利用酵母双杂交系统筛选与鉴定石斑鱼EcBAG3互作因子

doi:10.13560/j.cnki.biotech.bull.1985.2021-1368

生物技术通报 ›› 2022, Vol. 38 ›› Issue (8): 77-83.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1368

利用酵母双杂交系统筛选与鉴定石斑鱼EcBAG3互作因子

蔡佳¹^,²(), 梁振宇¹, 黄瑜¹, 鲁义善¹, 施钢¹, 简纪常¹

1.广东海洋大学水产学院广东省水产经济动物病原生物学及流行病学重点实验室广东省水产经济动物病害控制重点实验室南方海洋科学与工程广东省实验室（湛江）,湛江 524088
2.广西科学院广西北部湾海洋研究中心广西海洋天然产物与组合生物合成化学重点实验室,南宁 530007

收稿日期:2021-10-29 出版日期:2022-08-26 发布日期:2022-09-14
作者简介:蔡佳,男,博士,副教授,研究方向：鱼类免疫学、水生动物病毒致病机理;E-mail： matrix924@foxmail.com
基金资助:
国家自然科学基金项目(U20A2065);国家自然科学基金项目(32002426);国家自然科学基金项目(32073006);广西自然科学基金(2020GXNSFAA297243);南方海洋科学与工程广东省实验室（湛江）（湛江湾实验室）自主立项项目(ZJW-2019-06);广东海洋大学“冲一流”专项资金项目(231419013);广东海洋大学“冲一流”专项资金项目(231419017);广东海洋大学“冲一流”与“创新强校工程”科研项目(2019KTSCX056);广西红树林滨海湿地生态保护与可持续利用人才小高地人才资助项目(BGMRC202101)

Screening and Identifing the Interacting Proteins of Grouper（Epinephelus coioides）EcBAG3 Using Yeast Two-hybrid System

CAI Jia¹^,²(), LIANG Zhen-yu¹, HUANG Yu¹, LU Yi-shan¹, SHI gang¹, JIAN Ji-chang¹

1. Fisheries College,Guangdong Ocean University,Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals,Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes,Guangdong Southern Marine Science and Engineering Laboratory（Zhanjiang）,Zhanjiang 524088
2. Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry,Guangxi Beibu Gulf Marine Research Center,Guangxi Academy of Sciences,Nanning 530007

Received:2021-10-29 Published:2022-08-26 Online:2022-09-14

摘要/Abstract

摘要：

以参与石斑鱼抗病毒免疫反应的EcBAG3蛋白为研究对象,采用酵母双杂交等技术筛选与鉴定EcBAG3的互作蛋白。首先构建了石斑鱼脾脏细胞的酵母双杂交cDNA文库,文库的容量为5.6×10⁶ CFU,插入片段的平均长度在750 bp以上,重组率为100%。进一步利用此文库筛选EcBAG3的互作因子,初步得到了83个阳性克隆。对这些克隆进行测序与一对一互作验证,最终获得7个候选互作蛋白,功能预测结果显示,互作蛋白涉及免疫调控、转录调节、胁迫响应、信号转导等方面的功能,为进一步解析石斑鱼BAG3在病毒感染中的作用机制奠定了基础。

关键词: 石斑鱼, 酵母双杂交, BAG3, 互作蛋白

Abstract:

The EcBAG3 protein involved in the antiviral immune response of grouper（Epinephelus coioides）was used as the research object,and the interacting proteins of EcBAG3 were screened and identified by yeast two-hybrid technology. Firstly,a yeast two-hybrid cDNA library of grouper（Epinephelus coioides）spleen cells was constructed. The capacity of the library was 5.6×10⁶ CFU,the average length of the inserted cDNA fragments was over 750 bp,and the recombination rate was 100%. Furthermore,the interaction factors of Ec-BAG3 were screened from this library and 83 positive clones were initially obtained. Finally 7 candidate interacting proteins were confirmed through sequencing and one-to-one interaction verification. The functional prediction showed that the interacting proteins were involved in immune regulation,transcriptional regulation,stress response,signal transduction,etc.,which laid a foundation to further understand the mechanism of EcBAG3 during viral infection.

