LAG3慢病毒质粒构建及其稳定转染细胞系的建立

doi:10.13481/j.1671-587X.20220117

摘要/Abstract

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8 012和2 066 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。

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

Abstract: 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 EcoRⅠ and 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 8 012 and 2 066 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.

Key words: Lymphocyte activation gene 3, Lentiviral expression vectorr, Fluorescent protein, Cell transfection

中图分类号:

Q782

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. LAG3慢病毒质粒构建及其稳定转染细胞系的建立[J]. 吉林大学学报(医学版), 2022, 48(1): 136-141.

Yuxuan LIU,Lili HUANG,Fuxu YANG,Kaiyi FANG,Nannan HU,Yeteng MU,Chong GUO,Wei XIA,Xingang GUAN. Construction of LAG3 lentiviral plasmid and establishment of its stable transfection cell line[J]. Journal of Jilin University(Medicine Edition), 2022, 48(1): 136-141.

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