Abstract: 【Abstract】  Objective   To screen and identify new specific antigen gene from a cDNA library of adult Clonorchis sinensis, and investigate the immunogenicity of the recombinant proteins.   Methods   The λZAP cDNA library of adult C. sinensis was immunoscreened with pooled sera of clonorchiasis patients. The positive clones were sequenced and analyzed. The sequence encoding the mature peptide was cloned into prokaryotic expression vector pET28b（+）. The recombinant plasmid was transformed into E. coli BLR21 （DE3） or BLR21 （DE3） pLysS and followed by expression of the protein induced by IPTG. The recombinant protein was purified by His-bind-resin （Ni-NTA） affinity chromatography and identified by Western blotting. BALB/c mice were immunized with purified recombinant pET28b-Cs2 protein, and the sera from immunized mice were analyzed for specific antibodies by ELISA.  Results   A total of 44 positive clones were isolated from the C. sinensis cDNA library. Three clones containing specific tandem repeats of PPMP amino acid sequence were named as C. sinensis PPMP antigen genes. The genes containing KPPMPGDRDA, QPPMPGGRDA were named as type PPMPⅠ and type PPMPⅡ anti-gens, respectively. Sequence analysis revealed that these PPMP genes were a novel specific C. sinensis antigen gene family. Two new genes, PPMPⅠ Cs2 and PPMPⅡ Cs3, were expressed in E. coli, and SDS-PAGE showed that the two recombinant proteins were about Mr 22 000 and Mr 39 000. The two soluble recombinant proteins were recognized by pooled sera of clonorchiasis patients. A high level of specific IgG against the recombinant proteins （maximum dilution 1 ∶ 64 000） was produced in immunized mice.   Conclusion   A novel PPMP gene family of C. sinensis has been identified, and its re-combinant proteins show high immunogenicity.

Key words: Clonorchis sinensis, Immuonscreen, Cloning, Sequence analysis, Prokaryotic expression, Immunogenicity