携带hTERT-P2A-EGFP基因真核表达质粒的构建和鉴定

doi:10.13481/j.1671-587x.20170201

吉林大学学报(医学版) ›› 2017, Vol. 43 ›› Issue (02): 213-219.doi: 10.13481/j.1671-587x.20170201

• 基础研究 • 下一篇

携带hTERT-P2A-EGFP基因真核表达质粒的构建和鉴定

陈晓娜¹, 王晓丹^2,3, 孙丽光^2,3, 方芳¹, 崔巍巍¹, 杨永广^2,3, 刘娅¹

1. 吉林大学公共卫生学院营养与食品卫生学教研室, 吉林长春 130021;
2. 吉林大学第一医院转化医学研究院, 吉林长春 130061;
3. 吉林大学免疫学研究所, 吉林长春 130061

收稿日期:2016-09-14 出版日期:2017-03-28 发布日期:2017-03-31
通讯作者: 刘娅,教授,博士研究生导师(Tel:0431-85166352,E-mail:liuya@jlu.edu.cn);杨永广,教授,博士研究生导师(Tel:0431-88783477,E-mail:yongg@jlu.edu.cn) E-mail:liuya@jlu.edu.cn;yongg@jlu.edu.cn
作者简介:陈晓娜(1989-),女,河南省郑州市人,在读医学硕士,主要从事营养与食品卫生方面的研究。
基金资助:
国家自然科学基金资助课题（81202379）

Construction and identification of eukaryotic expression plasmid carrying hTERT-P2A-EGFP

CHEN Xiaona¹, WANG Xiaodan^2,3, SUN Liguang^2,3, FANG Fang¹, CUI Weiwei¹, YANG Yongguang^2,3, LIU Ya¹

1. Department of Nutrition and Food Hygiene, School of Public Health, Jilin University, Changchun 130021, China;
2. Academy of Translational Medicine, First Hospital, Jilin University, Changchun 130061, China;
3. Institute of Immunology, Jilin University, Changchun 130061, China

Received:2016-09-14 Online:2017-03-28 Published:2017-03-31

摘要/Abstract

摘要： 目的：构建真核表达质粒hTERT-P2A-EGFP，探讨其在HEK293FT细胞中的表达和转染效率。方法：利用pBABE-puro-hTERT和pRRLSIN-cPPT-MSCV-EGFP质粒构建重组质粒。以该质粒为模板采用PCR法获取hTERT、P2A和EGFP基因，采用重叠PCR法获得目的片段hTERT-P2A-EGFP，经酶切后目的片段与真核表达载体pRRLSIN-cPPT-MSCV-EGFP连接，得到并鉴定含有hTERT-P2A-EGFP基因的重组质粒。经脂质体介导重组质粒转染到HEK293FT细胞，荧光显微镜观察绿色荧光蛋白（GFP）在细胞中的表达。结果：PCR检测目的基因hTERT、P2A和EGFP的片段长度分别为3400、110和720 bp。酶切后目的基因片段hTERT-P2A-EGFP约4300 bp。测序分析，目的基因1547位点发生了突变。利用定点突变技术成功诱变，再次测序后目的基因序列与 GenBank 公布的基因序列完全一致。重组质粒转染HEK293FT细胞后在荧光显微镜下可以观察到GFP。流式细胞术检测，重组质粒转染HEK293FT细胞的效率为44.8%。结论：成功构建携带目的基因hTERT-P2A-EGFP的重组质粒，且可用于细胞转染。

关键词: 人端粒酶逆转录酶, 质粒构建, 绿色荧光蛋白

Abstract: Objective: To construct the eukaryotic expression plasmid carrying hTERT-P2A-EGFP, and to explore its expression and transfection efficiency in the HEK293FT cells. Methods: The recombinant plasmid was constructed by using pBABE-puro-hTERT and pRRLSIN-cPPT-MSCV-EGFP plasmids. The hTERT,P2A,and EGFP genes were obtained using pBABE-puro-hTERT as template by PCR. And the correct hTERT was inserted into pRRLSIN-cPPT-MSCV-EGFP vector. Then the recombinant plasmid containing hTERT-P2A-EGFP gene was obtained and identified. The HEK293FT cells were transfected by the recombinant plasmid, and the expression of green fluorescence protein(GFP) was observed by fluorescence microscope. Results: The PCR results showed that the fragments of hTERT, P2A, and EGFP were 3 400, 110 and 720 bp. And the length of gene fragment(hTERT-P2A-EGFP)was 4 300 bp by enzyme digestion. The results of sequencing showed that the 1 547 site of the target gene was mutated. Using site-directed mutagenesis, the 1 547 site was successfully mutated. And the target gene sequence was completely identical with the sequence published in GenBank. The recombinant plasmid was transfected into the HEK293FT cells, and GFP was observed in the cells.The results of flow cytometry showed that the transfection efficiency of recombinant plasmid was 44.8%. Conclusion: The recombinant plasmid carrying hTERT-P2A-EGFP gene is successfully constructed, and it can be used for cell transfection.

