旋毛虫核酸酶Ts87降解小鼠中性粒细胞胞外捕获网DNA骨架的研究  

TRICHINELLA SPIRALIS NUCLEASE TS87 DEGRADES THE DNA BACKBONE OF MOUSE NEUTROPHILS EXTRACELLULAR TRAP

作　　者：张明明 郝瑜琬 程喻力[1] 黄京京 贾智惠 诸欣平[1] ZHANG Ming-ming;HAO Yu-wan;CHENG Yu-li;HUANG Jing-jing;JIA Zhi-hui;ZHU Xin-ping(Department of Medical Microbiology and Parasitology,School of Basic Medical Sciences,Capital Medical University,Beijing 100069,China;Chinese Center for Disease Control and Prevention,Chinese Center for Tropical Diseases Research,WHO Centre for Tropical Diseases,National Center for International Research on Tropical Diseases,Ministry of Science and Technology,National Health Commission Key Laboratory of Parasite and Vector Biology,Shanghai 200025,China)

机构地区：[1]首都医科大学基础医学院,病原生物学系寄生虫学教研室,北京100069 [2]中国疾病预防控制中心寄生虫病预防控制所,国家热带病研究中心,WHO热带病合作中心,科技部国家级热带病国际联合研究中心,国家卫生健康委员会寄生虫病原与媒介生物学重点实验室,上海200025

出　　处：《寄生虫与医学昆虫学报》2025年第1期1-9,15,共10页Acta Parasitologica et Medica Entomologica Sinica

基　　金：国家自然科学基金项目(No.81672042,No.82202549)。

摘　　要：目的本研究旨在探索旋毛虫Ts87蛋白是否可以通过对小鼠NETs DNA骨架的降解作用实施宿主免疫逃避。方法首先,利用生物信息学软件分析Ts87蛋白的理化性质,通过序列同源性比对确认其属于核酸酶家族。随后,通过分子克隆及蛋白纯化技术,获得高纯度的Ts87蛋白,并证实其具有核酸酶活性。将Ts87与已知酶活性位点的人溶酶体DNaseⅡα以及结构已解析的泰国伯克霍尔德氏菌DNaseⅡ的一级序列进行比对,预测出其可能的活性位点,并通过定点突变技术获得Ts87酶活突变体,酶活性实验证实其功能。在体外检测小鼠骨髓中性粒细胞胞外核酸量及免疫荧光染色小鼠NETs,来证实Ts87蛋白酶活突变体降解NETs能力是否有所下降。最后,Ts87免疫小鼠后,通过免疫荧光染色小鼠肠道NETs的MPO与CitH3表达量,观察小鼠体内NETs的降解情况及虫荷。结果成功获得高纯度Ts87蛋白,并证实其具有核酸酶活性,在体外可以降解小鼠NETs,且Ts87突变体降解NETs的能力下降。体内实验结果表明,免疫小鼠产生抗Ts87抗体后,体内NETs降解减少,利于NETs对虫体攻击,使小鼠体内虫荷降低。结论旋毛虫能利用Ts87核酸酶逃避NETs的捕获,该研究为旋毛虫病的防治提供潜在的疫苗与药物靶点。Objective This study aimed to investigate whether Trichinella spiralis Ts87 protein can facilitate immune evasion by degrading the mouse neutrophil extracellular traps(NETs).Methods First,we used bioinformatics tools to analyze the physicochemical properties of Ts87 and,through sequence homology alignment,confirmed its classification within the nuclease family.Molecular cloning and protein purification techniques were then employed to obtain high-purity Ts87 protein,and its nuclease activity was confirmed.Additionally,we compared the primary sequence of Ts87 with known active sites from human lysosomal DNase IIαand the structurally characterized Burkholderia thailandensis DNase II.This enabled us to predict potential active sites.Ts87 enzymatic mutants were generated using site-directed mutagenesis,and their functions were confirmed through enzymatic activity experiments.The extracellular nucleic acid content of mouse bone marrow neutrophils and the immunofluorescence staining of mouse NETs were assessed in vitro to verify whether the Ts87 protease mutants exhibited a decreased ability to degrade NETs.Finally,after immunizing mice with Ts87,calculated larvae burden and the degradation of NETs in vivo were observed by immunostaining the MPO and CitH3 expression in the intestinal NETs of the mice.Results High-purity Ts87 protein was successfully obtained and confirmed to possess nuclease activity,capable of degrading NETs in vitro.The Ts87 mutants exhibited reduced capacity in degrading NETs.In vivo experiments in mice demonstrated that the production of anti-Ts87 antibodies in immunized mice reduced the degradation of NETs,facilitating NET-mediated attack on the parasites and resulting in decreased larvae burden in the mice.Conclusions Trichinella spiralis can utilize Ts87 nuclease to evade capture by NETs,providing potential vaccine and drug targets for the prevention and treatment of trichinellosis.

关 键 词：旋毛虫核酸酶Ts87 中性粒细胞胞外捕获网 免疫逃避 

分 类 号：R383.1[医药卫生—医学寄生虫学]