MAVS全长及其截短体真核表达载体的构建和鉴定

doi:10.3969/j.issn.1004-1524.20240298

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (1): 49-60.DOI: 10.3969/j.issn.1004-1524.20240298

MAVS全长及其截短体真核表达载体的构建和鉴定

玉妹^#(), 毕冬琳^#(), 郑天倚, 李琼毅^*()

西北民族大学生命科学与工程学院,甘肃兰州 730030

收稿日期:2024-03-28 出版日期:2025-01-25 发布日期:2025-02-14
作者简介:玉妹(2003—),女,广西南宁人,本科生,研究方向为分子生物学。E-mail:YuMiky03@hotmail.com;
毕冬琳(1998—),女,云南昆明人,硕士研究生,研究方向为预防兽医。E-mail:2114560019@qq.com
#为共同第一作者。
通讯作者: *李琼毅,E-mail:lqy@xbmu.edu.cn
基金资助:
甘肃省自然科学基金(23JRRA720);西北民族大学实验室开放项目(SYSKF-2023102)

Construction and identificaition of eukaryotic expression vectors for MAVS full-length and its truncate

YU Mei^#(), BI Donglin^#(), ZHENG Tianyi, LI Qiongyi^*()

Faculty of Life Sciences and Engineering, Northwest Minzu University, Lanzhou 730030, China

Received:2024-03-28 Online:2025-01-25 Published:2025-02-14

摘要/Abstract

摘要：

建立线粒体抗病毒蛋白(mitochondrial antiviral-signaling protein,MAVS)全长及截短体的真核表达载体,并检测其在HEK293细胞中的表达,为后续探索MAVS在病毒逃逸机制研究中的作用提供重要的实验材料。首先,根据NCBI数据库中人源MAVS的全长序列,设计截短体引物,通过PCR从cDNA模板中扩增出MAVS全长及不同截短体的基因片段,并将其克隆到pcDNA3.1-3xflag载体中。随后,将构建好的载体转染到HEK-293细胞中,利用RT-PCR技术检测扩增产物大小,与目的大小进行对比,用Western blot技术检测MAVS蛋白在293细胞中的表达情况。结果表明,成功构建了全长MAVS(MAVS-FL)及其截短体MAVS-△C、MAVS-△CP、MAVS-PBD、MAVS-△Tm的真核表达载体,Western blot结果表明,构建的载体蛋白在HEK-293中能够有效表达。本研究成功构建了MAVS全长及其截短体的真核表达载体,并在HEK-293细胞中表达了这些蛋白,为后续研究MAVS蛋白的功能和抗病毒机制提供了实验材料。

关键词: 线粒体抗病毒蛋白, 真核表达载体, 截短体构建, 蛋白表达

Abstract:

The eukaryotic expression vectors for the full-length and truncated forms of the mitochondrial antiviral-signaling protein (MAVS) was established and their expression in HEK293 cells was detected, which provides important experimental materials for subsequent investigations into the viral escape mechanism mediated by MAVS. Firstly, the full-length human MAVS sequence from the NCBI database was analyzed to design primers for generating truncated forms. Gene fragments corresponding to the full-length and various truncated forms of MAVS were amplified from cDNA templates using PCR and cloned into the pcDNA3.1-3xflag vector. Subsequently, the constructed vectors were transfected into HEK-293 cells. RT-PCR was employed to verify the size of the amplified products, which were compared with the target sizes, and Western blot analysis was used to detect the expression of MAVS proteins in the 293 cells. Eukaryotic expression vectors for full-length MAVS (MAVS-FL) and its truncates MAVS-△C, MAVS-△CP, MAVS-PBD, and MAVS-△Tm were successfully constructed. Western blot results confirmed that the proteins encoded by these vectors were effectively expressed in HEK-293 cells. In this study, we successfully constructed eukaryotic expression vectors for the full-length and truncated forms of MAVS and expressed these proteins in HEK-293 cells. This work provides important experimental materials for further study of the functions and antiviral mechanisms of MAVS proteins.

