A search for a synthesized vaccine candidate intended for periodontal diseases ; on the basis of the conformation dependent characteristics of the dominant epitope of periodontal pathogen, Porphyromonas gingivalis.

牙龈卟啉单胞菌是一种重要的牙周病原菌，其菌毛介导了该菌与宿主组织的黏附。这项研究的目的是验证产生一种化学合成的疫苗的可能性，该疫苗诱导能够抑制牙龈假单胞菌附着宿主组织的能力的抗体。首先，建立了一组抗牙龈假单胞菌菌毛的单抗。这些单抗都能识别菌毛的高阶结构，但不能识别一级或二级结构。接下来，构建了噬菌体展示多肽文库，该文库将18个随机多肽插入到M13噬菌体的GP3基因中。根据与其中一种单抗的反应性进行筛选，鉴定出一个可能与牙龈假单胞菌菌毛的高级结构相似的序列。大量制备和纯化该噬菌体克隆，并用该噬菌体颗粒免疫小鼠。免疫小鼠…更多的人产生针对插入序列和天然牙龈假单胞菌菌毛的血清抗体。为了稳定所选择的多肽的构象表位，通过基因工程使其在噬菌体上环化。由于不可能化学合成该多肽，因此删除或替换了序列中一些被认为对抗原性不重要的氨基酸残基，并化学合成了一个可溶的环肽。然而，这些尝试并没有提高多肽的抗原性。据报道，在噬菌体上表达的几个具有功能的多肽序列在制备成多肽时不起作用，也不可能表达为可溶性多肽，这是有报道的。建立了大肠杆菌麦芽糖结合蛋白(MBP)的多肽融合表达系统。据报道，该系统有效地改善了上述问题。为此，我们克隆了MBP基因，并构建了表达载体。18mer线状肽和11mer环肽在MBP和18mer上成功表达，但11mer肽不能与所用单抗反应。较少

Porphyromonas gingivalis is an important periodontal pathogen, and its fimbriae mediate the adherence of this bacterium to the host tissues. The aim of this study was to verify the possibility to generate a chemically synthesized vaccine which induces antibodies capable of inhibiting the ability of P. gingivalis to adhere host tissues. First, a panel of monoclonal antibodies (mAb) reacting to P. gingivalis fimbriae were established. All these mAb recognized higher order structure of fimbriae but not the primary or secondary structures. Next, a phage-displayed peptide library was constructed which contained an insertion of 18mer random peptide into the gp3 gene of M13 bacteriophage. Screening was performed according to the reactivity with one of the mAb, and a sequence probably mimicking the higher order structure of P. gingivalis fimbriae was identified. This phage clone was prepared and purified in a large quantity, and mice were immunized with the phage particles. The immunized mice … More produced serum antibodies specific for the inserted sequence and for native P. gingivalis fimbriae. To stabilize the conformational epitope of the selected peptide, the peptide was made cyclic on the phage by genetic engineering. As it was impossible to chemically synthesize the peptide, some amino acid residues in the sequence which were thought to be not important for the antigenicity were deleted or replaced, and a soluble cyclic peptide was chemically synthesized. However, these attempts did not improve the antigenicity of the peptide. It has been reported the similar cases that several peptide sequences expressed on bacteriophages and selected for the functionality do not function when they are prepared as peptides themselves, nor the sequences are impossible to express as soluble peptides. An expression system as a peptide-fusion with Escherichia coli maltose binding protein (MBP) was established. This system has been reported effective in improving the above mentioned problem. The MBP gene was cloned for this purpose, and an expression vector was newly constructed. The 18mer linear peptide and 11mer cyclic peptide was successfully expressed on MBP and 18mer but not 11mer peptide reacted with the mAb used. Less