[Cloning, expression and evaluation on effect in serological diagnosis of cysteine protease of Clonorchis sinensis].
Kľúčové slová
Abstrakt
OBJECTIVE
To clone and express the cysteine protease of Clonorchis sinensis and evaluate its effect on immunodiagnosis of human clonorchiasis.
METHODS
Based on a cysteine protease gene fragment of C. sinensis (CS-CP, GenBank accession: AF093242), a pair of primers were designed and amplified from total cDNA of C. sinensis, and the gene was cloned into plasmid pPIC9K and expressed in Pichia pastoris GS115. The expressed product was purified. Ten BALA/c mice were immunized with the purified CS-CP, and the anti CS-CP antibody in the sera of immunized mice was tested with ELISA. Finally, its effect on serodiagnosis was evaluated with Dot-ELISA and Western blot.
RESULTS
The CS-CP gene (approximately 927 bp) was successfully amplified, cloned and expressed in Pichia pastoris GS115. The sera of mice immunized with purified rCS-CP could be recognized by the soluble antigen of C. sinensis adult worms and its special anti CS-CP antibody titer was high (1: 64 000). The rCS-CP was probed by Dot-ELISA and Western-blot with sera from patients with clonorchiasis and other parasitic infections, and it had a sensitivity of 91.7% (55/60) in diagnosis of clonorchiasis and a specifity of 97.6% (82/84) for healthy population. The rCS-CP had no cross-reaction with the patients of schistosomiasis japonica, but had a cross-reactivity of 20.0% (2/10) with the patients with paragonimiasis westermani.
CONCLUSIONS
rCS-CP possesses a favorable diagnostic effect and it is a better serodiagnostic one among recombinant antigens.
