| Listeria monocytogenes (LM)is a gram-positive, facultative intracellular bacterial pathogen causing meningitis, miscarriage and septicemia of both humans and animals. P60 is a important antigen confering protective immunity for LM. Inactivation of P60 is thought of the key reason resulting in decrease of protection of LM inactivated vaccine.In order to enchance immune potential of LM inactivated vaccine, two experiments were performed in present study.1 Cloning and expression of the gene of protein P60, a invasion-related protein from L. monocytogenes and its purification.Iap gene encoding P60 was amplified by PCR and constructed into prokaryotic expressing vector pET-30a, then transformed into E.coli BL21(DE3). SDS-PAGE analysis showed that the expressed fusion-protein named as His-P60 (P60) was 46 ku and expressed in the form of inclusion body. Western blot showed that the P60 could be recognized by antiserum against whole cell bacteria of L. monocytogenes. P60 was then purified by His affinity chromatography to a concentration as 3.5 mg/mL.2 Determination of enhancing effects of P60 from L monocytogenes on protective potential of inactivated vaccine.The vaccine was developed by mixing P60 (1 mg/mL) with LM inactivated bacteria, adjuvanted with Montanide ISA206. Then BALB/c mice were vaccinated (0.2 mL). Subsequently antibody titers, lymphocytes stimulating index, T lymphocyte subpopulation percentage as well as the level of IL-4 and IFN-γwere tested by ELASA, MTT, Flow Cytometry and ELISA respectively. Mice were then injected with 2×106 (medial lethal dose) L. monocytogenes at day 21st after the second vaccination. Reproduction rate of the bacteria in spleen of mice were detected 48 hours after chanllege. 10 mice chosen randomly and recorded the death time and number of mice in two weeks. Meanwhile, P60 subunit vaccine (1 mg/mL), LM inactivated vaccine (1010 CFU/mL) and PBS vaccine were all used as control. The results showed:①At 7 days after the first vaccination, Antibody titer of P60-LM group was obviously higher than LM, P60 and PBS groups (p < 0.05); at 14 days, the difference was significant (p < 0.01).②21 days after the second vaccination, lymphocytes stimulating index of P60-LM group (1.975±0.024) was significantly higher than LM (1.539±0.032) and PBS (1.046±0.027) groups, with an increasing rate of 26.11% and 8.45% (p < 0.05), while there was no obvious difference between P60-LM inactivated bacteria and P60 group (p > 0.05).③percentages of CD3+ and CD4+ lymphocytes were significantly higher than those of the other three groups while CD8+ lower than others (p < 0.05). Among them, CD4+ was increased by 9.72%, 11.36% and 38.64% respectively while CD8+ was decreased by 60.46%, 22.89% and 18.67%, compared to the PBS, LM and P60 groups.④7 days after the first vaccination, the level of IL-4 was decreased except PBS group (p < 0.05), and IFN-γhad no obvious difference among groups except PBS group (p > 0.05). While the ratio of IFN-γ/ IL-4 of P60-LM and P60 groups were increased and there were obvious differences compared to other groups (p < 0.05).⑤when challenged with live LM bacteria, only P60-LM group showed no death. At day 14th after challenge, survival rate was 6/10 for group P60, 4/10 for LM, 0 for group PBS and 9/10 for group P60-LM. At 48 h after challenge, average number of LM (CFU/g) recovered from spleen of mice was 1.24*108 in PBS group, significantly higher than other groups (p<0.01). It was 118.33 times over group P60 (1.02*105), 1392.16 over group LM (1.2*106) and 194254.45 over group P60-LM (73.1).Boosting potential of P60 using P60-LLO-LM vaccine was also determined by same experiments as in BALB/c mice. Similar results were obtained.Conclusion: Present sdudy showed that adding P60 P60inactivated vaccine can, improve the activation and multiplication of lymphocytes in spleen of mouse; can stimulate T cells to differentiate toward CD4+ Th-type; induce Th cells to differentiate toward Th1-type; generally enhance both cellular and humoral immunity; and increase protective potential of LM inactivated vaccine. |
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