序列优化的小鼠IL-33基因在哺乳动物细胞中的有效分泌表达

目的: 白细胞介素(IL)-33具有重要的免疫调控作用,在疾病中扮演着重要的角色。本文旨在通过基因优化实现IL-33在哺乳动物细胞中的高效表达,为疾病机理研究以及疫苗免疫佐剂应用等提供基础。方法: 根据小鼠白细胞介素-33成熟肽(mIL-33)的氨基酸序列,以哺乳动物细胞基因表达密码子偏好性进行基因优化设计;化学合成优化的mIL-33基因片段,通过搭桥PCR将编码人CD8α信号肽的核酸序列分别与优化或未优化的mIL-33基因连接,并与绿色荧光蛋白(EGFP)基因分别构建到双表达单元质粒 pBudCE4.1的不同启动子下;重组质粒经 lipofectamine 3000 和PEI转染293FT 细胞;以 Western blot和ELISA检测重组蛋白的表达;收集表达的mIL-33刺激巨噬细胞Raw264.7,ELISA检测培养上清的TNFα水平,以证明IL-33的生物学活性。结果: 重组质粒经酶切鉴定及测序分析证实构建成功; lipofectamine 3000转染效率较PEI转染更高;Western blot和ELISA 结果显示密码子优化的mIL-33表达水平较未优化序列更高,在EF-1α启动子和CMV启动子指导下mIL-33在293FT 细胞表达水平相当,CD8α信号肽成功引导mIL-33的分泌,产物具生物学活性。结论: 密码子优化操作显著改善了 mIL-33在哺乳动物细胞中的表达水平,为进一步研究奠定了基础。

Efficient Secretory Expression of Optimized Mouse Interleukin-33 Gene in Mammalian Cells

Objective: Interleukin (IL)-33 has important immunoregulatory effects and plays an important role in the disease. The aim of this paper is to achieve high-efficient expression of IL-33 in mammalian cells through gene optimization, and provide an important basis for disease mechanism research and vaccine immunoadjuvant application. Methods: The gene optimization for mammalian cell expression was carried out according to codon preference. The optimized and not optimized mIL-33 gene sequences were chemically synthesized. The human CD8αsignal peptide sequence was ligated to 5'end of the mIL-33 genes and led to fused CD8α+mIL-33 (Not optimized) and CD8α+mIL-33 (optimized) gene fragments by bridge PCR. The gene sequences of CD8α+ mIL-33 (Not optimized) or CD8α+mIL-33 (optimized) and EGFP expressing green fluorescent protein were respectively constructed into different expression units of plasmid pBudCE4.1 which has double expression units, and then 293FT cells were transfected with the recombinant plasmids using lipofectamine 3000 or PEI. The expression of recombinant proteins was detected by Western blot and ELISA. The expressed mIL-33s were used to stimulate macrophage Raw264.7, and the TNFα level of the culture supernatant was detected by ELISA to confirm the biological activity of expressed products. Results: The constructed recombinant plasmids were confirmed by restriction endonuclease digestion and sequencing analyses. The transfection efficiency of lipofectamine 3000 was higher than that of PEI. Western blot and ELISA showed higher levels of expression and secretion of IL-33 using optimized gene version. The expression level of mIL-33 in 293FT cells under the control of EF-1α promoter or CMV promoter was comparable. Expressed mIL-33 showed dose-dependent biological activity to stimulate TNF-α production in RAW264.7. Conclusion: The codon optimization significantly improved the secretory expression of mIL-33 with biological activity in mammalian cells, which laid a foundation for further research.