ALEX1基因过表达慢病毒载体的构建和鉴定

发布时间：2018-01-08 09:22

本文关键词：ALEX1基因过表达慢病毒载体的构建和鉴定　出处：《中国免疫学杂志》2016年05期 　论文类型：期刊论文

【摘要】：目的:探讨构建Arm重复蛋白1(ALEX1)基因过表达慢病毒表达载体,感染乳腺癌细胞系SK-BR3后观察ALEX1的表达,为研究ALEX1在乳腺癌中的作用及机制奠定基础。方法:利用DNA重组技术将ALEX1基因插入到慢病毒穿梭质粒LV5中,获得重组慢病毒质粒V-ALEX1,经酶切鉴定及DNA测序鉴定后,用Lipofectamine 2000将重组质粒和慢病毒包装质粒系统(p Gag/Pol、pRev、p VSV-G)转染293T细胞中包装成重组慢病毒颗粒,经流式细胞术测定重组慢病毒滴度。将成功包装的ALEX1过表达慢病毒载体(LV5-ALEX1)和阴性对照载体(LV5-NC),感染SK-BR3细胞(MOI为10)48~72 h,Realtime PCR和Western blot法分析ALEX1表达情况。结果:酶切鉴定结果显示:产生约1 500 bp的片段,片段大小与ALEX1基因cDNA大小一致。DNA测序比对说明测序结果与预期ALEX1基因序列完全一致,证实ALEX1基因正确插入载体中,成功构建ALEX1基因过表达载体。经293T细胞包装后,成功获得病毒滴度为4.25×10~8TU/ml的重组慢病毒LV5-ALEX1。Realtime PCR和western blot结果显示:与感染LV5-NC的SK-BR3细胞相比,LV5-ALEX1感染可明显增加ALEX1 mRNA及蛋白的表达水平。结论:成功构建ALEX1基因过表达慢病毒载体,ALEX1基因过表达慢病毒能够感染乳腺癌SK-BR3细胞,可使外源基因获得稳定过表达。
[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.

【作者单位】：重庆市北碚区中医院检验科;
【基金】：辽宁省科技厅项目(2015020353)
【分类号】：R3416
【正文快照】： 1本文为辽宁省科技厅项目(2015020353)。Arm家族蛋白是指含有Armadillo repeat结构的一类蛋白。Armadillo repeat结构是首次在果蝇的极性基因中发现,该基因在果蝇的早期细胞极性形成、维持上皮组织的完整性和早期胚胎形成过程中具有重要作用[1]。ALEX1(Arm proteins lost in e

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