E3泛素连接酶FBXL8对A亚群禽白血病病毒复制的影响

doi:10.16431/j.cnki.1671-7236.2024.04.037

Abstract

Abstract: 【Objective】 The objective of this study was to prepare chicken E3 ubiquitin ligase FBXL8 polyclonal antibody and to investigate the impact of chicken-derived FBXL8 on the replication of Avian leukosis virus subgroup A (ALV-A) in chicken embryonic fibroblasts (DF-1).【Method】 The FBXL8 gene was amplified by PCR using DF-1 cell cDNA as a template,followed by construction of the recombinant plasmid pET-28a-FBXL8.The recombinant plasmid pET-28a-FBXL8 was transformed into E.coli expression strain RIL for induction and purification.The recombinant protein His-FBXL8 was utilized to immunize 10-month-old New Zealand White rabbits in order to prepare polyclonal antibodies.Western blotting and indirect ELISA were used to identify the immunogenicity and titer of the polyclonal antibody.Overexpression primers and interference sequences targeting FBXL8 gene were designed according to the published FBXL8 gene sequence.Furthermore,the overexpression plasmid psi-flag-FBXL8 and interference plasmid pLKO.1-FBXL8-shRNA were constructed.Western blotting was used to detect the expression effect.Successful overexpression plasmid and interference plasmid were constructed to transfect DF-1 cells,followed by infection with ALV-A after 24 hours.The expression levels of structural proteins P27 and GP85 of ALV-A were detected by semi-quantitative PCR and Western blotting.【Result】 The FBXL8 gene fragment with the size of 1 182 bp was successfully amplified by PCR,and the prokaryotic expression recombinant plasmid pET-28a-FBXL8 was successfully constructed.The induced expression fusion protein His-FBXL8 was 43 ku.Western blotting and indirect ELISA results showed that the prepared rabbit polyclonal antibody of FBXL8 could specifically recognize foreign protein,and the titer of the antibody was 1∶ 64 000.FBXL8 overexpression plasmid and interference plasmid were successfully constructed,which could effectively overexpress and knock down FBXL8 in DF-1 cells.Semi-quantitative PCR and Western blotting results showed that overexpression of FBXL8 inhibited the replication of ALV-A,while knockdown of FBXL8 promoted its replication.【Conclusion】 The FBXL8 polyclonal antibody had good specificity and reactivity.In DF-1 cells,FBXL8 negatively regulated the replication of ALV-A.The results provided an important reference for further exploring the biological characteristics of FBXL8,and laid a foundation for revealing the molecular mechanism of FBXL8 against ALV-A infection.

Key words: FBXL8; Avian leukosis virus subgroup A (ALV-A); polyclonal antibody; virus replication

CLC Number:

S852.65⁺9.6

ZHENG Shiling, CHEN Xueyang, LIU Jing, FANG Chun, LIANG Xiongyan, YANG Yuying. Effect of E3 Ubiquitin Ligase FBXL8 on the Replication of Avian Leukosis Virus Subgroup A[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1696-1705.

