Development of peptide component vaccine against P.gingivalis

针对重组40 kda外膜蛋白（OMP）的多克隆抗体和单克隆抗体（MAb）已被用于研究宿主防御牙龈假单胞菌感染的潜在贡献。通过对存活细菌和3h -胸腺嘧啶结合到新合成的DNA的实验，我们发现当豚鼠补体存在时，这些抗体对牙龈假单胞菌有显著的杀伤活性。此外，我们用标记为^<14> c -乙酸钠的细菌进行放射性杀菌试验，证实了在补体源存在的情况下暴露于单抗的牙龈p.s龈381的细胞裂解。这些数据表明，40kDa OMP将是开发抗牙龈假单胞菌疫苗的有效靶点。接下来，我们从完整的DNA序列中分析40 kda的OMP氨基酸序列。平均选取6个肽段作为肽组分疫苗，合成这些肽段（约20个残基）。抗40 kda OMP的多克隆抗体识别合成的6个肽段中的2个。合成多抗原肽（MAP），免疫兔。抗血清能识别MAP抗原，但不能识别40kda的OMP。因此，我们将肽与二氢叶酸还原酶偶联并再次免疫。抗血清反应40 kda的OMP。研究结果表明，合成的多肽可用于牙周病组份疫苗的研制。

Polyclonal antibody and monoclonal antibodies (MAb) directed against a recombinant 40-kDa outer membrane protein (OMP) have been used to investigate the potential contribution of host defense against infection of P.gingivalis. By the assay for the surviving bacteria and the incorporation of 3H-thymidine into newly synthesized DNA,we found significant killing activity for P.gingivalis by these antibodies when guinea pig complement was present. Additionally, we confirmed the cell lysis of P.gingivalis 381 exposed to the MAb in the presence of complement sources in a radioactive bacericidal assay using bacteria labelled with ^<14>C-sodium acetate. These data suggest that 40kDa OMP will be effective target to develop the vaccine against P.gingivalis. Next we analyzed amino acid sequences of 40-kDa OMP from complete DNA sequences. 6 peptides were selected by mean as peptide component vaccine and synthesized these peptides (about 20 reidues). Polyclonal antibody against 40-kDa OMP recognized 2 among 6 peptides synthesized. Then we synthesized multi-antigen peptides (MAP) and immunized to rabbits. Antisera recogniezed the MAP antigen, however did not recognized 40-kDa OMP.Therefore, we conjugated the peptide to dihydrofolate reductase and immunized again. The antiserem reacted to 40-kDa OMP.In conclusion therefore, synthesized peptide will be usefull to develop the component vaccine against periodontal disease.