[1]李全维,高明慧,寇卜心,等.TP53BP2基因真核表达载体的构建及在人胚肾Expi293F细胞中蛋白表达、纯化及活性鉴定[J].现代检验医学杂志,2024,39(06):11-17.[doi:10.3969/j.issn.1671-7414.2024.06.002]
　LI Quanwei,GAO Minghui,KOU Puxin,et al.Construction of Eukaryotic Expression Vector of TP53BP2 Gene and Its Expression, Purification and Activity Identification in Human Embryonic Kidney Expi293F Cells[J].Journal of Modern Laboratory Medicine,2024,39(06):11-17.[doi:10.3969/j.issn.1671-7414.2024.06.002]
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TP53BP2基因真核表达载体的构建及在人胚肾Expi293F细胞中蛋白表达、纯化及活性鉴定()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第39卷
期数:
2024年06期
页码:
11-17
栏目:
论著
出版日期:
2024-11-15

文章信息/Info

Title:
Construction of Eukaryotic Expression Vector of TP53BP2 Gene and Its Expression, Purification and Activity Identification in Human Embryonic Kidney Expi293F Cells
文章编号:
1671-7414（2024）06-011-07
作者:
李全维; 高明慧; 寇卜心; 柴梦音; 石 英; 刘晓霓
（北京肝病研究所/ 首都医科大学附属北京佑安医院，北京 100069）
Author(s):
LI Quanwei;  GAO Minghui;  KOU Puxin;  CHAI Mengyin;  SHI Ying;  LIU Xiaoni
（Beijing Institute of Hepatology/Beijing You’an Hospital, Capital Medical University, Beijing 100069, China）
关键词:
肿瘤抑制因子p53 结合蛋白2; 人全长肿瘤抑制因子P53 结合蛋白2 真核表达; 人胚肾细胞Expi293; 瞬时转染; 蛋白纯化; 活性鉴定
分类号:
Q786；Q503
DOI:
10.3969/j.issn.1671-7414.2024.06.002
文献标志码:
A
摘要:
目的 构建人肿瘤抑制因子p53 结合蛋白2（tumor suppressor p53-binding protein 2，TP53BP2）的重组真核表达载体，转染人胚肾Expi293F 细胞，获得高纯度的重组人全长TP53BP2 蛋白并对其进行生物学活性鉴定。方法 利用UniProt 网站查询TP53BP2 基因序列，并进行Expi293F 表达系统序列优化，通过同源重组连接至pcDNA3.1(+)-P2AeGFP载体并进行双酶切和测序鉴定，通过转染试剂聚乙烯亚胺（polyethylenimine, PEI）将pcDNA3.1(+)-P2A-eGFPTP53BP2质粒瞬时转染至Expi293F 细胞，荧光显微镜观察转染效率，收集实验组及对照组细胞，利用免疫印记试验（Western blot，WB）检测TP53BP2 重组蛋白表达水平。通过His 标签纯化试剂盒及Superdex 200 10/300GL 层析柱进行蛋白纯化，十二烷基硫酸钠聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDSPAGE）对纯化后重组蛋白进行鉴定。利用免疫共沉淀（Co-immunoprecipitation，Co-IP）检测重组人全长TP53BP2 蛋白与p65 蛋白结合情况。利用免疫荧光（immunofluorescence，IF）检测重组人全长TP53BP2 蛋白与p65 蛋白共定位。利用表面等离子体共振（surface-plasmon resonance，SPR）技术，检测纯化后的重组人全长TP53BP2 蛋白与TP53BP2抗体的相互作用。结果 经测序和双酶切鉴定，重组质粒pcDNA3.1(+)-P2A-eGFP-TP53BP2 构建成功。经荧光显微镜观察结果显示转染效率约为60%，WB 结果表明TP53BP2 蛋白在Expi293F 细胞中过表达，证明转染成功。SDS-PAGE结果表明纯化后重组蛋白纯度在90% 以上，证明纯化成功。Co-IP 结果表明，TP53BP2 重组蛋白可与p65 蛋白相互作用。IF 结果表明，His 标签蛋白、TP53BP2 蛋白及p65 蛋白存在共定位，表明三者之间存在相互作用。SPR 结果表明，纯化的重组人TP53BP 蛋白与TP53BP2 抗体具有较好的结合活性。以上结果均证明重组人全长TP53BP2 蛋白具有生物学活性。结论 成功构建了TP53BP2 基因真核表达载体并在人胚肾Expi293F 细胞中成功表达出具有生物学活性的重组人全长TP53BP2 蛋白，为进一步研究TP53BP2 的结构和功能奠定了基础。
Abstract:
