克雷白氏肺炎桿菌所表現的第三型線毛在細菌進行感染時扮演黏附宿主細胞的重要角色，同時也是引起宿主體內強大免疫反應的結構之ㄧ。目前已知此多分子聚合而成的胞器，主要經由chaperone-usher pathway組裝而成。為了找出線毛單元體上參與線毛組裝堆疊的重要胺基酸，我們於MrkA單元體上不同位置進行定點突變，並測試其組裝成線毛的能力。結果顯示位於MrkA中段的Ile-82與
Cys-87，可能分別參與了MrkA單元體之間的交互作用以及維持MrkA結構之雙硫鍵的形成，因此在線毛堆疊上扮演了非常重要的角色。另外在MrkA的N端與C端也發現了六個胺基酸, Val-32, Phe-34, Asp-40, Val-45, Gly-189及Tyr-201, 在置換成丙胺酸後破壞了第三型線毛原有的功能，因此在線毛結構的維持上亦具有一定的重要性。本研究的另一個目的為建構第三型線毛作為疫苗呈現的系統。實驗室之前建構了4株插入突變株，分別於MrkA的不同位置插入含有18個胺基酸的豬內源反轉錄病毒(PERV)外套膜抗原決定位。我們確認其線毛的表現與抗原決定位的呈現效率之後，將其中一株突變株D27-E的線毛純化並進行抗原性測試。最後證實了此重組線毛可成功地於老鼠體內產生針對PERV外套膜蛋白的抗體。本研究顯示了第三型線毛呈現抗原決定位的潛力，在未來的疫苗製作上具有很大的發展性。

Type 3 fimbriae produced by Klebsiella pneumoniae are required for initiating infection by attaching to host tissues and responsible to the strong immunogenicity of bacteria. This macromolecular organelle is assembled via the chaperone-usher pathway. The first aim of this study is to identify residues in the major pilin MrkA important for pilus assembly. Site-directed mutagenesis was performed on MrkA and whether the MrkA of these mutants can be assembled into fimbriae were evaluated. The data showed that two amino acids at the middle region of MrkA, Ile-82 and Cys-87, play critical roles in pilus assembly. Six amino acids in the N- and C- terminal regions of MrkA, including Val-32, Phe-34, Asp-40, Val-45, Gly-189 and Tyr-201, disrupt normal pilus functions and may relate to maintaining stability of pilus structures. The second aim of this study is to construct a vaccine displaying fimbriae. Previously, our lab had created 4 insertion mutants containing 18-amino-acid porcine endogenous retrovirus (PERV) envelop epitopes inserted in 4 different positions of MrkA respectively. After confirming the fimbriation and epitope display efficiencies of these mutants, the fimbriae of one mutant strain, D27-E, was purified. Immunization of mice with the purified recombinant fimbriae successfully elicited antibodies that specifically recognized PERV envelope protein, revealing the potential of type 3 fimbriae as vaccine display systems.

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