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| Cloning, Sequence Analysis and Codon Optimization of Chicken (Gallus gallus) IL-17 Gene to Improve the Expression Level |
| SUN Peng1, ZHU Ai-Chen1, LIANG Tian1, WNAG Ming-Di1, ZHAO Yu-Long3, HUO Shu-Ying1*, ZHANG Jian-Lou1,2* |
1 College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China;
2 Hebei Veterinary Biotenology Innovation Center, Baoding 071001, China;
3 Ruipu (Baoding) Biological Pharmaceutical Co., Ltd., Baoding 071000, China |
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Abstract Interleukin-17 (IL-17) is an early initiator of inflammation induced by T cells. It can promote the release of proinflammatory cytokines and stimulate the inflammatory response. At the same time, IL-17 also has the role of resisting some pathogens, so it has the role of biological adjuvant. It is unclear whether chicken (Gallus gallus) IL-17 has a biological adjuvant effect. In order to study the biological adjuvant effect of chicken IL-17, it is necessary to prepare the recombinant protein of chicken IL-17. In this study, the IL-17 cDNA sequence of chicken was amplified by reverse transcription polymerase chain reaction (RT-PCR), and its gene sequence and amino acid sequence were analyzed with biological software. In order to improve the expression level of chicken IL-17 gene in eukaryotic cells, the codon of chicken IL-17 gene was optimized according to the preferred codons of human (Homo sapiens) and chicken, and the wild-type and codon- optimized eukaryotic expression vectors were constructed respectively, and were expressed in human embryonic kidney (HEK) 293T cells. The results showed that the amplified chicken IL-17 gene sequence had 2 base differences and 1 amino acid mutation in the signal peptide sequence compared with that in GenBank. Therefore, the amino acid sequence of mature chicken IL-17 protein was not changed, indicating that the chicken IL-17 gene was polymorphic. Bioinformatics analysis showed that chicken IL-17 gene contained a typical signal peptide sequence to mediate the secretory expression of chicken IL-17, and the protein contained a N-glycosylation site. The transient expression in HEK 293T cells showed that codon optimization could significantly improve the expression level of chicken IL-17. The expression level was increased from 13 µg/ T75 Cell culture flask of wild-type IL-17 gene to 45 µg/T75 Cell culture flask of codon optimized IL-17 gene, which was 3.5 times higher, Moreover, the optimized recombinant protein could stimulate the proliferation of lymphocytes and induce IL-6 production in chicken fibroblasts. The results showed that codon optimization could obviously increase the expression of IL-17 gene in eukaryotic cells and the optimized IL-17 protein still had biological activity. This study provides favorable conditions for further study on the biological adjuvant effect of chicken IL-17.
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Received: 11 April 2022
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Corresponding Authors:
*huoshuying@163.com; zhangjianlou@hebau.edu.cn
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