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Objective To construct a lipL32/1‐lipL21‐OmpL1/2 fusion gene and its prokaryotic expression system,and to establish an enzyme‐linked immunosorbent assay (ELISA) using the rLipL32/1‐LipL21‐OmpL1/2 fusion antigen of Leptospira interrogans for sensitive and specific detection of IgM in the serum of patients with leptospirosis.Methods lipL32/1‐lipL21‐OmpL1/2 fusion genes were constructed using a primer‐linking PCR.The target recombinant protein antigens,rLipL32/1,rLipL21,rOmpL1/2 and rLipL32/1‐LipL21‐OmpL1/2,were expressed and the purified antigens were then immobilized to the surface of microplate wells for ELISA‐based detection ofIgM in the sera of leptospirosis patients.Results Of 493 acute leptospirosis patients,95.7% and 97.8% were positive by rLipL32/1‐LipL21‐ OmpL1/2‐IgM‐ELISA using different serum dilutions,which was higher than the rLipL32/1‐IgM‐ELISA (93.1% and 90.3%),rLipL21‐IgM‐ELISA (90.3% and 87.0%),and rOmpL1‐IgM‐ELISA (85.6% and 81.1%) (P<0.01).All IgM‐ELISAs tested negative against 56 non‐leptospirosis patients with typhoid fever,hemorrhagic fever or dengue fever.Conclusion Trigeminal fusion antigen increases ELISA sensitivity and the rLipL32/1‐LipL21‐OmpL1/2‐ IgM‐ELISA is a sensitive and specific serological diagnostic method for clinical leptospirosis.
Objective To construct a lipL32 / 1-lipL21-OmpL1 / 2 fusion gene and its prokaryotic expression system, and establish an enzyme-linked immunosorbent assay (ELISA) using the rLipL32 / 1-LipL21-OmpL1 / 2 fusion antigen of Leptospira interrogans for sensitive and specific detection of IgM in the serum of patients with leptospirosis. Methods lipL32 / 1-lipL21-OmpL1 / 2 fusion genes were constructed using a primer-linking PCR.The target recombinant protein antigens, rLipL32 / 1, rLipL21, rOmpL1 / 2 and rLipL32 / 1-LipL21-OmpL1 / 2, were expressed and the purified antigens were then immobilized to the surface of microplate wells for ELISA-based detection of IgG in the sera of leptospirosis patients. Results of 493 acute leptospirosis patients, 95.7% and 97.8 % were positive by rLipL32 / 1-LipL21-OmpL1 / 2-IgM-ELISA using different serum dilutions, which was higher than the rLipL32 / 1-IgM- ELISA (93.1% and 90.3% and 87.0%), and rOmpL1-IgM-ELISA (85.6% and 81. 1%) (P <0.01) .All IgM-ELISAs tested negative against 56 non-leptospirosis patients with typhoid fever, hemorrhagic fever or dengue fever. Confocal Trigeminal fusion antigen increases ELISA sensitivity and the rLipL32 / 1-LipL21- OmpL1 / 2- IgM-ELISA is a sensitive and specific serological diagnostic method for clinical leptospirosis.