靶向融合防龋DNA疫苗经肌肉免疫动物的实验研究

目的　观察靶向融合防龋DNA疫苗pGJA P在大鼠肌肉组织中的原位表达。检测靶向融合防龋DNA疫苗pGJA P经股四头肌注射途径免疫大鼠后体内抗体水平和防龋效果 ,并与融合防龋DNA疫苗pGLUA P进行比较。在小鼠体内长期观察抗体动态变化。方法　质粒pGJA P经股四头肌注射途径免疫大鼠 ,免疫组织化学方法检测重组蛋白的原位表达。质粒pGJA P和pGLUA P经股四头肌注射途径免疫定菌鼠 ,ELISA法检测血清IgG和唾液IgA抗体水平 ,Keyes记分法评估定菌鼠磨牙患龋情况。质粒pGJA P和pGLUA P经股四头肌注射途径免疫BALB/c小鼠 ,ELISA法检测血清IgG和唾液IgA动态变化。结果　大鼠股四头肌组织检测到表达的重组蛋白。大鼠pGJA P免疫组血清抗PAcIgG水平 (1∶2 0 0 0 0 0 )和抗GTFIgG水平 (1∶5 80 0 0 )均显著高于pGLUA P免疫组 (1∶2 30 0 0和 1∶110 0 0 )(均P <0 0 1)。大鼠pGJA P免疫组唾液抗PAcIgA水平 (1∶8)和抗GTFIgA水平 (1∶6 )均显著高于pGLUA P免疫组 (1∶2和 1∶2 ) (均P <0 0 1)。pGLUA P免疫组未能引起有效的唾液IgA反应。pGJA P免疫组龋齿记分显著低于pGLUA P免疫组和空白组 (P <0 0 1)。小鼠pGJA P免疫组有效血清IgG、唾液IgA和pGLUA P免疫组有效血清IgG均持续至本实验结束 ,pGJA P免疫组血清IgG和唾液Ig

Immunization with targeted fusion anticaries DNA vaccine via intramuscular route:experiment with murine

OBJECTIVES: To observe the expression of a targeted fusion anticaries DNA vaccine pGJA-P in muscular in vivo. To compare the levels of specific antibodies and anticaries efficacy generated by pGJA-P and fusion anticaries DNA vaccine pGLUA-P in gnotobiotic rats, and observe the kinetics of antibody responses in BALB/c mice. METHODS: (1) Twelve 28-day-old female Wistar rats were randomly divided into 2 groups of 6 rats to be injected with the plasmid pGJA-P containing the signal peptide and extracellular regions of human CTLA(4), hinge and Fc regions of human IgG, the glu sequence of gtfB gene and A-P fragment of pac gene of Streptococcus mutans or the eukaryotic expression plasmid pCI into the quadriceps muscle of thigh respectively. Three days after the rats were killed and specimens of quadriceps muscles of thigh were taken. Immunohistochemical SP staining was used to examine the in situ expression of pGJA-P. (2) Twenty-four 18-day-old female Wistar rats were randomly divided into 4 groups of 6 rats. The rats were fed with cariogenic food. During the age of 20 - 22 days cariogentic food containing broad-spectrum antibiotics was fed. Then aseptic cotton stick was used to swab the oral cavity and be smeared onto the solid medium so as to observe the growth of bacteria under anaerobic culture for 48 hours. During the age of 24 - 26 days, S. mutans Ingbritt cultured anaerobically was swab onto the surface of teeth of the rats twice with an interval of 30 minutes. After the inoculation aseptic cotton stick was used to wipe the oral cavity and be smeared onto the solid medium so as to observe the growth of bacteria under anaerobic culture for 48 hours. When the gnotobiotic rats were 28 days old they were injected with pGJA-P, pGLUA-P, fusion anticaries DNA vaccine against both PAc, cell surface protein antigen, and glucosyltransferase (GTF), pCI or normal saline into the quadriceps muscle of thigh respectively, 2 weeks later a booster shot was given. When the rats were 63 days old their saliva and blood samples were collected. The serum IgG and salivary IgA were assayed by using ELISA. The gnotobiotic rats were killed and their maxillary bone the mandibles were isolated. The anticaries effect was evaluated by Keyes caries scores. (3) Twenty-four 4-week-old BALB/c mice were randomly divided into 4 groups of 6 mice: to be injected with pGJA-P, pGLUA-P, pCI, or normal saline respectively into the quadriceps muscles of thigh, 2 weeks later a booster shot was given. Before the injection and every 2 weeks after the immunization specimens of saliva and blood were collected. The serum IgG and salivary IgA were assayed by using ELISA. RESULTS: (1) Recombinant protein could be detected in the quadriceps muscles of the rats immunized with pGJA-P, but not in the muscles of the rats immunized with pCI. (2) The levels of serum anti-PAc IgG (1:200 000) and anti-GTF IgG (1:58 000) of the rats immunized with pGJA-P were significantly higher than those of the rats immunized with pGLUA-P (1:23 000 and 1:11 000 respectively) (both P < 0.01). The levels of salivary anti-PAc IgA (1:8) and anti-GTF IgA (1:6) of the rats immunized with pGJA-P were significantly higher than those of the rats immunized with pGLUA-P (1:2 and 1:2 respectively) (both P < 0.01). The Keyes scores of the pGJA-P group were significantly lower than those of the pGLUA-P group and the control groups (all P < 0.01). The effective serum IgG and salivary IgA in the pGJA-P group and effective serum IgG in the pGLUA-P group all persisted to the end of the experiment. (3) Two weeks after the initial immunization the serum anti-PAc IgG level of the mice immunized with pGJA-P increased remarkably, 4 times that of the mice immunized with pGLUA-P, and 33 times those of the mice injected with pCI or normal saline. Two weeks after the booster immunization, the serum anti-PAc IgG level of the mice immunized with pGJA-P was 14 times that of the mice immunized with pGLUA-P, and 117 times those of the mice injected with pCI or normal saline. The serum anti-PAc IgG immunized with pGJA-P reached its peak 10 weeks after the initial immunization, 4 times that of the mice immunized with pGLUA-P, and 160 times those of the mice injected with pCI or normal saline. The serum anti-PAc IgG of the mice immunized with pGLUA-P reached its peak at 16 weeks, however, significantly lower than the peak of the mice immunized with pGJA-P (P < 0.01). The serum anti-Pac IgG levels of the mice injected with pCI or with normal saline were not significantly different (P > 0.05). Since the second week after the initial immunization, significant difference in the serum anti-PAc IgG level could be seen between the mice immunized with pGJA-P or the mice immunized with pGLUA-P, and between the mice immunized with pGJA-P and the mice immunized with pGLUA-P and those injected with pCI or normal saline (all P < 0.01). Six weeks after the initial immunization the salivary anti-PAc IgA level of the mice immunized with pGJA-P was 18 times those of the mice injected with pCI or with normal saline (both P < 0.01), 10 weeks after the salivary anti-PAc IgA level of the mice immunized with pGJA-P reached its peak, 24 times those of the mice immunized with pCI or normal saline without a significant difference between the latter 2 groups (P > 0.05). No effective salivary IgA response was seen in the mice immunized with pGLUA-P. CONCLUSION: pGJA-P can be expressed in vivo. Immunization with pGJA-P intramuscularly induces effective mucosal and systematic humoral responses. It is an effective DNA vaccine against dental caries.