Thelohanellus kitauei, is a member of obligate parasitic myxozoans, which causes intestinal giant-cystic disease of common carp (Cyprinus carpio) and has resulted in significant economic losses in carp farms. Cystatin secreted by parasites can regulate the immune response of host to facilitate parasite’s survival. In this study, the secretory TK-cystatin gene, encoding a protein of 120 amino acid residues (13.65 kDa), was cloned from T. kitauei genome. Phylogenetic analysis showed that TK-cystatin gene is closely related to the cystatin-A from Hydra vulgaris. Multiple sequence alignment revealed that TK-cystatin had three conserved motifs: N-terminal G¹⁹G²⁰, Q⁷³VVAG⁷⁷, and C-terminal L¹⁰²P¹⁰³. Molecular docking between TK-cystatin and three cysteine proteases showed a lower binding energy (− 13 KJ/mol) with cathepsin L whereas a higher binding energy (− 8.6 KJ/mol) with cathepsin B. TK-cystatin gene was expressed in Escherichia coli. Activity assays revealed that TK-cystatin has stronger inhibitory activity on endopeptidases (papain and cathepsin L) and weaker inhibitory activity on exopeptidase (cathepsin B). TK-cystatin was stable under the condition of acidity or alkalinity or below 57 °C. This study laid a foundation for the design and development of the anti-T. kitauei vaccine in carp culture in the future.

Thelohanellus kitauei是专性寄生粘液虫的一员，它会引起鲤鱼（ Cyprinus carpio ）肠道巨囊病，给鲤鱼养殖场造成重大经济损失。寄生虫分泌的半胱氨酸蛋白酶抑制剂可以调节宿主的免疫反应，以利于寄生虫的生存。在本研究中，从T. kitauei基因组中克隆了分泌型 TK-cystatin 基因，编码 120 个氨基酸残基（13.65 kDa）的蛋白质。系统发育分析表明TK-cystatin基因与水螅中的cystatin-A密切相关。多重序列比对显示TK-胱抑素具有三个保守基序：N端G ¹⁹ G ²⁰ 、Q ⁷³ VVAG ⁷⁷和C端L ¹⁰² P ¹⁰³ 。 TK-胱抑素和三种半胱氨酸蛋白酶之间的分子对接显示与组织蛋白酶 L 的结合能较低 (− 13 KJ/mol)，而与组织蛋白酶 B 的结合能较高 (− 8.6 KJ/mol)。TK-胱抑素基因在大肠杆菌中表达。活性测定表明，TK-cystatin 对内肽酶（木瓜蛋白酶和组织蛋白酶 L）具有较强的抑制活性，对外肽酶（组织蛋白酶 B）具有较弱的抑制活性。 TK-胱抑素在酸性、碱性条件下及57℃以下稳定。本研究为今后鲤鱼养殖抗北卫毛虫疫苗的设计和开发奠定了基础。