Key words: grouper（Epinephelus coioides）, yeast two hybrid, BAG3, interacting proteins

蔡佳, 梁振宇, 黄瑜, 鲁义善, 施钢, 简纪常. 利用酵母双杂交系统筛选与鉴定石斑鱼EcBAG3互作因子[J]. 生物技术通报, 2022, 38(8): 77-83.

CAI Jia, LIANG Zhen-yu, HUANG Yu, LU Yi-shan, SHI gang, JIAN Ji-chang. Screening and Identifing the Interacting Proteins of Grouper（Epinephelus coioides）EcBAG3 Using Yeast Two-hybrid System[J]. Biotechnology Bulletin, 2022, 38(8): 77-83.

图/表 6

图1 总RNA琼脂糖凝胶检测 M：DL2000 DNA marker;1：GS细胞提取的总RNA

Fig. 1 Agarose gel electrophoresis for the detection of total RNA M：DL2000 DNA marker. 1：Total RNA extracted from GS cells

图2 文库部分插入片段琼脂糖凝胶检测 M：DL2000 DNA marker;1-24：插入cDNA片段的PCR 产物

Fig. 2 Agarose gel electrophoresis for the detection of partial inserted cDNA fragments in library M：DL2000 DNA marker. 1-24：PCR products of inserted cDNA fragments

图3 pGBKT7-EcBAG3自激活检测

Fig. 3 Self-activation verification of pGBKT7-EcBAG3

图4 SD/-Leu-Trp-Ade-His/X-α-Gal/Aba平板上筛选获得的部分阳性克隆

Fig. 4 Partial positive clones screened from SD/-Leu-Trp-Ade-His/X-α-Gal/Aba plates

表1 酵母双杂交阳性克隆比对结果

Table 1 Alignments of positive clones screened through yeast two hybrid

候选蛋白 Candidate protein	NCBI登录号 Accession number of NCBI
Heat Shock Cognate 71 kD Protein（Hsc71）	XP_033491860.1
Heat Shock Protein 70（Hsp70）	AWD76391.1
Filamin-A	XP_033484911.1
Collagen,Type V,Alpha 2a	XP_033473099.1
Zinc Finger Protein 217（ZNF217）	XP_033484552.1
CCN Family Member 1（CCN1）	XP_033476535.1
Suppressor Of Cytokine Signaling 9（SOCS9）	XP_033486547.1

图5 EcBAG3与候选互作蛋白验证阳性对照：pGBKT7-53+pGADT7-T;阴性对照：pGBKT7-lam+pGADT7;阴性对照：pGBKT7-EcBAG3+pGADT7

Fig. 5 Verification of EcBAG3 and candidate interacting protein Positive control：pGBKT7-53+pGADT7-T. Negative control：pGBKT7-lam+pGADT7. Negative control：pGBKT7-EcBAG3+pGADT7

