目的 从5个功能不同的华支睾吸虫候选蛋白中筛选出适用于华支睾吸虫病早期血清学诊断的最佳抗原。方法 选取公开数据库NCBI序列读取档案库的华支睾吸虫转录组数据并结合蛋白功能信息，筛选出果糖-1,6-二磷酸醛缩酶（FBA）、半胱氨酸蛋白酶1（CP1）、增殖细胞核抗原（PCNA）、尼曼匹克C2型蛋白（NPC2）和神经前体细胞表达发育下调蛋白8（NEDD8）等5个候选诊断抗原。分析抗原在华支睾吸虫新脱囊幼虫和成虫阶段的表达情况，利用免疫表位数据库和蛋白抗原空间表位预测工具预测抗原表位，并从华支睾吸虫cDNA中PCR扩增对应基因，构建pET-302/NT-His原核表达载体。在大肠埃希菌中经异丙基-β-D-硫代半乳糖苷诱导表达并通过镍-次氮基三乙酸亲和层析纯化重组蛋白。采集经口灌胃感染法感染华支睾吸虫囊蚴后6周的新西兰白兔和BALB/c小鼠阳性血清、华支睾吸虫病早期患者血清（感染后2周~6周）、晚期患者血清（感染后6周以上），及健康对照人群血清和其他寄生虫感染者血清。采用间接ELISA法检测血清中特异性IgM，以华支睾吸虫成虫抗原作为阳性对照，并以阳性血清与阴性血清的标准化450 nm吸光度（ΔA₄₅₀值）的比值（P/N）作为评价指标。使用GraphPad Prism 10软件计算敏感性、特异性，绘制受试者工作特征（ROC）曲线并评估曲线下面积（AUC）。结果 转录组学分析表明，5个候选蛋白在新脱囊幼虫和成虫阶段均呈高水平表达。结构及表位预测提示候选蛋白均含多种线性和构象性B细胞表位，关键残基位于表位暴露区域，具有良好抗原潜力。FBA、CP1、PCNA、NPC2和NEDD8等5种蛋白均成功重组表达并纯化，十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）显示条带清晰、相对分子质量与理论一致，重组蛋白的纯度分别为86.2%、90.6%、97.1%、82.5%、93.6%，蛋白浓度分别为1 780、514、594、375、1 263 μg/mL。ELISA结果显示，PCNA和NEDD8的IgM反应最强，其P/N值（兔/小鼠）分别为1.675/4.176与1.521/4.076；CP1、FBA和NPC2的反应性相对较弱（1.478/1.870、0.952/2.531、1.040/2.413）。对患者血清的检测结果显示，PCNA、NEDD8及其组合抗原在早期患者中ΔA₄₅₀值明显升高（0.304 ± 0.302、0.506 ± 0.383和0.553 ± 0.358），均高于健康对照组（0.028 ± 0.017、0.066 ± 0.044、0.066 ± 0.041）（Z = 3.924、3.763、4.116，P < 0.01），也高于晚期感染组患者（0.035 ± 0.030、0.070 ± 0.060、0.076 ± 0.071）（Z = 3.890、4.116、4.386，P < 0.01）。在早期感染患者中，PCNA、NEDD8及其组合抗原的敏感性分别为10/14、11/14、12/14，特异性均为95.5%（21/22）。ROC曲线分析显示，PCNA、NEDD8及其组合抗原的AUC分别为0.873 4、0.879 9和0.915 6。结论 PCNA和 NEDD8作为诊断抗原可用于华支睾吸虫早期感染的血清IgM检测，其联合应用可进一步提升早期诊断性能，具有开发为血清学早期诊断试剂的潜力。

Objective To identify the optimal antigen for early serological diagnosis of clonorchiasis screened from five functionally distinct Clonorchis sinensis candidate proteins. Methods The transcriptome data of C. sinensis were retrieved from Sequence Read Archive in NCBI, together with protein function information, were used to screen five candidate diagnostic antigens, including fructose-1,6-bisphosphate aldolase (FBA), cysteine protease 1 (CP1), proliferating cell nuclear antigen (PCNA), niemann-pick type C2 protein (NPC2), and neural precursor cell expressed developmentally downregulated protein-8 (NEDD8). The expression of antigens was detected in larvae freshly released from C. sinensis metacercariae and adults. Antigen epitopes were predicted using immune epitope databases and spatial epitope prediction tools for protein antigen, and corresponding genes were amplified from C. sinensis cDNA using PCR assay to construct a prokaryotic expression vector pET-302/NT-His. Following induction in Escherichia coli with isopropyl-β-D-thiogalactoside (IPTG), the recombinant protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Serum samples were collected from New Zealand white rabbits and BALB/c mice infected with C. sinensis metacercariae via oral gavage for 6 weeks, early-stage clonorchiasis patients (infected for 2 to 6 