Construction of Chicken (Gallus gallus) TET2 and Truncated Eukaryotic Expression Vector and Its Effects on Innate Immune Response
MA Ke-Jiao1,2, CAI Qing-Qing1, WANG Jia-Xing1,2, WANG Qiang-Zhou1, BAI Hao1, CHEN Shi-Hao1,*, CHANG Guo-Bin1
1 Institute of Epigenetics and Epigenomics, Yangzhou University, Yangzhou 225009, China; 2 College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
Abstract Ten-eleven translocation 2 (TET2) regulates the innate immune response of mammals in 2 ways: Methylation-dependent and methylation-independent. However, the innate immune regulation of chicken (Gallus gallus) TET2 has not been elucidated. The aim of this study was to clone chicken TET2 gene and construct the eukaryotic expression vector of TET2 and its truncated form, and preliminarily explore the effect of TET2 and its functional domain on chicken innate immune response. According to the chicken TET2 genome (GenBank No. NM_001277794.1) information, primers were designed to clone the full-length CDS sequence. The full length of TET2 was divided into N-terminal N1126 and C-terminal CD 2 truncated fragments, and then the truncated gene fragment was amplified. It was connected to the pCAGGS-Myc eukaryotic expression vector by homologous recombination. The recombinant plasmid pCAGGS-Myc-TET2 and its truncated form were transfected into chicken embryo fibroblasts DF-1, respectively. The protein expression and cellular localization of chicken TET2 and its truncated form were detected by Western blot and immunofluorescence. The effects of overexpression of pCAGGS-Myc-TET2 and truncated vectors on the expression of genes related to the innate immune response induced by ploy (I:C) were detected by qRT-PCR. PCR results showed that the full-length sequence of chicken TET2 and its truncated N1126 and CD were successfully cloned. Bioinformatics analysis showed that Anser cygnoides and Anas platyrhynchos were most closely related to G. gallus TET2. The results of Western blot showed that the full-length and truncated TET2 fused with Myc tag could be expressed normally in DF-1 cells. The results of immunofluorescence showed that the TET2 were localized in the nucleus. The results of qRT-PCR showed that overexpression of full-length and truncated chicken TET2 could significantly promote the expression of melanoma differentiation associated gene 5 (MDA5) and tripartide motif containing 25 (TRIM25) induced by poly (I:C)(P<0.05). However, there was no significant effect on the expression of interferon regulatory factor 7 (IRF7)(P>0.05).Compared with overexpression of N1126 truncation, overexpression of chicken TET2 full-length and CD domain significantly promoted poly (I:C)-induced IFN-β expression (P<0.05). In this study, the effects of TET2 and its truncated mutants on chicken innate immune response were preliminarily explored, which provides a reference for the study of the molecular mechanism of chicken TET2 regulating innate immune response.
MA Ke-Jiao,CAI Qing-Qing,WANG Jia-Xing, et al. Construction of Chicken (Gallus gallus) TET2 and Truncated Eukaryotic Expression Vector and Its Effects on Innate Immune Response[J]. 农业生物技术学报, 2024, 32(10): 2371-2380.
[1] 陈世豪, 潘诗雨, 赵睿涵, 等. 2021. 鸡SAMD9L基因真核表达载体构建及其对ALV-J病毒复制的影响[J]. 扬州大学学报(农业与生命科学版), 42(6): 54-59. (Chen S H, Pan S Y, Zhao R H, et al.2021. Construction of eukaryotic expression vector of chicken SAMD9L gene and its effect on ALV-J replication[J]. Journal of Yangzhou University (Agricultural and Life Science Edition), 42(6): 54-59.) [2] 沈兆基, 谢春嫡, 沙嘎那日•吉日木图, 等. 2023. 非洲猪瘟病毒MGF505-5R基因分子特征及蛋白表达载体构建[J]. 农业生物技术学报, 31(05): 1100-1110. (Shen Z J, Xie C D, Shagainar J, et al.2023. Molecular characterization and protein expression vector construction of African swine fever virus MGF505-5R gene[J]. Journal of Agricultural Biotechnology, 31(05): 1100-1110) [3] 王强州, 潘诗雨, 方梦雅, 等. 2023. 