Development of peptide component vaccine against P.gingivalis

针对重组40 kDa外膜蛋白(OMP)的多克隆抗体和单抗(MAb)已被用来研究宿主对牙龈假单胞菌感染的潜在防御作用。在豚鼠补体存在的情况下，通过对存活细菌的检测和~3H-胸腺嘧啶核苷掺入新合成的DNA的实验，我们发现这些抗体对牙龈假单胞菌有显著的杀灭活性。此外，我们在放射性杀菌试验中证实了Pggivalis 381在补体来源存在的情况下暴露于MAb中的细胞裂解作用，该试验使用的是用^&lt；14&gt；C-乙酸钠标记的细菌。这些数据表明，40 kDa OMP将成为研制牙龈假单胞菌疫苗的有效靶点。接下来，我们从完整的DNA序列中分析了40 kDa OMP的氨基酸序列。用Mean法筛选出6个多肽作为多肽成分疫苗，合成了约20个氨基酸的多肽。抗40 kDa OMP多克隆抗体在合成的6个多肽中识别2个。然后合成多抗原肽(MAP)，免疫兔。抗血清可识别MAP抗原，但不能识别40 kDa的OMP，因此我们将其与二氢叶酸还原酶偶联并再次免疫。该抗血清可与40 kDa的OMP发生反应，为研制牙周病复合疫苗奠定了基础。

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.