Key words: mitochondrial antiviral protein, eukaryotic expression vector, truncated forms construction, protein expression

中图分类号:

玉妹, 毕冬琳, 郑天倚, 李琼毅. MAVS全长及其截短体真核表达载体的构建和鉴定[J]. 浙江农业学报, 2025, 37(1): 49-60.

YU Mei, BI Donglin, ZHENG Tianyi, LI Qiongyi. Construction and identificaition of eukaryotic expression vectors for MAVS full-length and its truncate[J]. Acta Agriculturae Zhejiangensis, 2025, 37(1): 49-60.

图/表 9

表1 MAVS及截短体表达载体引物表

Table 1 MAVS and truncated primer list

名称 Name	表达片段 Expressed fragment	产物大小 Product size/bp	引物序列 Primer sequence(5’-3’)
MAVS-FL	MAVS全长 Whole length of MAVS	1 620	F: GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTT GCCACCATGCCGTTTGCTGAAGACAAGA R: GTCCTTATCGTCGTCATCCTTGTAATCGAATTCGT GCAGACGCCGCCGGTACAGCACCACC
ΔC	103~540 aa	1 389	F: GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGC CACCATGAGGGGCTGTGAGCTAGTTGATCTCG R: GTCCTTATCGTCGTCATCCTTGTAATCGAATTCGTG CAGACGCCGCCGGTAC
ΔCP	173~540 aa	1 101	F: GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGC CACCATGCCAGATGGTGGCCCCCTGGAGTCCT R: GTCCTTATCGTCGTCATCCTTGTAATCGAATTCG TGCAGACGCCGCCGGTAC
PBD	173~514 aa	1 023	F: GACCCAAGCTGGCTAGCGTTTAAACTTAAG CTTGCCACCATGCCAGATGGTGGCCCCCTGGAGTCCT R: GTCCTTATCGTCGTCATCCTTGTAATCGAATTC CCCAGGTGAGGGCCTGTGGCATGG
ΔTm	1~514 aa	1 542	F: GACCCAAGCTGGCTAGCGTTTAAACTTAAGC TTGCCACCATGCCGTTTGCTG R: GTCCTTATCGTCGTCATCCTTGTAATCGAATTCC CCAGGTGAGGGCCTGTGGCATGG

图1 MAVS基因全长的PCR扩增产物

Fig.1 PCR amplification products of full-length of MAVS gene

图2 MAVS基因全长及截短体双酶切电泳结果

Fig.2 Electrophoresis results of full-length and truncated bodies of MAVS gene by double enzyme digestion a, MAVS-FL; b, MAVS-ΔC; c, MAVS-ΔC; d, MAVS-PBD; e, MAVS-ΔTM. Lane 1, Plasmid; Lane 2, Plasmid digested with HindⅢ-XhoⅠ; M, DNA Marker.