基于CRISPR-Cas9技术构建TET2基因敲除的鸡胚成纤维细胞系[J]. 中国农业科技导报, 25(11): 227-233. (Wang Q Z, Pan S Y, Fang M Y, et al.2023. Construction of chicken embryo filrovlast cell line with TET2 gene knockout based on CRISPR-Cas9 technology[J]. Journal of Agricultural Science and Technology, 25(11): 227-233.) [4] 吴怡晨, 凌志强. 2013. TET家族DNA羟化酶与5-hmC在肿瘤中的作用机制的研究进展[J]. 中国细胞生物学学报, 35(12): 1806-1812. (Wu Y C, Ling Z Q.2013. Research advance of TET family DNA hydroxylase and 5-hmC in neoplasm[J]. Chinese Journal of Cell Biology, 35(12): 1806-1812.) [5] 熊俊, 朱冰. 2017. TET家族蛋白介导的DNA氧化的调控与其生物学功能[J]. 生命科学, 29(10): 926-933. (Xiong J, Zhu B.2017. TET-mediated DNA oxidation and its biological functions[J]. Chinese Bulletin of Life Sciences, 29(10): 926-933.) [6] Barber M R, Aldridge J R, Webster R G, et al.2010. Association of RIG-I with innate immunity of ducks to influenza[J]. Proceedings of the National Academy of Sciences of the USA, 107(13): 5913-5918. [7] Bowman R L, Levine R L2017. TET2 in normal and malignant hematopoiesis[J]. Cold Spring Harbor Perspectives in Medicine, 7(8): a026518. [8] Chen S, Wang D, Liu Y, et al.2020. Targeting the histone methyltransferase disruptor of telomeric silencing 1-like restricts Avian leukosis virus subgroup J replication by restoring the innate immune response in chicken macrophages[J]. Frintiers in Microbiology, 11: 603131. [9] Chen S, Hu X, Cui I H, et al.2019. An endogenous retroviral element exerts an antiviral innate immune function via the derived lncRNA lnc-ALVE1-AS1[J]. Antiviral Research, 170: 104571. [10] Cong B, Zhang Q, Cao X.2021. The function and regulation of TET2 in innate immunity and inflammation[J]. Protein Cell, 12(3): 165-173. [11] Ito S, D'Alessio A C, Taranova O V, et al.2010. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification[J]. Nature, 466(7310): 1129-1133. [12] Li D, Chen J, Pei D.2018. The battle between TET proteins and DNA methylation for the right cell[J]. Trends in Cell Biology, 28(12): 973-975. [13] Hu L L, Li Z, Cheng J D.2013. Crystal structure of TET2-DNA complex: Insight into TET-mediated 5mC oxidation[J]. Cell, 155(7): 1545-1555. [14] Ma S, Wan X, Deng Z, et al.2017. Epigenetic regulator CXXC5 recruits DNA demethylase Tet2 to regulate TLR7/9-elicited IFN response in pDCs[J]. Journal of Experimental Medicine, 214(5): 1471-1491. [15] Matthias L, Artur S, Gert Z, et al.2012. Chicken cells sense Influenza A virus infection through MDA5 and CARDIF signaling involving LGP2[J]. Journal of Virology, 86(2): 705-717. [16] Nakagawa T, Lv L, Nakagawa M.et al.2015. CRL4VprBP E3 ligase promotes monoubiquitylation and chromatin binding of TET dioxygenases[J]. Molecular Cell, 57(2): 247-260. [17] Nishiyama A, Nakanishi M.2021. Navigating the DNA methylation landscape of cancer[J]. Trends in Genetics, 37(11): 1012-1027. [18] Thoresen D, Wang W, Galls D, et al.2021. The molecular mechanism of RIG-Ⅰ activation and signaling[J]. Immunological Reviews, 304(1): 154-168. [19] Winkler R, Gillis E, Lasman L, et al.2019. m(6)A modification controls the innate immune response to infection by targeting typeⅠinterferons[J]. Nature Immunology, 20(2): 173-182. [20] Wu X, Zhang Y.2017. TET-mediated active DNA demethylation: Mechanism, function and beyond[J]. Nature Reviews Genetics, 18(9): 517-534. [21] Xu Y P, Lv L, Liu Y, et al.2019. Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy[J]. Journal of Clinical Investigation, 130(10): 4316-4331. [22] Yue X, Lio C J, Samaniego-Castruita D, et al.2019. Loss of TET2 and TET3 in regulatory T cells unleashes effector function[J]. Nature Communications, 10(1): 2011. [23] Zhong C, Zhu J.2015. Tet2: Breaking down barriers to T cell cytokine expression[J]. Immunity, 42(4): 593-595.
