Evaluation Of Leptospira Outer Membrane Protein Serological Diagnostic Value And The CTB-OmpL1Vaccine Protective Effect

Background and Objective:Leptospira represented in pathogenic Leptospira widely distributed intropical, subtropical and temperate regions, the zoonosis caused bypathogenic Leptospira was called leptospirosis. Leptospirosis is one of themost important infectious diseases which need to prevention and control.There are some shortcomings in the currently diagnosis methods andvaccine of leptospirosis. Recent studies show that the outer membraneprotein of Leptospira is an important component for the outer membrane ofLeptospira, which are pathogenic and immunogenic. To improve thesensitivity and specificity of the serological tests, LipL21, Loa22, LipL32and LigACon4-8were constructed and expressed. These four recombinantproteins were used as antigen to establish the ELISA for detecting theantibody in equine serum and canine serumn. And the LUMINEX methordwas established to detect the leptospirosis antibody in canine serum, whichlay a foudation for development of new serological diagnosis methods forleptospirosis detection. CTB-OmpL1recombinant lactobacillus oralvaccine was constructed by molecular biological method, and we evaluatedthe immune protective effect of the oral vaccine in hamster model, which islaying the foundation for the development of new vaccines. Contents and methods:1. LipL21, Loa22, LipL32and LigACon4-8gene were amplified fromgenomic DNA of leptospira, and we construct prokaryotic expressionvector LipL21-PET28/Rosetta, Loa22-PET28/Rosetta, LipL32-PGEX4T2/BL21, LigACon4-8-PET28/Rosetta, Mass-inducedexpression of recombinant bacteria, purified the recombinant protein byNi+NTA column and cation/ion column, and identificantify the proteinby Wester blot.2. The ELISA was established for detecting the antibody in equine andcanine serum by using recombinant proteins LipL21, Loa22, LipL32and LigACon4-8as antigens, and we detected a large number of clinicalequine serum and canine serums, which compared with the traditionalMAT results to evaluate its diagnostic value.3. The Luminex method was established for detecting the antibody incanine serum by using recombinant proteins LipL21, Loa22, LipL32and LigACon4-8as antigens, and we detected a large number of clinicalcanine serums, which compared with the traditional MAT results toevaluate its diagnostic value.4. Leptospira recombinant protein CTB-OmpL1was constructed andexpressed by genetic engineering techniques, transfer of plasmid toLactococcus lactis NZ3900to get the oral vaccine, and establish thehamster model of leptospirosis infection to evaluate its immuneprotection.Results:1. The recombinant expression plasmid LipL21-PET28/Rosetta, Loa22-PET28/Rosetta, LipL32-PGEX4T2/BL21, LigACon4-8-PET28/Rosettawere constructed successfully. Dual-enzyme digestion identificationresults showed that the genes are complete. The gene sequence results show that the sequences are consistent with the Genebank datumbasically. We got high concentration of purified protein, which is2-5mg/ml in concentration and90%in purity. The wester blot showed thatthese four recombinant proteins can reaction with equine or canineserum of infection leptospirosis.2.130MAT negtive and176MAT positive equine serums were detected byELISA which using LipL21, Loa22, LipL32and LigACon4-8recombinant proteins as antigens. The ELISA results showed that thesensitivity were84.66%,77.84%,77.84%,82.39%respectively, andthe specificity were83.85%,92.31%,86.15%,86.15%.3.102MAT negtive and203MAT positive canine serums were detectedby ELISA that LipL21, Loa22, LipL32and LigACon4-8recombinantproteins were used as antigens. The ELISA results showed that thesensitivity were99.5%,89.7%,92.6%,97.5%respectively, and thespecificity were84.3%,81.4%,84.3%,84.3%respectively.4.20MAT negtive and203MAT positive canine serums were detected byLuminex methord that LipL21, Loa22, LipL32and LigACon4-8recombinant proteins were used as antigens. The Luminex resultsshowed that the sensitivity were89.66%,75.86%，82.27%,78.33%respectively, and the specificity were85.0%，80.0%，90.0%，80.0%respectively.5. Rrecombinant CTB-OmpL1lactobacillus oral vaccine was constructed,and the hamsters model of infected with leptospirosis was established.The oral vaccine can induce to good immune protection for vaccinedhamsters, their survival rate up to87.5%after challenging. Conclusion:LipL21, Loa22, LipL32and LigACon4-8were expressed successfully, andthese high-purity recombinant proteins were produced and it had goodantigenicity. The ELISA detecting leptospirosis antibody in equine andcanine serum was established successfully, and the Luminex methoddetecting leptospirosis antibody in canine serum was establishedsuccessfully. A large number of equine and canine serum samples weretested by these methords, the results showed high sensitivity and specificity.Leptospira recombinant CTB-OmpL1oral vaccine was constructedsuccessfully, and showed a good immune protection for hamster model,which lay a foundation development of new vaccines.