2015C32020)
【分類號】：Q78

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1 孫志華;杜軍偉;劉娟;許長萌;韓玉霞;關團;陳創(chuàng)夫;張輝;;FTH1在布魯氏菌侵染宿主細胞中的作用[J];石河子大學學報(自然科學版);2014年02期

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