鸡<em>IL</em>-<em>17</em>基因的克隆、序列分析及密码子优化提高表达水平

doi:10.3969/j.issn.1674-7968.2023.07.012

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摘要白细胞介素-17 (interleukin-17, IL-17)是 T 细胞诱导炎症的早期启动因子,可促进促炎性细胞因子的释放和刺激炎症反应,同时 IL-17 也能够抵抗某些病原,因此具有生物佐剂作用。鸡(Gallus gallus)的 IL-17 是否具有生物佐剂作用尚不清楚。为探讨鸡 IL-17 的生物佐剂作用,需要制备鸡 IL-17 的重组蛋白。为此本研究通过 RT-PCR 扩增了鸡的 IL-17 cDNA 序列,利用生物学软件对其基因序列及氨基酸序列进行比较分析。为提高鸡 IL-17 基因在真核细胞中的表达水平,依据人(Homo sapiens)和鸡偏爱的密码子对 IL-17 基因进行密码子优化,分别构建了野生型和密码子优化的真核表达载体,并在人胚胎肾(human embryonic kidney, HEK) 293T细胞进行了表达。实验结果显示,扩增的鸡IL-17 基因序列与 GenBank 的相比有 2 个碱基的差异和 1 个存在于信号肽序列中氨基酸的突变,所以未改变成熟鸡 IL-17 蛋白质氨基酸的序列,表明鸡的 IL-17 基因存在着多态性。生物信息学分析表明,鸡 IL-17 基因含有 1 个典型的信号肽序列以介导鸡 IL-17 的分泌表达,同时蛋白中含有 1 个 N-糖基化位点。通过在 HEK 293T 细胞的瞬时表达,证明密码子优化可明显提高鸡 IL-17 的表达水平,表达水平由野生型 IL-17 基因的 13 µg/T75 细胞培养瓶提高到密码子优化的 IL-17 基因的 45 µg/T75 细胞培养瓶,提高了 3.5 倍,同时优化后的重组蛋白可以刺激淋巴细胞的增殖和诱导鸡成纤维细胞产生 IL-6。本研究表明密码子优化可明显提高 IL-17 基因在真核细胞中的表达并且优化表达的 IL-17 蛋白仍具有生物活性。这为进一步研究鸡 IL-17 的生物佐剂作用提供了有利条件。

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关键词 ：鸡, 白细胞介素-17(IL-17), 密码子优化, 真核表达

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.

Key words： Chicken Interleukin-17 (IL-17) Codon optimization Eukaryotic expression

收稿日期: 2022-04-11

基金资助:河北省创新能力提升计划项目(20567668H); 河北省自然科学基金(C2021204105)

通讯作者: *huoshuying@163.com;zhangjianlou@hebau.edu.cn

引用本文:

孙鹏, 朱爱臣, 梁天, 王明迪, 赵玉龙, 霍书英, 张建楼. 鸡IL-17基因的克隆、序列分析及密码子优化提高表达水平[J]. 农业生物技术学报, 2023, 31(7): 1464-1476.
SUN Peng,ZHU Ai-Chen,LIANG Tian,WNAG Ming-Di,ZHAO Yu-Long,HUO Shu-Ying ,ZHANG Jian-Lou, . Cloning, Sequence Analysis and Codon Optimization of Chicken (Gallus gallus) IL-17 Gene to Improve the Expression Level. 农业生物技术学报, 2023, 31(7): 1464-1476.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.07.012 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I7/1464

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