关键词: 多顺反子, 口蹄疫病毒2A多肽, 巴斯德毕赤酵母, 高效表达

Abstract: In order to effectively express heterologous proteins in Pichia pastoris, the plasmid with bicistronic structure that connect two cephalosporin C (CPC) acylase genes with foot-and-mouth disease virus (FMDV) 2A sequence was constructed. After two-copy recombinant plasmid was integrated into the Pichia pastoris genome, positive recombinants were identified with selectable markers and PCR amplification. The optimal CPC acylase productivity of two-copy transformant reached 2810 U/L in shaking flask fermentation, the CPC acylase activity of two-copy transformant was 1.6 times of one-copy one at the same condition of cell concentrate. Thus, the 2A peptide with short structure and effective self-cleavage reaction not only achieved the goal of regulating multiple genes by a single promoter, but also provided a simple and effective thinking for improving expression of heterologous proteins by gene copy number.

Key words: multicistron, foot-and-mouth disease virus 2A, Pichia pastoris, effective expression