References

[1] 崔治中.禽白血病[M].北京:中国农业出版社,2015. CUI Z Z.Avian Leukosis[M].Beijing:China Agriculture Press, 2015.(in Chinese)
[2] CHIU E S,VANDEWOUDE S.Endogenous Retroviruses drive resistance and promotion of exogenous retroviral homologs[J].Annual Review of Animal Biosciences,2021,9:225-248.
[3] 赵振华,杨玉莹,成子强.禽白血病[M].北京:中国农业出版社,2006. ZHAO Z H,YANG Y Y,CHENG Z Q.Avian Leukosis[M].Beijing:China Agriculture Press,2006.(in Chinese)
[4] 赵振华,顾玉芳,王凤龙,等.禽骨髓细胞瘤病的病理学研究(初报)[J].动物医学进展,1999,3:85-86. ZHAO Z H,GU Y F,WANG F L,et al.Pathological study on avian myelocytomatosis (preliminary report)[J].Progress in Veterinary Medicine,1999,3:85-86.(in Chinese)
[5] LI Y,CUI S,LI W,et al.Vertical transmission of Avian leukosis virus subgroup J (ALV-J) from hens infected through artificial insemination with ALV-J infected semenz[J]. BMC Veterinary Research,2017,13(1):204.
[6] 王慧.禽白血病的流行与种群净化技术研究进展[J].湖南畜牧兽医,2022,5:44-46. WANG H.Research progress on the prevalence and population purification technology of avian leukosis[J].Hunan Journal of Animal Science & Veterinary Medicine,2022,5:44-46.(in Chinese)
[7] ÇETIN G,KLAFACK S,STUDENCKA-TURSKI M,et al.The ubiquitin-proteasome system in immune cells[J]. Biomolecules,2021,11(1):60.
[8] 艾永兴,赵程程,郑海南,等.病毒编码的泛素化酶与去泛素化酶的研究进展[J].畜牧与兽医,2012,44(S1):81-84. AI Y X,ZHAO C C,ZHENG H N,et al.Advances in virus-encoded ubiquitinating enzymes and deubiquitinating enzymes[J].Animal Husbandry & Veterinary Medicine,2012,44(S1):81-84.(in Chinese)
[9] 王兴明,王后坤,陈雪阳,等.E3泛素连接酶RNF165在A亚群禽白血病病毒复制中的作用[J].中国畜牧兽医,2023,50(9):3781-3789. WANG X M,WANG H K,CHEN X Y,et al.The role of E3 ubiquitin ligase RNF165 in the replication of subgroup A Avian leukosis virus[J]. China Animal Husbandry & Veterinary Medicine,2023,50(9):3781-3789.(in Chinese)
[10] 刘晶,陈雪阳,曾扬,等.E3泛素连接酶RNF185对A亚群禽白血病病毒体外复制的影响[J].中国畜牧兽医,2022,49(11):4364-4371. LIU J,CHEN X Y,ZENG Y,et al.Effect of E3 ubiquitin ligase RNF185 on in vitro replication of subgroup A Avian leukosis virus[J].China Animal Husbandry & Veterinary Medicine,2022,49(11):4364-4371.(in Chinese)
[11] CHEN X Y,WANG X M,YANG Y X,et al.A20 enhances the expression of the proto-oncogene C-Myc by downregulating TRAF6 ubiquitination after ALV-A infection[J].Viruses,2022,14(10):2210.
[12] FRESCAS D,PAGANO M.Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP:Tipping the scales of cancer[J].Nature Reviews Cancer,2008,8(6):438-449.
[13] WANG Z,LIU P,INUZUKA H,et al.Roles of F-box proteins in cancer[J].Nature Reviews Cancer,2014,14(4):233-247.
[14] YAO J,WANG X P,YANG J,et al.SCF-FBXL8 contributes to liver metastasis and stem-cell-like features in colorectal cancer cells by mediating ubiquitination and degradation of TP53[J]. Clinical and Translational Medicine,2023,13(3):e1208.
[15] CHANG S C,HSU W,SU E C,et al.Human FBXL8 is a novel E3 ligase which promotes BRCA metastasis by stimulating pro-tumorigenic cytokines and inhibiting tumor suppressors[J].Cancers,2020,12(8):2210.
[16] YOSHIDA A,CHOI J,JIN H R,et al.Fbxl8 suppresses lymphoma growth and hematopoietic transformation through degradation of cyclin D3[J].Oncogene,2021,40(2):292-306.
