Establishment and application of ELISAs based on rLipL32/1-LipL21-OmpL1/2 fusion antigen of Leptospira interrogans / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish ELISAs based on rLipL32/1-LipL21-OmpL1/2 fusion antigen of Leptospira interrogans for detecting specific IgG and IgM in serum of patients with leptospirosis.</p><p><b>METHODS</b>Microscope agglutination test(MAT) was performed to detect serum specimens from leptospirosis patients and to determine titers of rabbbit antiserum agaist rLipL32/1-LipL21-OmpL1/2 to reference standard strains of L. interrogans. By using rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 and rOmpL1/2 as the coated antigens, ELISAs for detecting specific serum IgM and IgG were established. The established ELISAs were applied to MAT-positive serum specimens from 107 patients with leptospirosis.</p><p><b>RESULT</b>The results of MAT confirmed that 66% (71/107) of the patients were infected with L.interrogans serogroup Icterohaemorrhagiae, and the rLipL32/1-LipL21-OmpL1/2 antiserum were able to agglutinate all 15 reference standard L.interrogans strains with 1 : 20approximate, equals1 : 160 titers. The positive rates of ELISAs using rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 or rOmpL1/2 as the antigen were 89.7%, 75.7%, 85.1% and 79.4% for detecting IgM, respectively, while 99.1%, 99.1%, 94.4% and 86.0% for detecting IgG, respectively. The positive detection rate of rLipL32/1-LipL21-OmpL1/2-IgM-ELISA was higher than those of the other three IgM detection ELISAs (P<0.05). The positive detection rate of rLipL32/1-LipL21-OmpL1/2-IgG-ELISA was higher than that of rOmpL1/2-IgG-ELISA (P<0.05), while there was no significant differnce with that of rLipL21-IgG-ELISA and rLipL32/1-IgG-ELISA (P>0.05).</p><p><b>CONCLUSION</b>The ELISAs using rLipL32/1-LipL21-OmpL1/2 as the antigen can be applied as a sensitive,specific and universal serological method for diagnosis of leptospirosis.rLipL32/1-LipL21-OmpL1/2-IgM-ELISA shows a definite value for early diagnosis of leptospirosis compared with the other ELISAs used in this study.</p>

Prokaryotic expression of trigeminy artificial fusion gene of Leptospira interrogans and the immunogenicity of its products / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct lipL32/1-lipL21-OmpL1/2 fusion gene of Leptospira interrogans and its prokaryotic expression system, and to identify the immunogenicity of its products.</p><p><b>METHODS</b>PCR using linking primers was applied to construct lipL32/1-lipL21-OmpL1/2 fusion gene and a prokaryotic expression system of the fusion gene was then established using routine genetic engineering technique. SDS-PAGE was used to examine output of the target recombinant protein rLipL32/1-LipL21-OmpL1/2. Double immunodiffusion and Western Blot assay were applied to identify immunogenicity of rLipL32/1-LipL21-OmpL1/2.</p><p><b>RESULT</b>lipL32/1-lipL21-OmpL1/2 fusion gene with correct sequence and its prokaryotic expression system E.coli BL21DE3pET42a-lipL32/1-lipL21-ompL1/2 was obtained in this study. The output of rLipL32/1-LipL21- OmpL1/2 after optimisation was 37.78 mg/L. The immunodiffusion titer of rabbit antiserum against rLipL32/1-LipL21-OmpL1/2 was 1:4. The rLipL32/1-LipL21-OmpL1/2 antiserum was able to recognize rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 and rOmpL1/2. Positive Western hybridization signals were found among rLipL32/1-LipL21-OmpL1/2 and rabbit antiserum against whole cell of strain 56601 and serum from patients infected with L.interrogans serogroups Icterohaemorrhagiae, Grippotyphosa, Autumnalis and Pomona.</p><p><b>CONCLUSION</b>The fusion gene lipL32/1-lipL21-OmpL1/2 and its prokaryotic expression system were successfully constructed in this study. The expressed fusion protein can be used as the antigen for developing universal genetic engineering vaccine and universal serological tests of leptospirosis.</p>