Abstract:

Objective To immunologically screen the cDNA library of adult Paragonimus westermani for the gene encoding thioredoxin peroxidase (TPx), clone and express the gene, and evaluate the immunodiagnostic value of TPx recombinant protein. Methods The λ ZAP cDNA library was immunologically screened with the pooled serum of P. westermani patients. The obtained positive clones were sequenced and analyzed by multiple sequence alignment. The full length (rPwTPx) and N-terminal truncated (rPwTPx_1) sequences of PwTPX were subcloned into the prokaryotic plasmid pET28a (+), respectively, to construct the expression vectors rPwTPX and rPwTPX_1. The expression of the two constructed recombinant plasmids was induced by 1 mmol/L IPTG. Protein extraction reagents, lysozyme and nuclease were used to lyse and express the bacterial fluid. The recombinant proteins of rPwTPx and rPwTPx_1 were purified using the His-tagged affinity column (Ni-NTA). SDS-PAGE was used to analyzed the expression of the recombinant proteins. rPwTPx and rPwTPx_1 were used to test the sera of 36 paragonimiasis westermani patients, 15 patients with clonorchiasis sinensis, 15 schistosomiasis japonica patients, 15 fascioliasis gigantica patients, 15 cysticercosis patients, and 36 healthy donors, using the indirect ELISA method. Data were analyzed with the SAS 9.2 software. Results The TPx recombinant proteins rPwTPx and rPwTPx_1 were obtained through expression and purification, with relative molecular mass M_r of 25 000 and 22 000, respectively. The rPwTPx ELISA results showed that the A₄₅₀ values for the sera from patients of paragonimiasis westermani, healthy persons, clonorchiasis sinensis, schistosomiasis japonica, fascioliasis gigantic, and cysticercosis patients were 0.150 ± 0.092, 0.036 ± 0.014, 0.043 ± 0.019, 0.047 ± 0.013, 0.060 ± 0.022 and 0.048 ± 0.021, respectively, The positive rate in serum of paragonimiasis westermani patients was 58.3% (21/36). There was no cross-reaction with sera of healthy donors, clonorchiasis sinensis, schistosomiasis japonica and cysticercosis patients. The cross-reaction with sera of fascioliasis gigantic patients was 2 samples. The ELISA using rPwTPx_1 as the antigen presented the A₄₅₀ values to the above testing sera was 0.144 ± 0.092, 0.022 ± 0.009, 0.027 ± 0.015, 0.033 ± 0.022, 0.036 ± 0.015 and 0.032 ± 0.018, respectively. The positive rate of paragonimiasis westermani patients sera was 88.9% (32/36), while the sera of healthy donors showed no cross-reaction. Only 1 serum sample of 15 clonorchiasis sinensis patients had cross-reaction. The cross-reaction with sera of schistosomiasis japonica, fascioliasis gigantic and cysticercariasis patients were 3, 2 and 2 samples. The total diagnostic coincidence rate, sensitivity and specificity of ELLSA using rPwTPx and rPwTPx_1 as antigen were 87.1%, 58.3%, 97.9%, and 90.9%, 88.9%, 91.7%, respectively. The sensitivity of the rPwTPx_1 ELISA was significantly higher than that of the rPwTPx ELISA (P < 0.05). Crude antigen-ELISA showed that the A₄₅₀ value of serum samples from the 36 healthy donors was 0.012, and S was 0.006. The sensitivity, specificity and total diagnostic coincidence rate of crude antigen were 100%, 83.3% and 87.9%, which did not significantly differ from those of rPwTPx and rPwTPx_1 (P > 0.05). Conclusion The gene encoding TPx was screened and expressed, and two structural types of recombinant protein were obtained, including full length and truncated types, which recombinant protein shows proper sensitivity and high specificity for the serodiagnosis of P. westermani infection.

Key words: Paragonimus westermani, Thioredoxin peroxidase, Immunoscreen, Expression, ELISA