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| 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 |
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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.
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Received: 10 November 2023
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Corresponding Authors:
* mrrchen@yzu.edu.cn
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