参考文献 31

[1]	Qin QW, Chang SF, Ngoh-Lim GH, et al. Characterization of a novel Ranavirus isolated from grouper Epinephelus tauvina[J]. Dis Aquat Org, 2003, 53:1-9. doi: 10.3354/dao053001 URL
[2]	Liu H, Teng Y, Zheng XC, et al. Complete sequence of a viral nervous necrosis virus(NNV)isolated from red-spotted grouper(Epinephelus akaara)in China[J]. Arch Virol, 2012, 157(4):777-782. doi: 10.1007/s00705-011-1187-5 URL
[3]	Huang XH, Huang YH, Ouyang ZL, et al. Singapore grouper Iridovirus, a large DNA virus, induces nonapoptotic cell death by a cell type dependent fashion and evokes ERK signaling[J]. Apoptosis, 2011, 16(8):831-845. doi: 10.1007/s10495-011-0616-y URL
[4]	Li C, Wang LQ, Liu JX, et al. Singapore grouper Iridovirus(SGIV)inhibited autophagy for efficient viral replication[J]. Front Microbiol, 2020, 11:1446. doi: 10.3389/fmicb.2020.01446 URL
[5]	Huang YH, Zhang Y, Liu ZT, et al. Autophagy participates in lysosomal vacuolation-mediated cell death in RGNNV-infected cells[J]. Front Microbiol, 2020, 11:790. doi: 10.3389/fmicb.2020.00790 URL
[6]	Li C, Liu JX, Zhang X, et al. Red grouper nervous necrosis virus(RGNNV)induces autophagy to promote viral replication[J]. Fish Shellfish Immunol, 2020, 98:908-916. doi: 10.1016/j.fsi.2019.11.053 URL
[7]	Huang YH, Huang XH, Yang Y, et al. Involvement of fish signal transducer and activator of transcription 3(STAT3)in nodavirus infection induced cell death[J]. Fish Shellfish Immunol, 2015, 43(1):241-248. doi: 10.1016/j.fsi.2014.12.031 URL
[8]	Bruno AP, De Simone FI, Iorio V, et al. HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels[J]. Cell Cycle, 2014, 13(23):3640-3644. doi: 10.4161/15384101.2014.952959 URL
[9]	Rosati A, Graziano V, De Laurenzi V, et al. BAG3:a multifaceted protein that regulates major cell pathways[J]. Cell Death Dis, 2011, 2:e141. doi: 10.1038/cddis.2011.24 URL
[10]	Shi HY, Xu HD, Li ZJ, et al. BAG3 regulates cell proliferation, migration, and invasion in human colorectal cancer[J]. Tumour Biol, 2016, 37(4):5591-5597. doi: 10.1007/s13277-015-4403-1 URL
[11]	Behl C. Breaking BAG:the co-chaperone BAG3 in health and disease[J]. Trends Pharmacol Sci, 2016, 37(8):672-688. doi: 10.1016/j.tips.2016.04.007 URL
[12]	Rosati A, Leone A, Del Valle L, et al. Evidence for BAG3 modulation of HIV-1 gene transcription[J]. J Cell Physiol, 2007, 210(3):676-683. doi: 10.1002/jcp.20865 URL
[13]	Rosati A, Khalili K, Deshmane SL, et al. BAG3 protein regulates caspase-3 activation in HIV-1-infected human primary microglial cells[J]. J Cell Physiol, 2009, 218(2):264-267. doi: 10.1002/jcp.21604 URL
[14]	Kyratsous CA, Silverstein SJ. The co-chaperone BAG3 regulates Herpes Simplex Virus replication[J]. PNAS, 2008, 105(52):20912-20917. doi: 10.1073/pnas.0810656105 pmid: 19088197
[15]	盛慧, 陈姗姗, 艾聪聪, 等. 利用酵母双杂交技术筛选卵菌效应因子互作蛋白概述[J]. 山东农业大学学报:自然科学版, 2019, 50(3):357-360.
	Sheng H, Chen SS, Ai CC, et al. A review of interaction proteins of oomycete effectors screened by yeast two hybrid system[J]. J Shandong Agric Univ:Nat Sci Ed, 2019, 50(3):357-360.
[16]	Brooks D, Naeem F, Stetsiv M, et al. Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover[J]. PLoS Genet, 2020, 16(4):e1008700. doi: 10.1371/journal.pgen.1008700 URL