To construct a recombinant eukaryotic expression vector of human tumor suppressor p53-binding protein 2 (TP53BP2) and transfect human embryonic kidney Expi293F cells. High-purity recombinant human full-length TP53BP2 protein was obtained and its biological activity was identified. Methods The TP53BP2 gene sequence was queried on the UniProt website, and the Expi293F expression system was optimized. The TP53BP2 gene was connected to pcDNA3.1(+)-P2AeGFP vector by homologous recombination, and identified by double enzyme digestion and sequencing. Transect pcDNA3.1(+)- P2A-eGFP-TP53BP2 plasmid into Expi293F cells of Polyethylenimine (PEI), observe the transfection efficiency with a fluorescence microscope, collected cells from the experiment group and control group. The expression level of TP53BP2 recombinant protein was detected by Western blot (WB). Protein was purified by His label purification kit and Superdex 200 10/300 GL chromatographic column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified recombinant protein was identified by SDS-PAGE. Combining recombinant human full-length TP53BP2 protein with p65 protein was investigated for Co-immunoprecipitation (Co-IP) precipitation. Recombinant human full-length TP53BP2 protein was colocalized with p65 protein by Immunofluorescence (IF). The surface plasmon resonance (SPR) technique was used to detect the interaction between purified recombinant human full-length TP53BP2 protein and TP53BP2 antibody. Results The recombinant plasmid pcDNA3.1(+)-P2A-eGFP-TP53BP2 was successfully constructed by sequencing and double digestion. The fluorescence microscopy results showed that the transfection efficiency was about 60%. WB showed that the TP53BP2 protein was overexpressed in Expi293F cells, which proved that transfection was successful. SDS-PAGE results showed that the purity of the purified recombinant protein was above 90%, which proved that the purification was successful. Co-IP results showed that the TP53BP2 could interact with p65 protein. The results of IF showed that His tag protein, TP53BP2 protein, and p65 protein were co-located, indicating the interaction between the three proteins. SPR results showed that the purified TP53BP2 recombinant protein had good binding activity with the TP53BP2 antibody. These results all prove that the recombinant human full-length TP53BP2 protein has biological activity. Conclusion The eukaryotic expression vector of TP53BP2 gene was successfully constructed and the recombinant full-length human TP53BP2 protein with biological activity was successfully expressed in human embryonic kidney Expi293F cells. It lays a foundation for further study on the structure and function of TP53BP2.

