Kazal-type serine proteinase inhibitors (KPIs) function in physiological and immunological processes requiring proteinase action. In the present study, the first Cherax quadricarinatus KPI gene (designated CqKPI) was identified and characterized. The open reading frame of CqKPI contains 405 nucleotides and encodes a protein of 134 amino acids. CqKPI has two Kazal domains comprising 44 amino acid residues with the conserved amino acid sequence C-X₃-C-X₇-C-X₆-Y-X₃-C-X₆-C-X₁₂-C. Each Kazal domain has six conserved cysteine residues, which can form a structural conformation of three pairs of disulfide bonds stabilizing the Kazal domain. CqKPI exhibited high similarity with previously identified KPIs from crayfish hemocytes. The results of tissue distribution showed that CqKPI had the highest expression level in hemocytes, and this was in agreement with phylogenic relationships. Recombinant CqKPI (rCqKPI) was heterologously expressed in Escherichia coli and purified for further study. The proteinase inhibition assays suggested that rCqKPI could potently inhibit elastase and weakly inhibit trypsin, subtilisin A, and proteinase K, but not α-chymotrypsin. It can firmly bind to Bacillus hwajinpoensis, Staphylococcus aureus, and Vibrio parahaemolyticus, with weak binding to Candida albicans. In addition, CqKPI inhibited bacterial secretory proteinase activity and inhibited the growth of B. hwajinpoensis and C. albicans. These data suggest that CqKPI might be involved in anti-bacterial immunity, acting as an inhibitor of the proteinase cascade in the resistance to invasion of pathogens.

Kazal 型丝氨酸蛋白酶抑制剂 (KPI) 在需要蛋白酶作用的生理和免疫过程中发挥作用。在本研究中，第一个Cherax quadricarinatus KPI 基因（命名为CqKPI）被鉴定和表征。CqKPI 的开放阅读框包含 405 个核苷酸，编码 134 个氨基酸的蛋白质。CqKPI 有两个 Kazal 结构域，包含 44 个氨基酸残基，具有保守的氨基酸序列 CX ₃ -CX ₇ -CX ₆ -YX ₃ -CX ₆ -CX ₁₂-C。每个 Kazal 结构域都有六个保守的半胱氨酸残基，它们可以形成稳定 Kazal 结构域的三对二硫键的结构构象。CqKPI 与先前从小龙虾血细胞中鉴定的 KPI 表现出高度相似性。组织分布结果表明，CqKPI在血细胞中的表达量最高，这与系统发育关系一致。重组 CqKPI (rCqKPI) 在大肠杆菌中异源表达并纯化用于进一步研究。蛋白酶抑制试验表明 rCqKPI 可以有效地抑制弹性蛋白酶，并弱抑制胰蛋白酶、枯草杆菌蛋白酶 A 和蛋白酶 K，但不能抑制 α-胰凝乳蛋白酶。能与华津浦芽孢杆菌、金黄色葡萄球菌、和副溶血性弧菌，与白色念珠菌结合较弱。此外，CqKPI 抑制细菌分泌蛋白酶活性并抑制B. hwajinpoensis和C. albicans的生长。这些数据表明，CqKPI 可能参与抗菌免疫，在抵抗病原体入侵中充当蛋白酶级联的抑制剂。