weeks), late-stage clonorchiasis patients (infected for more than 6 weeks), healthy controls and individuals with other parasitic infections. Serum specific IgM antibody was detected using indirect enzyme-linked immunosorbent assay (ELISA), with C. sinensis adult antigen as a positive control, and the ratio of absorbance of positive serum to negative serum (P/N) at 450 nm (ΔA₄₅₀ value) served as an evaluation indicator. The sensitivity and specificity were estimated with the software GraphPad Prism 10, and the area under the receiver operating characteristic (ROC) curves (AUC) was estimated. Results Transcriptomic analysis showed that the five candidate proteins were highly expressed in both larvae freshly released from C. sinensis metacercariae and adults. Structural and epitope predictions suggested that antigen epitopes contained multiple linear and conformational B cell epitopes, and their key residues were located in the epitope-exposed region, indicating high antigenic potential. Recombinant expression of five proteins (FBA, CP1, PCNA, NPC2, and NEDD8) was successfully completed, and these five recombinant proteins were successfully purified. SDS-PAGE displayed clear bands, with relative molecular masses consistent with theoretical values. The purities of recombinant proteins were 86.2%, 90.6%, 97.1%, 82.5%, and 93.6%, and the concentrations of recombinant proteins were 1 780, 514, 594, 375, and 1 263 μg/mL, respectively. ELISA measured the strongest IgM response to PCNA and NEDD8, with P/N values (rabbit/mouse) of 1.675/4.176 and 1.521/4.076, respectively. The IgM responses to CP1, FBA and NPC2 were relatively weak, with P/N values (rabbit/mouse) of 1.478/1.870, 0.952/2.531, 1.040/2.413, respectively. The ΔA₄₅₀ values for PCNA (0.304 ± 0.302), NEDD8 (0.506 ± 0.383), and their combinations (0.553 ± 0.358) were all higher in detection of serum samples from early-stage clonorchiasis patients relative to health controls [(0.028 ± 0.017), (0.066 ± 0.044) and (0.066 ± 0.041)] (Z = 3.924, 3.763 and 4.116; all P < 0.01) or late-stage patients [(0.035 ± 0.030), (0.070 ± 0.060) and (0.076 ± 0.071)] (Z = 3.890, 4.116 and 4.386; all P < 0.01). The sensitivities of PCNA, NEDD8 alone and in combinations were 10/14, 11/14, and 12/14 for diagnosis of serum samples from early-stage clonorchiasis patients, and the specificity was all 95.5% (21/22). ROC curve analysis showed that the AUC of PCNA, NEDD8 alone and in combinations were 0.873 4, 0.879 9, and 0.915 6, respectively. Conclusion PCNA and NEDD8 may be used as diagnostic antigens for detection of serum IgM antibody from individuals with early-stage C. sinensis infections. PCNA-NEDD8T combinations may improve the early diagnostic performance, which has potential for early immunodiagnosis of clonorchiasis.