本文關(guān)鍵詞：ALEX1基因過表達慢病毒載體的構(gòu)建和鑒定　出處：《中國免疫學(xué)雜志》2016年05期 　論文類型：期刊論文

  更多相關(guān)文章： ALEX 慢病毒 構(gòu)建 鑒定

【摘要】：目的:探討構(gòu)建Arm重復(fù)蛋白1(ALEX1)基因過表達慢病毒表達載體,感染乳腺癌細胞系SK-BR3后觀察ALEX1的表達,為研究ALEX1在乳腺癌中的作用及機制奠定基礎(chǔ)。方法:利用DNA重組技術(shù)將ALEX1基因插入到慢病毒穿梭質(zhì)粒LV5中,獲得重組慢病毒質(zhì)粒V-ALEX1,經(jīng)酶切鑒定及DNA測序鑒定后,用Lipofectamine 2000將重組質(zhì)粒和慢病毒包裝質(zhì)粒系統(tǒng)(p Gag/Pol、pRev、p VSV-G)轉(zhuǎn)染293T細胞中包裝成重組慢病毒顆粒,經(jīng)流式細胞術(shù)測定重組慢病毒滴度。將成功包裝的ALEX1過表達慢病毒載體(LV5-ALEX1)和陰性對照載體(LV5-NC),感染SK-BR3細胞(MOI為10)48~72 h,Realtime PCR和Western blot法分析ALEX1表達情況。結(jié)果:酶切鑒定結(jié)果顯示:產(chǎn)生約1 500 bp的片段,片段大小與ALEX1基因cDNA大小一致。DNA測序比對說明測序結(jié)果與預(yù)期ALEX1基因序列完全一致,證實ALEX1基因正確插入載體中,成功構(gòu)建ALEX1基因過表達載體。經(jīng)293T細胞包裝后,成功獲得病毒滴度為4.25×10~8TU/ml的重組慢病毒LV5-ALEX1。Realtime PCR和western blot結(jié)果顯示:與感染LV5-NC的SK-BR3細胞相比,LV5-ALEX1感染可明顯增加ALEX1 mRNA及蛋白的表達水平。結(jié)論:成功構(gòu)建ALEX1基因過表達慢病毒載體,ALEX1基因過表達慢病毒能夠感染乳腺癌SK-BR3細胞,可使外源基因獲得穩(wěn)定過表達。
[Abstract]:Objective: to construct the Arm repeat protein 1 (ALEX1) gene lentiviral expression vector, to observe the expression of ALEX1 in breast cancer cell line SK-BR3 after infection, and lay the foundation for the research on the mechanism of ALEX1 in breast cancer. Methods: the recombinant DNA ALEX1 gene inserted into lentiviral shuttle plasmid LV5, obtained the recombinant lentiviral plasmid V-ALEX1 by restriction enzyme digestion and DNA sequencing, the recombinant plasmid and lentiviral packaging plasmid system using Lipofectamine 2000 (P Gag/Pol, pRev, P, VSV-G) was transfected into 293T cells for package of recombinant lentiviral particles, the determination of the titer of recombinant lentivirus by flow cytometry. The successful packaging overexpression of ALEX1 lentiviral vector (LV5-ALEX1) and negative control vector (LV5-NC), SK-BR3 infected cells (MOI 10) 48~72 h, ALEX1 Realtime PCR and Western blot analysis method. Results: the expression of enzyme digestion showed that: About 1500 fragments of BP ALEX1 gene fragment size and cDNA size.DNA sequencing that was completely consistent with the expected results confirmed that ALEX1 gene sequence of ALEX1 gene was correctly inserted into the vector and successfully constructed ALEX1 gene expression vector. After packaging with 293T cell, the virus titer of success is 4.25 * 10~8TU/ml LV5-ALEX1.Realtime recombinant lentivirus and PCR western blot results showed that: compared with LV5-NC infected SK-BR3 cells, LV5-ALEX1 infection can significantly increase the expression level of ALEX1 protein and mRNA. Conclusion: the successful construction of ALEX1 gene lentiviral vector, ALEX1 gene overexpression of lentivirus can infect breast cancer SK-BR3 cells, can obtain the stable over expression of exogenous gene.

【作者單位】： 重慶市北碚區(qū)中醫(yī)院檢驗科;
【基金】：遼寧省科技廳項目(2015020353)
【分類號】：R3416
【正文快照】： 1本文為遼寧省科技廳項目(2015020353)。Arm家族蛋白是指含有Armadillo repeat結(jié)構(gòu)的一類蛋白。Armadillo repeat結(jié)構(gòu)是首次在果蠅的極性基因中發(fā)現(xiàn),該基因在果蠅的早期細胞極性形成、維持上皮組織的完整性和早期胚胎形成過程中具有重要作用[1]。ALEX1(Arm proteins lost in e

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