Molecular cloning and characterization of a novel Clonorchis sinensis antigenic protein containing tandem repeat sequences

Objective To find and clone new antigen genes from the λ-ZAP cDNA expression library of adult Clonorchis sinensis,and determine the immunological characteristics of the recombinant proteins. Methods The cDNA expression library of adult C. sinensis was screened by pooled sera of clonorchiasis patients. The sequences of the positive phage clones were compared with the sequences in EST database,and the full-length sequence of the gene(Cs22 gene)was obtained by RT-PCR. cDNA fragments containing 2 and 3 times tandem repeat sequences were generated by jumping PCR. The sequence encoding the mature peptide or the tandem repeat sequence was respectively cloned into the prokaryotic expression vector pET28a(+),and then transformed into E. coli Rosetta DE3 cells for expression. The recombinant proteins(rCs22-2r,rCs22-3r,rCs22M-2r,and rCs22M-3r) were purified by His-bind-resin(Ni-NTA) affinity chromatography. The immunogenicity of rCs22-2r and rCs22-3r was identified by ELISA. To evaluate the immunological schistosomiasis patients,15 paragonimiasis westermani patients and 13 cysticercosis patients were examined by ELISA. To locate antigenic determinants,the pooled sera of clonorchiasis patients and healthy persons were analyzed for specific antibodies by ELISA with recombinant protein rCs22M-2r and rCs22M-3r containing the tandem repeat sequences.Results The full-length sequence of Cs22 antigen gene of C. sinensis was obtained. It contained 13 times tandem repeat sequences of EQQDGDEEGMGGDGGRGKEKGKVEGEDGAGEQKEQA. Bioinformatics analysis indicated that the protein(Cs22) belonged to GPI-anchored proteins family. The recombinant proteins rCs22-2r and rCs22-3r showed a certain level of immunogenicity. The positive rate by ELISA coated with the purified PrCs22-2r and PrCs22-3r for sera of clonorchiasis patients both were 45.7%(16/35),and 3.2%(1/31) for those of healthy persons. There was no cross reaction with sera of schistosomiasis and cysticercosis patients. The cross reaction with sera of paragonimiasis westermani patients was 1/15.The recombinant proteins rCs22M-2r and rCs22M-3r which only contained tandem repeats were specifically recognized by pooled sera of clonorchiasis patients. Conclusion The Cs22 antigen gene of Clonorchis sinensis is obtained,and the recombinant proteins have certain diagnostic value. The antigenic determinant is located in tandem repeat sequences.