图3 MAVS-ΔC截短体与全长核酸序列比对

Fig.3 Comparison of MAVS-ΔC truncate with full-length nucleic acid sequence

图4 MAVS-ΔCP截短体与全长核酸序列比对

Fig.4 Comparison of MAVS-ΔCP truncate with full-length nucleic acid sequence

图5 MAVS-PBD截短体与全长核酸序列比对

Fig.5 Comparison of MAVS-PBD truncate with full-length nucleic acid sequence

图6 MAVS-ΔTM截短体与全长核酸序列比对

Fig.6 Comparison of MAVS-ΔTM truncate with full-length nucleic acid sequence

图7 MAVS截短体结构图

Fig.7 Structural diagram of MAVS mutant

图8 Western blot检测MAVS截短体在293细胞上的表达

Fig.8 Western blot detection of MAVS truncate expression on 293 cells

参考文献 28

[1]	赵文杰. 线粒体抗病毒信号蛋白MAVS在非酒精性脂肪肝病中的功能研究[D]. 武汉: 武汉大学, 2023.
	ZHAO W J. The function of mitochondrial antiviral signal protein MAVS in nonalcoholic fatty liver disease[D]. Wuhan: Wuhan University, 2023. (in Chinese with English abstract)
[2]	钟霓, 王晨, 翁光秀, 等. ZNF205通过靶向RIG-I正调控RLR抗病毒信号通路[J]. 免疫学杂志, 2023, 39(8): 708-713.
	ZHONG N, WANG C, WENG G X, et al. ZNF205 positively regulates RLR antiviral signaling by targeting RIG-I[J]. Immunological Journal, 2023, 39(8): 708-713. (in Chinese with English abstract)
[3]	ONOMOTO K, ONOGUCHI K, YONEYAMA M. Regulation of RIG-I-like receptor-mediated signaling: interaction between host and viral factors[J]. Cellular & Molecular Immunology, 2021, 18(3): 539-555.
[4]	郑航, 孙英杰, 张频, 等. MAVS介导的抗病毒天然免疫信号通路的调控[J]. 中国动物传染病学报, 2018, 26(1): 81-88.
	ZHENG H, SUN Y J, ZHANG P, et al. The regulation of mavs-mediated antiviral innate immunity[J]. Chinese Journal of Animal Infectious Diseases, 2018, 26(1): 81-88. (in Chinese with English abstract)
[5]	董望. 线粒体抗病毒信号蛋白在猪瘟病毒感染中的作用及病毒免疫逃逸机制的研究[D]. 杨凌: 西北农林科技大学, 2018.
	DONG W. Effect of MAVS on classical swine fever virus infection and mechanism of immunosuppression[D]. Yangling: Northwest A & F University, 2018. (in Chinese with English abstract)
[6]	杨星星, 王凯, 陈晓娟, 等. 线粒体抗病毒信号蛋白(MAVS)在宿主天然免疫信号通路中的调节作用[J]. 生物化学与生物物理进展, 2013, 40(5): 397-405.
	YANG X X, WANG K, CHEN X J, et al. Functions and regulation of MAVS, the mitochondrial antiviral signaling protein in innate immunity[J]. Progress in Biochemistry and Biophysics, 2013, 40(5): 397-405. (in Chinese with English abstract)
[7]	毛旭明. 鹅MAVS介导的Ⅰ型干扰素通路激活与其抗病毒作用研究[D]. 扬州: 扬州大学, 2018.
	MAO X M. Acitivation of type Ⅰ interferon pathway mediated by goose MAVS and its antiviral effects evaluation[D]. Yangzhou: Yangzhou University, 2018. (in Chinese with English abstract)
[8]	CHEN Y Q, SHI Y H, WU J, et al. MAVS: a two-sided CARD mediating antiviral innate immune signaling and regulating immune homeostasis[J]. Frontiers in Microbiology, 2021, 12: 744348.
[9]	QI N, SHI Y H, ZHANG R, et al. Multiple truncated isoforms of MAVS prevent its spontaneous aggregation in antiviral innate immune signalling[J]. Nature Communications, 2017, 8: 15676.
[10]	POTTER J A, RANDALL R E, TAYLOR G L. Crystal structure of human IPS-1/MAVS/VISA/Cardif caspase activation recruitment domain[J]. BMC Structural Biology, 2008, 8: 11.
[11]	丁云磊, 孙英杰, 王晓旭, 等. RIG-I样受体信号通路及其调控研究进展[J]. 中国动物传染病学报, 2014, 22(5): 72-79.
	DING Y L, SUN Y J, WANG X X, et al. Advances in signaling pathways and regulation of RIG-I-like receptor[J]. Chinese Journal of Animal Infectious Diseases, 2014, 22(5): 72-79. (in Chinese with English abstract)
