中国生物工程杂志

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.