Cloning And Expression Of Leptospira Outer Membrane Lipoprotein Loa22 And Study On Immunoprotectivity

Objectives:(1) To construct prokaryotic expression plasmid pQE31–Loa22 containing the outer membrane lipoprotein Loa22 gene of Leptospira and express target protein.(2) To observe humoral or cellar immune responses in guinea-pigs induced by the Loa22 protein injected intramuscularly.(3) To study the immune protective effects of the Loa22 protein.Methods:The Loa22 gene was amplified by PCR from the genome of L.interrogans, strain 56601. The fragment of Loa22 gene was directly ligated into prokaryotic expression vector pQE31 to construct pQE31-Loa22. The recombinant plasmid pQE31-Loa22 was identified by DNA sequencing and restriction enzyme analysis and transformed into E.coliM15 to be expressed. The Loa22 protein was confirmed by SDS-PAGE and Western-blot. Guinea-pigs were immunized with Loa22 protein or PBS buffer intramuscularly at 2 week interval for three times. Two weeks after the last time of immunization, the guinea-pigs were challenged with L.interrogans, strain 56601. ELISA was used to detect the titer of the specific antibody in the sera of immuned guinea-pigs. The proliferation response of spleen cells was detected by MTT assay, and the stimulation index(SI) was counted. Cross-immuneresponse of L.interrogans, strain 56607, 56603 and 56609 with anti-Loa22 protein serum was tested by ELISA. Blocking of Leptospira adhering to cell by anti-Loa22 protein serum was observed by Fontana.Results:The Loa22 gene was successfully amplified and subcloned into prokaryotic expression vector pQE31. DNA sequencing and restriction enzyme analysis and showed that the prokaryotic expression recombinant pQE31-Loa22 was successfully constructed. Loa22 protein with molecular weight about 22KD was detected by SDS-PAGE or Western-blot in M15 transformed with pQE31-Loa22. The significant specific antibody titers were detected by ELISA. Two weeks after the last time of immunization, the highest titer was 1:8000. The proliferation response of spleen cells of guinea-pigs immuned with Loa22 protein (SI:3.25±0.37) were significantly higher than those of guinea-pigs immuned with PBS(SI:1.38±0.13)(P<0.01). The guinea-pigs immuned with Loa22 protein changed with L.interrogans, strain 56601 weren't infected, while the guinea-pigs immuned with PBS changed with L.interrogans, strain 56601 were infected. A high immune response of L.interrogans strain 56607, 56603and 56609 with anti-Loa22 protein serum was tested by ELISA. The titer was1/409600, 1/819200and 1/3376800 respectively. The rate of L.interrogans serovars lai strain 56601 adherence blocking to Raw184.7 was 90%~50%, as the anti-Loa22 protein serum was diluted by 1:50~1:400.Conclusions:(1) The Loa22 gene fragment was successfully amplified. The prokaryotic expression recombinant pQE31-Loa22 was successfully constructed, and Loa22 protein was successfully expressed.(2) Strong humoral and cellar immunity can be induced by Loa22 protein in guinea-pigs.(3) Specific immune response induced by the Loa22 protein could protect guinea-pigs from homogenous leptospira infection.(4) A high cross-immune response of various serotypic leptospira with anti-Loa22 protein serum was detected.(5) Anti- Loa22 protein serum could block Leptospira adherence.