[Cloning, expression and stage-specific analysis of Schistosoma japonicum P7 antigen and evaluation of its value in early diagnosis]

Abstract

Objective: To clone and express Schistosoma japonicum P7 antigen (GenBank accession No. EU121231), analyze stage-specific transcription and expression of the antigen, and evaluate its value in early diagnosis.

Methods: The positive clone (P7) screened from schistosomula cDNA library was amplified by PCR. The PCR product was subcloned into prokaryotic expression vector pET28a. The recombinant plasmids were identified by restrictive enzymes digestion. The positive recombinant plasmids were transformed into E. coli BL21 (DE3), induced by IPG for expression and purified. The diagnostic value of P7 recombinant protein was evaluated by Western blotting analysis. RT-PCR and Western blotting were used to investigate the differential transcription and expression of P7 during the developmental stages. The specific antibodies against P7 recombinant protein in the sera of S. japonicum-infected rabbits at 14 d postinfection, sera of schistosomiasis (28 cases), clonorchiasis (30 cases) and paragonimiasis (20 cases) patients, and sera of healthy people (30 cases) were detected by ELISA, respectively.

Results: The expression vector of p7/pET28a was established and the P7 recombinant protein (about Mr 20 100) was expressed in E. coli. Western blotting analysis showed that the recombinant protein was specifically recognized by immunized rabbit sera, and sera from mice on the 14th day post infection, but was not recognized by the sera of mice at 42 d post-infection. P7 mRNA was detected in cercariae, schistosomula and adult worms, while the protein was only found in schistosomula. The positive rate of rabbit sera collected at 14 d post-infection was 83.3% (15/18). The sensitivity and specificity of ELISA for diagnosis of schistosomiasis japonica were 75.0% (21/28) and 93.8% (75/80), respectively. And the P7 protein showed cross reaction with sera of clonorchiasis and paragonimiasis patients with positive rates of 6.7% (2/30) and 5.0% (1/20), respectively.

Conclusion: P7 antigen might be a potential candidate for early diagnosis of schistosomiasis.