[12]	HE Q Q, HUANG Y, NIE L Y, et al. MAVS integrates glucose metabolism and RIG-I-like receptor signaling[J]. Nature Communications, 2023, 14(1): 5343.
[13]	秦成峰, 秦鄂德. RIG-I样受体与RNA病毒识别[J]. 微生物学报, 2008, 48(10): 1418-1423.
	QIN C F, QIN Q. Retinoic acid-inducible gene-I-like receptors and RNA virus recognition—a review[J]. Acta Microbiologica Sinica, 2008, 48(10): 1418-1423. (in Chinese with English abstract)
[14]	KIM S S Y, SZE L, LIU C C, et al. The stress granule protein G3BP1 binds viral dsRNA and RIG-I to enhance interferon-β response[J]. Journal of Biological Chemistry, 2019, 294(16): 6430-6438.
[15]	SUN Q M, SUN L J, LIU H H, et al. The specific and essential role of MAVS in antiviral innate immune responses[J]. Immunity, 2006, 24(5): 633-642.
[16]	SETH R B, SUN L J, EA C K, et al. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-κB and IRF3[J]. Cell, 2005, 122(5): 669-682.
[17]	PEISLEY A, WU B, YAO H, et al. RIG-I forms signaling-competent filaments in an ATP-dependent, ubiquitin-independent manner[J]. Molecular Cell, 2013, 51(5): 573-583.
[18]	ANCHISI S, GUERRA J, GARCIN D. RIG-I ATPase activity and discrimination of self-RNA versus non-self-RNA[J]. mBio, 2015, 6(2): e02349.
[19]	JIANG F G, RAMANATHAN A, MILLER M T, et al. Structural basis of RNA recognition and activation by innate immune receptor RIG-I[J]. Nature, 2011, 479(7373): 423-427.
[20]	莫荣纤, 李洪珊, 李殿玉, 等. 泛素化修饰对维甲酸诱导基因I样受体家族(RLRs)信号通路分子调控作用的研究进展[J]. 微生物学报, 2024, 64(1):42-60.
	MO R X, LI H S, LI D Y, et al. Advances in the regulatory effects of ubiquitination on RLRs signaling pathways[J]. Acta Microbiologica Sinica, 2024, 64(1):42-60. (in Chinese with English abstract)
[21]	姚朵朵, 张须龙. 流感病毒感染的模式识别及下游相关信号通路[J]. 微生物学免疫学进展, 2018, 46(3): 60-66.
	YAO D D, ZHANG X L. Pattern recognition of influenza virus and downstream related signaling pathways[J]. Progress in Microbiology and Immunology, 2018, 46(3): 60-66. (in Chinese with English abstract)
[22]	FERREIRA A R, MAGALHÃES A C, CAMÕES F, et al. Hepatitis C virus NS3-4A inhibits the peroxisomal MAVS-dependent antiviral signalling response[J]. Journal of Cellular and Molecular Medicine, 2016, 20(4): 750-757.
[23]	付秋霞, 王礼翠, 贾帅争, 等. 基于NS3/4A切割MAVS的特性建立HCV丝氨酸蛋白酶活性监测细胞模型[C]// 中华医学会2010’全国临床微生物及感染免疫学术研讨会, 上海, 2010.
[24]	ZHANG Z D, XIONG T C, YAO S Q, et al. RNF115 plays dual roles in innate antiviral responses by catalyzing distinct ubiquitination of MAVS and MITA[J]. Nature Communications, 2020, 11(1): 5536.
[25]	ZHAO J, QIN C, LIU Y Z, et al. Herpes simplex virus and pattern recognition receptors: an arms race[J]. Frontiers in Immunology, 2021, 11: 613799.
[26]	GUPTA S, TERMINI J M, ISSAC B, et al. Constitutively active MAVS inhibits HIV-1 replication via type I interferon secretion and induction of HIV-1 restriction factors[J]. PLoS One, 2016, 11(2): e0148929.
[27]	ZHU J J, LI X, CAI X L, et al. Arginine monomethylation by PRMT7 controls MAVS-mediated antiviral innate immunity[J]. Molecular Cell, 2021, 81(15): 3171-3186.e8.
[28]	LI J X, ZHANG R, WANG C W, et al. WDR77 inhibits prion-like aggregation of MAVS to limit antiviral innate immune response[J]. Nature Communications, 2023, 14(1): 4824.

MAVS全长及其截短体真核表达载体的构建和鉴定

Construction and identificaition of eukaryotic expression vectors for MAVS full-length and its truncate

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