关键词: 光滑鳖甲, 丝氨酸蛋白酶抑制剂, 原核表达, 蛋白纯化, 功能验证

Abstract: 【Aim】 This study aims to clone and analyze the expression of a serine protease inhibitor gene from Anatolica polita borealis so as to verify its function. 【Methods】 A serine protease inhibitor gene from A. polita borealis was cloned using PCR and RACE methods. Bioinformatics approach was applied to analyze the basic properties of the gene and its coded protein, and to make the multiple sequence alignment with its homologous sequences. The recombinant expression vector pET-32a-ApSerpin-FA72 was constructed. The expression, purification and functional verification of the recombinant target protein were analyzed. 【Results】 A serine protease inhibitor gene was cloned from A. polita borealis and named ApSerpin-FA72 (GenBank accession no.: MF188125). Its opening reading frame (ORF) is 1 176 bp in length， encoding a 391 amino-acid residue with the predicted molecular weight of 43.7 kD and pI of 5.14. ApSerpin-FA72 is a hydrophilic protein with a signal peptide of 21 amino acids at the terminus. It might play roles in the stress response and immune response, and has the highest homology with Tribolium castaneum Serpin. The purified recombinant protein TrxA-ApSerpin-FA72 has the molecular weight of 63.7 kD. The functional verification results revealed that the recombinant protein TrxA-ApSerpin-FA72 had an inhibitory effect on the activities of trypsin and chymotrypsin. 【Conclusion】 The expression product of Anatolica polita serine protease inhibitor gene has an inhibitory effect on trypsin and chymotrypsin, indicating that it may inhibit the activity of digestive serine protease, and the verification of its functional activity can be helpful to further investigate the relationship between ApSerpin-FA72 and serine protease.

Key words: Anatolica polita borealis; serine protease inhibitor, prokaryotic expression, protein purification, functional verification