[17]	Meriin AB, Narayanan A, Meng L, et al. Hsp70-Bag3 complex is a hub for proteotoxicity-induced signaling that controls protein aggregation[J]. PNAS, 2018, 115(30):E7043-E7052.
[18]	Sherman MY, Gabai V. The role of Bag3 in cell signaling[J]. J Cell Biochem, 2021:jcb. 30111.
[19]	Cirone M. ER stress, UPR activation and the inflammatory response to viral infection[J]. Viruses, 2021, 13(5):798. doi: 10.3390/v13050798 URL
[20]	Münz C. Autophagy proteins in viral exocytosis and anti-viral immune responses[J]. Viruses, 2017, 9(10):288. doi: 10.3390/v9100288 URL
[21]	Mao JR, Lin E, He L, et al. Autophagy and viral infection[M]// Advances in Experimental Medicine and Biology. Singapore: Springer Singapore, 2019:55-78.
[22]	Iyer K, Chand K, Mitra A, et al. Diversity in heat shock protein families:functional implications in virus infection with a comprehensive insight of their role in the HIV-1 life cycle[J]. Cell Stress Chaperones, 2021, 26(5):743-768. doi: 10.1007/s12192-021-01223-3 URL
[23]	Rao YL, Wan QY, Su H, et al. ROS-induced HSP70 promotes cytoplasmic translocation of high-mobility group box 1b and stimulates antiviral autophagy in grass carp kidney cells[J]. J Biol Chem, 2018, 293(45):17387-17401. doi: 10.1074/jbc.RA118.003840 URL
[24]	Klimek C, Jahnke R, Wördehoff J, et al. The Hippo network kinase STK38 contributes to protein homeostasis by inhibiting BAG3-mediated autophagy[J]. Biochim Biophys Acta Mol Cell Res, 2019, 1866(10):1556-1566. doi: 10.1016/j.bbamcr.2019.07.007 URL
[25]	Sharma A, Batra J, Stuchlik O, et al. Influenza A virus nucleoprotein activates the JNK stress-signaling pathway for viral replication by sequestering host filamin A protein[J]. Front Microbiol, 2020, 11:581867. doi: 10.3389/fmicb.2020.581867 URL
[26]	Dotson D, Woodruff EA, Villalta F, et al. Filamin A is involved in HIV-1 vpu-mediated evasion of host restriction by modulating tetherin expression[J]. J Biol Chem, 2016, 291(8):4236-4246. doi: 10.1074/jbc.M115.708123 URL
[27]	Liu C, Qin XW, He J, et al. Roles of extracellular matrix components in Tiger frog virus attachment to fathead minnow(Pimephales promelas)cells[J]. Fish Shellfish Immunol, 2020, 107(pt a):9-15.
[28]	Millen S, Gross C, Donhauser N, et al. Collagen IV(COL4A1, COL4A2), a component of the viral biofilm, is induced by the HTLV-1 oncoprotein tax and impacts virus transmission[J]. Front Microbiol, 2019, 10:2439. doi: 10.3389/fmicb.2019.02439 URL
[29]	Huang SZ, Liu K, Cheng AC, et al. SOCS proteins participate in the regulation of innate immune response caused by viruses[J]. Front Immunol, 2020, 11:558341. doi: 10.3389/fimmu.2020.558341 URL
[30]	Monie DD, Correia C, Zhang C, et al. Modular network mechanism of CCN₁-associated resistance to HSV-1-derived oncolytic immunovirotherapies for glioblastomas[J]. Sci Rep, 2021, 11(1):11198. doi: 10.1038/s41598-021-90718-1 URL
[31]	Liu Y, Yin W, Wang JW, et al. KRAB-zinc finger protein ZNF268a deficiency attenuates the virus-induced pro-inflammatory response by preventing IKK complex assembly[J]. Cells, 2019, 8(12):1604. doi: 10.3390/cells8121604 URL