[17] 刘超,王后坤,朱丽霖,等.鸡RNF165蛋白多克隆抗体制备及其生物信息学分析[J].中国畜牧兽医,2022,49(11):4159-4167. LIU C,WANG H K,ZHU L L,et al.Preparation of polyclonal antibodies against chicken RNF165 protein and their bioinformatics analysis[J].China Animal Husbandry & Veterinary Medicine,2022,49(11):4159-4167.(in Chinese)
[18] LIANG X,GU Y,CHEN X,et al.Identification and characterization of a novel natural recombinant Avian leucosis virus from Chinese indigenous chicken flock[J].Virus Genes,2019,55(5):726-733.
[19] 李久庆,刘强,郭雷,等.一株新亚群禽白血病病毒全基因组序列分析[J].畜牧兽医学报,2017,48(9):1718-1723. LI J Q,LIU Q,GUO L,et al.Complete genome sequence analysis of a new subgroup of Avian leukemia virus[J].Chinese Journal of Animal and Veterinary Sciences,2017,48(9):1718-1723.(in Chinese)
[21] 黄小洁,刘丹,孔冬妮,等.禽网状内皮组织增生症病毒和A亚群禽白血病病毒共感染对疫苗免疫鸡生长性能和免疫器官的影响[J].中国兽药杂志,2022,56(2):9-18. HUANG X J,LIU D,KONG D N,et al.Effects of Avian reticuloendothelial dysplasia virus and subgroup A Avian leukemia virus co-infection on growth performance and immune organs of vaccine-immunized chickens[J].Chinese Journal of Veterinary Drug,2022,56(2):9-18.(in Chinese)
[22] 俞燕.地方品种鸡群禽白血病流行病学调查及检测净化技术研究[D].扬州:扬州大学,2020. YU Y.Epidemiological investigation of avian leukemia in local breed chickens and research on detection and purification technology[D].Yangzhou:Yangzhou University,2020.(in Chinese)
[23] 丁烨,任静宜,于洪强,等.病原相关分子模式和损伤相关分子模式在免疫炎症反应中的作用[J].国际口腔医学杂志,2016,43(2):172-176. DING Y,REN J Y,YU H Q,et al.Role of pathogen-associated molecular pattern and injury-associated molecular pattern in immune inflammatory response[J].International Journal of Stomatology,2016,43(2):172-176.(in Chinese)
[24] LI M,WU Y,CHEN J,et al.Innate immune evasion of Porcine epidemic diarrhea virus through degradation of the FBXW7 protein via the ubiquitin-proteasome pathway[J].Journal of Virology,2022,96(5):e0088921.
[25] LI Z,FAN S,WANG J,et al.Zebrafish F-box protein fbxo3 negatively regulates antiviral response through promoting K27-linked polyubiquitination of the transcription factors irf3 and irf7[J].The Journal of Immunology,2020,205(7):1897-1908.
[26] KAINULAINEN M,LAU S,SAMUEL C E,et al.NSs virulence factor of Rift Valley fever virus engages the F-Box proteins FBXW11 and β-TRCP1 to degrade the antiviral protein kinase PKR[J].Journal of Virology,2016,90(13):6140-6147.
[27] ZHOU X,WANG L,SHEN A,et al.Detection of ALV p27 in cloacal swabs and virus isolation medium by ELISA[J].BMC Veterinary Research,2019,15(1):383.
[28] ZHANG Y,YU M,XING L,et al.The bipartite sequence motif in the N and C termini of GP85 of subgroup J Avian leucosis virus plays a crucial role in receptor binding and viral entry[J].Journal of Virology,2020,94(22):e01232-20.
[29] BAJPAI S,JIN H R,MUCHA B,et al.Ubiquitylation of unphosphorylated c-myc by novel E3 ligase SCF-Fbxl8[J]. Cancer Biology and Therapy,2022,23(1):348-357.
[30] ROY S,BONDADA M S,ZHANG Y,et al.Proviral ALV-LTR sequence is essential for continued proliferation of the ALV-transformed B cell line[J].International Journal of Molecular Sciences,2022,23(19):11263.
[31] DHANASEKARAN R,DEUTZMANN A,MAHAUAD-FERNANDEZ W D,et al.The MYC oncogene—The grand orchestrator of cancer growth and immune evasion[J].Nature Reviews Clinical Oncologyat,2022,19(1):23-36.