Key words: green fluorescence protein, plasmid construction, human telomerase reverse transcriptase

中图分类号:

Q785

陈晓娜, 王晓丹, 孙丽光, 方芳, 崔巍巍, 杨永广, 刘娅. 携带hTERT-P2A-EGFP基因真核表达质粒的构建和鉴定[J]. 吉林大学学报(医学版), 2017, 43(02): 213-219.

CHEN Xiaona, WANG Xiaodan, SUN Liguang, FANG Fang, CUI Weiwei, YANG Yongguang, LIU Ya. Construction and identification of eukaryotic expression plasmid carrying hTERT-P2A-EGFP[J]. Journal of Jilin University Medicine Edition, 2017, 43(02): 213-219.

参考文献

[1] Wojtyla A, Gladych M, Rubis B. Human telomerase activity regulation [J].Mol Biol Rep, 2011, 38(5):3339-3349.
[2] Hayashi MT, Cesare AJ, Rivera T, et al. Cell death during crisis is mediated by mitotic telomere deprotection [J]. Nature, 2015, 522(7557):492-496.
[3] Dessauge F, Lollivier V, Ponchon B, et al. Effects of nutrient restriction on mammary cellturnover and mammary gland remodeling in lactating dairy cows [J]. J Dairy Sci, 2011, 94(9):4623-4635.
[4] Shukla S, Acharya S, Rajput D, et al. Telomere-the twilight to immortality [J]. J Assoc Physicians India, 2010, 58(31):553-560.
[5] Zvereva MI, Shcherbakova DM, Dontsova OA. Telomerase:structure, functions, and activity regulation[J]. Biochemistry (Mosc), 2010, 75(13):1563-1583.
[6] Osterhage JL, Friedman KL. Chromosome end maintenance by telomerase[J]. J Biol Chem,2009, 284(24):16061-16065.
[7] Domenech A, Raynes JG, Rodriquez EG, et al. Recombinant expression of goat milk serum amyloid A:preliminary studies of the protein and derived peptides on macrophage phagocytosis [J]. Protein Pept Lett, 2012, 19(3):299-307.
[8] Hasegawa T, Chosa N, Asakawa T, et al. Establishment of immortalized human periodontalligament cells derived from deciduous teeth [J]. Int J Mol Med, 2010, 26(5):701-705.
[9] Low KC, Tergaonkar V. Telomerase:central regulator of all of the hallmarks of cancer[J]. Trends Biochem Sci, 2013, 38(9):426-434.
[10] D'Souza Y, Chu TW, Autexier C. A translocation-defective telomerase with low levels of activity and processivity stabilizes short telomeres and confers immortalization[J]. Mol Biol Cell, 2013, 24(9):1469-1479.
[11] Miller J, Dakic A, Chen R, et al. HPV16 E7 protein and hTERT proteins defective for telomere maintenance cooperate to immortalize human keratinocytes[J]. PLoS Pathog, 2013, 9(4):e1003284.
[12] Artandi SE. Telomeres, telomerase, and human disease [J]. N Engl J Med, 2006, 355(12):1195-1197.
[13] Balducci L, Blasi A, Saldarelli M, et al. Immortalization of human adipose-derived stromal cells:production of cell lines with high growth rate, mesenchymal marker expression and capability to secrete high levels of angiogenic factors [J]. Stem Cell Res Ther, 2014, 5(3):63.
[14] Daniel M, Peek GW, Tollefsbol TO. Regulation of the human catalytic subunit of telomerase (hTERT)[J]. Gene, 2012, 498(2):135-146.
[15] Li NF, Broad S, Lu YJ, et al. Human ovarian surface epithelial cells immortalized with hTERT maintain functional pRb and p53 expression[J]. Cell Prolif, 2007, 40(5):780-794.
[16] Abdallah BM, Haack-Sørensen M, Burns JS, et al. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive proliferation[J]. Biochem Biophys Res Commun, 2005, 326(3):527-538.
[17] Vidale P, Magnani E, Nergadze SG, et al. The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts[J]. Chromosoma, 2012, 121(5):475-488.
[18] Yao CL, Hwang SM. Immortalization of human mesenchymal stromal cells with telomerase and red fluorescence protein expression[J]. Methods Mol Biol, 2012, 879:471-478.
[19] Ebe T. Green fluorescent protein as a marker gene expression[J]. Tanpakushitsu Kakusan Koso, 2007, 52(13 Suppl):1766-1767.
[20] 杨慧敏, 李文刚,吴高峰,等. 绿色荧光蛋白的研究进展[J]. 中国畜牧兽医, 2008, 35(8):29-32.
[21] 王晓丽, 邵卫星,单虎. 绿色荧光蛋白研究进展[J]. 动物医学进展, 2008, 29(1):56-59.
[22] 刘伟,王杰,幸永明,等.绿色荧光蛋白标记SOX9基因慢病毒载体的构建及在兔骨髓间充质干细胞中的表达[J]. 中国实验诊断学,2017,21(1):140-145.
[23] Chan HY, Sivakamasundari V, Xing X, et al. Comparison of IRES and F2A-based locus-specific multicistronic expression in stable mouse lines[J]. PLoS One, 2011, 6(12):e28885.
[24] Deng W, Yang D, Zhao B, et al. Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer[J]. PLoS One, 2011, 6(5):e19986.

