LAG3慢病毒质粒构建及其稳定转染细胞系的建立  

Construction of LAG3 lentiviral plasmid and establishment of its stable transfection cell line

作　　者：刘宇轩 黄莉莉 杨馥旭 方楷漪 胡楠楠 穆业腾 郭冲 夏薇 关新刚 LIU Yuxuan;HUANG Lili;YANG Fuxu;FANG Kaiyi;HU Nannan;MU Yeteng;GUO Chong;XIA Wei;GUAN Xingang(Key Laboratory of Pharmaceutical Biotechnology,School of Medical Technology,Beihua University,Jilin 132013,China)

机构地区：[1]北华大学医学技术学院医药生物工程重点实验室,吉林吉林132013

出　　处：《吉林大学学报（医学版）》2022年第1期136-141,共6页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科技发展计划项目(20200403118SF);吉林省教育厅科学技术研究项目(JJKH20200033KJ);吉林省卫健委卫生与健康技术创新项目(2020J023)。

摘　　要：目的:构建淋巴细胞活化因子3(LAG3)-mCherry红色荧光蛋白的LAG3-mCherry慢病毒表达载体,转染HEK293T细胞获得稳定表达LAG3-mCherry融合蛋白的细胞系,探讨LAG3-mCherry融合蛋白在HEK293T细胞中的定位。方法:将LAG3质粒和带有mCherry的慢病毒载体分别用限制性内切酶EcoRⅠ和NotⅠ进行双酶切,构建pEZ-LAG3-mCherry表达载体。将测序正确的质粒利用Lipofectamine 3000转染HEK293T细胞,利用荧光显微镜观察LAG3-mCherry在HEK293T细胞中的表达定位,Western blotting法检测LAG3-mCherry融合蛋白的表达情况。结果:酶切鉴定结果,电泳后重组质粒双酶切后可见约为8012和2066 bp大小的2条DNA条带,与pEZ-Lv-mCherry载体及LAG3基因片段大小相符;DNA测序结果,LAG3基因成功插入到慢病毒表达载体中。荧光显微镜观察,LAG3-mCherry融合蛋白主要在HEK293T细胞膜上表达,少量蛋白滞留在细胞质中。Western blotting法检测,在转染pEZ-LAG3-mCherry质粒中检测到LAG3对应特异性条带。结论:成功构建了LAG3-mCherry慢病毒表达载体pEZ-LAG3-mCherry。通过稳定转染成功制备了稳定表达LAG3-mCherry的细胞系。LAG3-mCherry融合蛋白主要分布于HEK293T细胞的细胞膜上。Objective:To construct the lentiviral vector of lymphocyte activation gene 3(LAG3)-mCherry red fluorescent protein(LAG3-mCherry)and establish the cell line stably expressed LAG3-mCherry after transfection of the HEK293T cells,and to investigate the cellular location of LAG3-mCherry fusion protein in the HEK293T cells.Methods:The LAG3 plasmid and lentiviral vector including mCherry gene were digested by endonucleases EcoRⅠand NotⅠ.The insert and vector fragments were extracted and ligated to construct the pEZ-LAG3-mCherry plasmid.The pEZ-LAG3-mCherry plasmid was sequenced and transfected into the HEK293T cells using Lipofectamine 3000.The expression location of LAG3-mCherry in the HEK293T cells was observed with fluorescence microscope and the expression of LAG3-mCherry fusion proteins in the cells were detected by Western blotting method.Results:The results of double digestion of recombinant plasmid showed that two DNA bands of about 8012 and 2066 bp were found in gel electrophoresis,which were consistent with the size of pEZ-Lv-mCherry vector and LAG3 gene fragment.The DNA sequencing results showed that the LAG-3 gene was successfully inserted into the lentiviral expression vector.The fluorescence microscope results demonstrated that most of LAG3-mCherry proteins were located on the cell membrane of HEK293T cells,while a few fusion proteins were found in the cytoplasm area.The results of Western blotting method showed a specific protein band of LAG3 in the HEK293T cells transfected with pEZ-LAG3-mCherry plasmid.Conclusion:The pEZ-LAG3-mCherry plasmid including LAG3-mCherry DNA sequence is successfully constructed and the cell line stably expressing LAG3-mCherry is established.The LAG3-mCherry fusion protein is mainly distributed on the cell membrane of HEK293T cells transfected with pEZ-LAG3-mCherry plasmid.

关 键 词：淋巴细胞活化因子3 慢病毒表达载体 红色荧光蛋白 细胞转染 

分 类 号：Q782[生物学—分子生物学]