Abstract：Abstract: In this study, a representative H3N2 influenza A strain was selected. Based on the analysis and prediction of the secondary structure of H3 protein and the codon usage frequency curve, the codons of H3 gene were optimized scientifically. Based on the principle that the intensity of codon optimization is inversely proportional to the disorder degree of local secondary structure, both expression efficiency and correct structure are considered. Then the optimized H3 sequence was cloned into the pPICZα expression vector and transferred into Pichia pastoris by electroporation. After methanol induction, the H3 protein was expressed and secreted efficiently in P. pastoris. The expression of H3 was identified by coomassian bright blue staining and Western blot, and high copy strains were screened. Finally, the strain was cultured and fermented in shaking flask. After induced by methanol at 30℃ for 72 h, the supernatant of fermentation was collected and concentrated by 10 kD membrane filtration. The purification of H3 recombinant protein was obtained by cobalt column affinity chromatography, and confirmed by coomassian bright blue staining and Western blot. The purified H3 antigen solution did not show haemagglutination activity, however, the nickel column bound H3 showed some to a certain extent. Our study laid a foundation for the recombinant production of H3 in P. pastoris and future vaccine development.
Key words: influenza A; H3N2; hemagglutinin; subunit vaccine; Pichia pastoris; codon bias
（Acta Laser Biology Sinica, 2021, 30(5): 422-428）
引用本文:
陈 溥，李安娜，汤岩松，龙浩雨，刘坤山，周 勉. 甲型流感H3抗原在毕赤酵母系统中的优化表达与纯化[J]. 激光生物学报, 2021, 30(5): 422-428.
CHEN Pu, LI Anna, TANG Yansong, LONG Haoyu, LIU Kunshan, ZHOU Mian. Optimized Expression and Purification of Influenza H3 Antigen in Pichia pastoris. journal1, 2021, 30(5): 422-428.