携带hTERT-P2A-EGFP基因真核表达质粒的构建和鉴定

Construction and identification of eukaryotic expression plasmid carrying hTERT-P2A-EGFP

RichHTML

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 0

[1]	律东, 梁春梅, 李明颖, 殷静雯, 罗旭东, 林举达, 马国达. miR-137过表达慢病毒的包装和鉴定[J]. 吉林大学学报(医学版), 2017, 43(04): 694-697.
[2]	王晓峰，周翔宇,张桂珍，王伟，杜珍武，张天夫. CD133基因启动子调控增强型绿色荧光蛋白基因在喉癌Hep-2细胞中的表达[J]. 吉林大学学报(医学版), 2013, 39(3): 498-502.
[3]	方艳秋\|刘磊\|谭岩\|蒋永兴. ODDD调控EGFP基因的乏氧特异表达载体的构建及其意义[J]. J4, 2012, 38(5): 870-875.
[4]	代炜\|张红艳\|李彦姝\|李妍\|孙长伏. PAK4基因RNAi慢病毒载体的构建与鉴定[J]. J4, 2012, 38(1): 11-14.
[5]	赵东海, 朱安峰, 杨淑艳, 钟秀宏, 张以忠, 赵丽微. 表达大鼠融合蛋白NKX2.5-pEGFP的骨髓间充质干细胞的建立[J]. J4, 2011, 37(4): 670-674.
[6]	冯颖, 石毅, 马杰, 焦平, 顾地周, 周余来. 脂质体介导人端粒酶逆转录酶基因构建永生化软骨细胞方法的建立及评价[J]. J4, 2010, 36(2): 233-237.
[7]	张惊宇, 杨安萍, 李金星, 刘路然, 赵虹, 杨子超. 大鼠海马CA1脑区慢病毒载体注射方法学改进[J]. J4, 2009, 35(5): 826-828.
[8]	白元松，孙延霞，刁建东，卢振霞. 绿色荧光蛋白基因在白血病细胞K562中的转导效率[J]. J4, 2009, 35(1): 135-137.
[9]	王华,尹晓光,王莉,吴秀丽,于永利,王丽颖. 用线性载体稳定转染P815细胞的脂质体转染方法的优化[J]. J4, 2008, 34(4): 721-724.
[10]	吴昊，吴江，杨宇，孙欣，. 携带信号肽NT4-GFP-穿膜肽Ant融合基因的重组腺相关病毒载体的构建及意义[J]. J4, 2007, 33(5): 798-802.
[11]	张春福，宋燕，谭岩，刘力华，刘立民，陈丽，孙志奇. 抗大肠癌siRNA载体上报告基因的构建与鉴定[J]. J4, 2007, 33(2): 392-395.
[12]	杨宇，吴江，杨欣，孙欣，吴昊，王全颖，杨广笑. 携强绿色荧光蛋白重组慢病毒的构建及其在原代培养SD大鼠皮层神经细胞中的表达[J]. J4, 2007, 33(2): 237-240.
[13]	沈小梅，张巨艳，成蓓. 大鼠CnA/EGFP基因共表达的真核表达质粒的构建[J]. J4, 2007, 33(1): 25-28.
[14]	梁爽，于振香，赵丹，杨柯，赵雪俭, 杨宝学. 大鼠睾丸AQP8和绿色荧光蛋白融合蛋白表达载体构建[J]. J4, 2006, 32(2): 218-220.
[15]	张绍昆，高萍，张琪，刘一，徐莘香. 人胰岛素样生长因子-1及绿色荧光蛋白双表达载体的构建[J]. J4, 2005, 31(1): 152-154.