Effect of E3 Ubiquitin Ligase FBXL8 on the Replication of Avian Leukosis Virus Subgroup A

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	QIN Yifei, CHEN Rong, SHI Zhendan. Cloning of VDR Gene and Preparation and Identification of Its Polyclonal Antibody in Geese [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(3): 1194-1202.
[2]	REN Jianle, LIN Yiting, JI Kang, TAN Shanshan, CHEN Xinxin, JIN Yi, WANG Ying, NIU Sheng, LIANG Libin, LI Junping, ZHAO Yujun, TIAN Wenxia. Preparation of Polyclonal Antibody and Development of an Indirect ELISA Method for Senecavirus A VP2 Protein [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 678-688.
[3]	WANG Yongfei, DENG Bowen, LIU Xiaoyan, HAERLEHA·Amantai, GUO Jiadong, ZHOU Zhengguo, CAI Jiang, LI Youwen. Prokaryotic Expression of EF-Tu Protein of Mycoplasma ovipneumoniae and Preparation of Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 689-699.
[4]	WANG Xingming, WANG Houkun, CHEN Xueyang, FANG Chun, LIANG Xiongyan, YANG Yuying. Role of E3 Ubiquitin Ligase RNF165 in the Replication of Avian Leukosis Virus Subgroup A [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3781-3789.
[5]	REN Shengjie, SHEN Hongli, XIANG Xun, DAI Feiyan, ZHU Maoyin, WU Jiali, HU Zhihui, DUAN Gang, CHANG Hua. Polyclonal Antibody Preparation of TLR7 Protein and Its Expression Characteristics Analysis in DF-1 Cells of Larus ridibundus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2865-2875.
[6]	SUO Nanji, LI Peixuan, LIU Zhuanxia, WANG Tingwei, LIU Kejiang, XIE Dongxu, ZENG Weiwei, WANG Wenhui. Prokaryotic Expression and Polyclonal Antibody Preparation of Cyp-A in Bactrian Camel [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2896-2905.
[7]	LUO Shimei, ZHAO Yao, MA Xianping, ZHANG Jinyang, YI Huashan. Prokaryotic Expression and Polyclonal Antibody Preparation of Bluetongue Virus NS4 Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2923-2930.
[8]	JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.
[9]	OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHANG Jie. Truncated Expression of ORF2 Gene of Porcine Parvovirus Type 6 and Preparation of Its Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2487-2495.
[10]	WANG Xueping, ZHANG Xiaojun, LI Xiaoyue, WANG Guodong, ZHANG Fuliang, SONG Yuwei, ZHANG Mingliang, MA Lei. Prokaryotic Expression of Hexon Protein of Highly Pathogenic Fowl Adenovirus Serotype 4 and Preparation of Polyclonal Antibodies [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2053-2060.
[11]	HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.
[12]	WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.
[13]	NIU Xiaojie, XU Mingguo, XIAO Li, LIU Wenhao, CHEN Chuangfu, WANG Yong. Expression and Purification of IPAJ Protein of Salmonella Pullorum, Preparation of Polyclonal Antibody and Establishment of ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1218-1228.
[14]	WU Jun, HE Wenwen, PU Hao, JIN Min, SHI Wenyu, MA Aijun, LUO Tingxiang, YANG Depeng, Bayinchahan, HU Ercha. Expression, Bioinformatics Analysis of aqp3 Truncated Gene of Dermacentor marginatus and Preparation of Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4589-4599.
[15]	CHEN Rujing, WU Xuemin, CHEN Qiuyong, YAN Shan, WU Renjie, CHE Yongliang, WANG Longbai, ZHOU Lunjiang. Prokaryotic Expression and Polyclonal Antibody Preparation of Porcine Circovirus Type 4 Rep Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 4188-4195.