参考文献/References:

[1] HUO Yunfei, CAO Ke, KOU Buxin, et al. TP53BP2: roles in suppressing tumorigenesis and therapeutic opportunities[J]. Genes Diseases, 2023, 10(5): 1982-1993.
[2] MILLER P, AKAMA-GARREN E H, OWEN R P, et al. p53 inhibitor iASPP is an unexpected suppressor of KRAS and inflammation-driven pancreatic cancer[J]. Cell Death and Differentiation, 2023, 30(7): 1619-1635.
[3] YANG Tongwang, ZHU Cunle, SHI Ying, et al. RDIVpSGP motif of ASPP2 binds to 14-3-3 and enhances ASPP2/k18/14-3-3 ternary complex formulation to promote BRAF/MEK/ERK signal inhibited cell proliferation in hepatocellular carcinoma[J]. Cancer Gene Therapy, 2022, 29(11): 1616-1627.
[4] LIANG Beibei, JIANG Yuan, SONG Shaohua, et al. ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT/β-catenin/HK2 axis[J]. Journal of Cellular and Molecular Medicine, 2023, 27(5): 659-671.
[5] XU Ping, SHI Honglin, JIAO Yan, et al. TP53BP2/ASPP2 inhibits autophagy of HepG2 cells by activating mTOR pathway in a p53-independent manner[J]. Chinese Journal of Cellular and Molecular Immunology, 2019, 35(11);986-991.
[6] TSINTARI V, WALTER B, FEND F, et al. Alternative splicing of Apoptosis Stimulating Protein of TP53-2 (ASPP2) results in an oncogenic isoform promoting migration and therapy resistance in soft tissue sarcoma (STS)[J]. BMC Cancer, 2022, 22(1): 725.
[7] LIU Dian, ERTAY A, HILL C, et al. ASPP1 deficiency promotes epithelial-mesenchymal transition, invasion and metastasis in colorectal cancer[J]. Cell death Disease, 2020, 11(4): 224.
[8] PANG Yi, PAN Lianhong, ZHANG Yonghui, et al. TP53BP2 decreases cell proliferation and induces autophagy in neuroblastoma cell lines[J]. Oncology Letters, 2019, 17(6): 4976-4984.
[9] 张明珠, 朱景怡, 张智宇, 等. 多功能蛋白ASPP2 在肿瘤发生发展中的作用[J]. 生命科学, 2024, 36(3):353-359. ZHANG Mingzhu, ZHU Jingyi, ZHANG Zhiyu,et al. The role of multifunctional protein ASPP2 in tumorigenesis and development [J]. Chinese Bulletin of Life Sciences, 2024, 36(3): 353-359.
[10] WU Tianqi, SONG Hongming, XIE Dan, et al. Silencing of ASPP2 promotes the proliferation,migration and invasion of triple-negative breast cancer cells via the PI3K/AKT pathway[J]. International Journal of Oncology, 2018, 52(6): 2001-2010.
[11] PATEL K D, BARASIYA Y V, PATEL J B, et al. Apoptosis stimulating protein of p53 (ASPP) 1 and ASPP2 m-RNA expression in oral cancer [J]. Archives of Oral Biology, 2020, 119: 104920.
[12] JIANG Feiyun, BIAN Ganxia, LI Jiehua. ASPP2 promotes cell apoptosis in cervical cancer through inhibiting autophagy [J]. Experimental and Therapeutic Medicine, 2022, 24(6): 726.
[13] KONNO T, KOHNO T, OKADA T, et al. ASPP2 suppression promotes malignancy via LSR and YAP in human endometrial cancer[J]. Histochemistry and Cell Biology, 2020, 154(2): 197-213.
[14] YANG Tongwang, GAO Yuxue, LIU Daojie, et al. ASPP2 enhances chemotherapeutic sensitivity through the down-regulation of XIAP expression in a p53 Independent manner in hepatocellular carcinoma[J]. Biochemical and Biophysical Research Communications, 2019, 508(3): 769-774.
[15] 南萍, 李秉超, 柳雅立, 等. 谷胱甘肽-S- 转移酶-ASPP2 融合蛋白的原核表达及纯化[J]. 东北农业大学学报, 2009, 40(11):65-68. NAN Ping, LI Bingchao, LIU Yali, et al. Prokaryotic expression and purification of GST-ASPP2 fusion proteins [J]. Journal of Northeast Agricultural University, 2009, 40(11): 65-68.
[16] ROYER C, SANDHAM E, SLEE E, et al. ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis[J]. Nature Communications, 2022, 13(1): 941.
[17] BERTRAN M T, MOUILLERON S, ZHOU Yanxiang,et al. ASPP proteins discriminate between PP1 catalytic subunits through their SH3 domain and the PP1 C-tail[J]. Nature Communications, 2019, 10(1): 771.
[18] SMIRNOV A, MAGRI A, LOTZ R, et al. ASPP2 binds to hepatitis C virus NS5A protein via an SH3 domain/PxxP motif-mediated interaction and potentiates infection [J]. Journal of General Virology, 2023, 104: 001895.
[19] KATZ C, BENYAMINI H, ROTEM S, et al. Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2[J]. Proceedings of the National Academy of Sciences USA,2008, 105(34): 12277-12282.
[20] MISHRA V. Affinity Tags for protein purification[J].Current Protein & Peptide Science, 2020, 21(8): 821-830.
[21] 徐杨, 杜惠芬, 李克生, 等. 再生基因 4(REG4) 真核表达载体的构建及其蛋白在 HEK 293T 细胞中的表达?纯化[J]. 现代检验医学杂志, 2023, 38(4): 35-38. XU Yang, DU Huifen, LI Kesheng, et al. Construction of eukaryotic expression vector of regenerating gene 4(REG4)and expression and purification of its protein in HEK 293T cells [J]. Journal of Modern Laboratory Medicine, 2023, 38(4): 35-38.
[22] KESIDIS A, DEPPING P, LOD? A, et al. Expression of eukaryotic membrane proteins in eukaryotic and prokaryotic hosts [J]. Methods, 2020, 180: 3-18.

备注/Memo

备注/Memo:
基金项目： 北京市自然科学基金资助项目（编号：7192084）；首都卫生发展科研专项（编号：2020-2-1152）；北京市属医学研究所公益发展改革试点项目（就医研2021-10）。
作者简介：李全维（1994-），男，硕士，在读研究生，主要研究方向：肝癌发生发展分子机制，E-mail：quanweili@mail.ccmu.edu.cn。
通讯作者： 刘晓霓（1972-），女，博士，研究员，副教授，主要研究方向：肝癌发生发展分子机制，E-mail：liuxiaoni888@ccmu.edu.cn。
石英（1976-），女，博士，研究员，副教授，主要研究方向：肝癌发生发展分子机制，E-mail：yingshi@ccmu.edu.cn。
更新日期/Last Update: 2024-11-15