Screening and efficacy evaluation of PCNA and NEDD8 as early serological diagnostic antigens for clonorchiasis

doi:10.12140/j.issn.1000-7423.2026.02.014

Abstract

Abstract:

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.

Key words: Clonorchis sinensis, Early diagnosis, PCNA, NEDD8, IgM

CLC Number:

R532.23

LI Yuanyuan, GAO Yuwei, YI Haibo, DU Xinyue, ZHOU Pincheng, WU Chenyun, ZHU Tingjun, GUO Simin, QIAN Menbao, WANG Zhaojun. Screening and efficacy evaluation of PCNA and NEDD8 as early serological diagnostic antigens for clonorchiasis[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2026, 44(2): 244-251.

Figures/Tables 5

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References 33

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蛋白名称 Protein name	基因 ID Gene ID	引物（5'→3'） Primer (5'→3')	内切酶 Endonuclease	长度/bp Length/bp
FBA	CLF_105235	F：GCGG GAATTCAATGTCTCTACCAAAGTACTTGC	EcoRⅠ	1 089
FBA	CLF_105235	R：CGTA CCTAGGTCAGTAAGCATGGCTTGGG	AvrⅡ
CP1	AY273802.1	F：CCGG GAATTCAAGAACTACTCAAGTTGAGCC	BamHⅠ	927
CP1	AY273802.1	R：AACC GGATCCCTATTTGATAATCGCTG	EcoRⅠ
PCNA	CLF_109756	F：TTAA CTCGAGATGTTCGAGGCCCGCCTCAA	XhoⅠ	783
PCNA	CLF_109756	R：CGCG CCTAGGTTAATCAGCATCATCTTCGATCTT	AvrⅡ
NPC2	CLF_109784	F：TATA CTCGAGCAATGGATGAACCCACTA	XhoⅠ	402
NPC2	CLF_109784	R：ATAT GGATCCTTAGTCGTGGATTACCGC	BamHⅠ
NEDD8	CLF_112635	F：CGCG GAATTCAATGTTGATCAAGGTGAAAACCCT	EcoRⅠ	237
NEDD8	CLF_112635	R：ATAT CCTAGGTCAACGAGAGCCGCCACGTA	AvrⅡ

抗原 Antigen	真阳性/例 True positive/case	假阳性/例 False positive/case	假阴性/例 False negative/case	真阴性/例 True negative/case	曲线下面积（95% CI）AUC (95% CI)	截断值Cut-off	敏感性（95% CI） Sensitivity（95% CI）	特异性/%（95% CI）Specificity/%（95% CI）
PCNA	10	1	4	21	0.873 4（0.726 9~1.000）	0.062 3	10/14（45.4%~88.3%）	95.5（78.2%~99.8%）
NEDD8	11	1	3	21	0.879 9（0.733 9~1.000）	0.169 3	11/14（52.4%~92.4%）	95.5（78.2%~99.8%）
PCNA + NEDD8	12	1	2	21	0.915 6（0.784 0~1.000）	0.144 7	12/14（60.1%~97.5%）	95.5（78.2%~99.8%）