Table_1_Characterization of Two Trichinella spiralis Adult-Specific DNase II and Their Capacity to Induce Protective Immunity.DOCX

Deoxyribonuclease II (DNase II) is a widespread endonuclease, which can degrade the DNA. Trichinella spiralis adult-specific DNase II-1 (TsDNase II-1) and DNase II-7 (TsDNase II-7) were identified in excretory–secretory (ES) or surface proteins of adult worm (AW) and intestinal infective larvae (IIL) using immunoproteomics with early infection sera. The aim of this study was to characterize the two T. spiralis DNase II enzymes and to investigate their role as potential vaccine candidate target molecules. The cDNA sequences of the two DNase II enzymes from 3 days old AWs of T. spiralis were cloned and expressed. The sequencing results showed that the complete cDNA sequences of the two DNase II enzymes were 1221 and 1161 bp long, and the predicted open reading frames encoded 347 and 348 amino acids, respectively. On Western blot analysis, natural TsDNase II-1 and TsDNase II-7 in the crude extracts of IIL, AWs, and newborn larvae (NBL) and AW ES proteins were recognized by both anti-rTsDNase II-1 and anti-rTsDNase II-7 sera. Indirect immunofluorescence test and qPCR showed that the two DNase II enzymes were highly expressed at AW and NBL stages and were mainly located at the cuticle and stichosome of the nematode. Vaccination with the two recombinant DNase II enzymes triggered prominent humoral responses that exhibited significant immune protection against T. spiralis larval infection, as demonstrated by the notable reduction in intestinal AW and muscle larva burdens. Specific antibodies to the two molecules evidently inhibited the in vitro parasite invasion of enterocytes and participated in the killing of NBL by an antibody-dependent cell-mediated cytotoxicity (ADCC) mode. The enzymes DNase II-1 and DNase II-7 are the potential target molecules for anti-Trichinella vaccine for blocking both larval invasion and development.

脱氧核糖核酸酶II（Deoxyribonuclease II, DNase II）是一类广泛分布的内切核酸酶，可降解DNA。本研究通过感染早期血清开展免疫蛋白质组学分析，从旋毛虫（Trichinella spiralis）成虫（adult worm, AW）与肠感染期幼虫（intestinal infective larvae, IIL）的排泄分泌（excretory–secretory, ES）蛋白及表面蛋白中，鉴定得到两种成虫特异性DNase II：TsDNase II-1与TsDNase II-7。本研究旨在对这两种旋毛虫DNase II酶进行功能表征，并探究其作为疫苗候选靶标分子的潜力。研究人员从旋毛虫3日龄成虫中克隆并表达了这两种DNase II酶的cDNA序列。测序结果显示，二者的完整cDNA序列长度分别为1221 bp与1161 bp，预测的开放阅读框分别编码347和348个氨基酸。蛋白质印迹分析表明，在肠感染期幼虫、成虫、新生幼虫（newborn larvae, NBL）的粗提物以及成虫排泄分泌蛋白中，天然存在的TsDNase II-1与TsDNase II-7均可被抗重组TsDNase II-1血清与抗重组TsDNase II-7血清识别。间接免疫荧光试验与实时定量PCR（qPCR）结果显示，这两种DNase II酶在成虫与新生幼虫阶段高表达，且主要定位于该线虫的角质层与咽管。用这两种重组DNase II酶免疫接种，可触发显著的体液免疫应答，对旋毛虫幼虫感染展现出良好的免疫保护效果，具体表现为肠道成虫荷虫量与肌幼虫荷虫量显著降低。针对这两种分子的特异性抗体可在体外有效抑制寄生虫对肠上皮细胞的入侵，并通过抗体依赖性细胞介导的细胞毒作用（antibody-dependent cell-mediated cytotoxicity, ADCC）途径参与杀灭新生幼虫。综上，DNase II-1与DNase II-7可作为抗旋毛虫疫苗的潜在靶标分子，能够同时阻断幼虫的入侵与发育过程。