利用酵母双杂交系统筛选与鉴定石斑鱼EcBAG3互作因子

Screening and Identifing the Interacting Proteins of Grouper（Epinephelus coioides）EcBAG3 Using Yeast Two-hybrid System

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 31

相关文章 15

编辑推荐

Metrics

本文评价

[1]	王子颖, 龙晨洁, 范兆宇, 张蕾. 利用酵母双杂交系统筛选水稻中与OsCRK5互作蛋白[J]. 生物技术通报, 2023, 39(9): 117-125.
[2]	温晓蕾, 李建嫄, 李娜, 张娜, 杨文香. 小麦叶锈菌与小麦互作的酵母双杂交cDNA文库构建与应用[J]. 生物技术通报, 2023, 39(9): 136-146.
[3]	王艺清, 王涛, 韦朝领, 戴浩民, 曹士先, 孙威江, 曾雯. 茶树SMAS基因家族的鉴定及互作分析[J]. 生物技术通报, 2023, 39(4): 246-258.
[4]	侯筱媛, 车郑郑, 李姮静, 杜崇玉, 胥倩, 王群青. 大豆膜系统cDNA文库的构建及大豆疫霉效应子PsAvr3a互作蛋白的筛选[J]. 生物技术通报, 2023, 39(4): 268-276.
[5]	王涛, 漆思雨, 韦朝领, 王艺清, 戴浩民, 周喆, 曹士先, 曾雯, 孙威江. CsPPR和CsCPN60-like在茶树白化叶片中的表达分析及互作蛋白验证[J]. 生物技术通报, 2023, 39(3): 218-231.
[6]	刘娟, 朱春晓, 肖雪琼, 莫陈汨, 王高峰, 肖炎农. 淡紫紫孢菌亲环蛋白PlCYP6 互作蛋白的筛选[J]. 生物技术通报, 2021, 37(7): 137-145.
[7]	杨华杰, 周玉萍, 范甜, 吕天晓, 谢楚萍, 田长恩. 拟南芥IQM4互作蛋白的筛选和鉴定[J]. 生物技术通报, 2021, 37(11): 190-196.
[8]	郝小花, 戴佳利, 暨文劲, 黄丹, 李东屏, 田连福. 水稻籽粒低镉蛋白LCD互作蛋白的筛选与鉴定[J]. 生物技术通报, 2020, 36(11): 21-29.
[9]	钟李婷, 陈秀珍, 唐云, 李俊仁, 王小兵, 刘彦婷, 周璇璇, 詹若挺, 陈立凯. 广藿香FPPS重组蛋白表达及互作蛋白筛选分析[J]. 生物技术通报, 2019, 35(12): 10-15.
[10]	贾建磊, 陈倩, 靳继鹏, 袁赞, 张利平. 绵羊BMPR1B基因真核表达及产物互作蛋白的鉴定[J]. 生物技术通报, 2019, 35(12): 94-104.
[11]	卢晓颖, 黄宝松, 马骞, 陈刚, 王忠良, 黄建盛, ERICAMENYOGBE, 谢瑞涛, 邓文鑫. 杂交石斑鱼干扰素调节因子3（IRF3）基因的克隆及表达分析[J]. 生物技术通报, 2019, 35(10): 144-151.
[12]	王艺桥,赵新杰,牛芳芳,郭小华,杨博,江元清. 拟南芥中WRKY31转录因子的转录活性与互作蛋白分析[J]. 生物技术通报, 2018, 34(5): 101-109.
[13]	李书鹏, 杨秀芬, 袁京京, 邱德文. 蛋白激发子Hrip1互作蛋白的筛选及其原核表达[J]. 生物技术通报, 2017, 33(6): 182-189.
[14]	袁敏,王莉,葛伟娜,张岚. 拟南芥FD与14-3-3/GRF7蛋白的相互作用研究[J]. 生物技术通报, 2017, 33(5): 117-122.
[15]	潘现飞,施泉,林新春,徐英武,曹友志. 利用酵母双杂交系统筛选雷竹SOC1相互作用蛋白[J]. 生物技术通报, 2016, 32(8): 207-213.