Cloning and expression of meningococcal antigens and preparation of the antibodies

腦膜炎雙球菌（Neisseria meningitidis, NM）是一種帶有莢膜多醣的革蘭氏陰性菌，是造成孩童細菌性腦膜炎的主要病因。人類的鼻腔是NM唯一的天然棲息地。若棲息於鼻腔黏膜上皮細胞的NM 穿過上皮組織進入循環的血液、再穿過血腦屏障到達腦脊液則會引發腦膜炎。為了適應不同的微環境以及逃避免疫系統的辨識，大部分的NM表面抗原在感染過程中會有時期或抗原性變異(phase or antigenic variation)，因此瞭解這些變化對於解開NM致病機制以及疫苗研發將有莫大助益。在監測抗原的表現上，抗體是一個很有用的試劑。本論文研究以大腸桿菌生產五種的重組NM蛋白質(包括RplJ、 Opc、Opa、H.8以及Laz)，並以其免疫小鼠。此五種蛋白中Opc只表現在某些菌株。為了評估得到的抗血清的特異性，以各抗血清為探針對七株不同的NM菌進行Western blotting。結果在7株菌株都可偵測到RplJ、 Opa、H.8以及Laz，但只有一株菌表現Opc，證明抗血清對變性後的NM表現目標蛋白質具有特異性。這些抗血清是否適用於其他免疫學方法則有待證實。Neisseria meningitidis (NM), a capsulated Gram-negative bacterium, is the leading cause of bacterial meningitis in children. The sole natural habitat and primary reservoir for NM is the human nasopharynx. After colonization on the epithelial cells lining the mucosal surfaces of the nasopharynx, NM needs to cross the epithelium, gaining entry into the bloodstream, and crossing blood-brain barrier to the cerebrospinal fluid to cause meningitis. Most NM surface antigens exhibit phase or/and antigenic variations to adapt different microenvironments and to escape from the immune recognition. Therefore, it is important to understanding the expression profiles of the surface antigens during infection, which should comprehend our understanding of meningococcal pathogenesis and consequently help the vaccine design. Antibody is a useful reagent to monitor the expression of a specific antigen. In this thesis study, the recombinant meningococcal RplJ, Opc, Opa, H.8 antigen, and Laz proteins were produced in E. coli and used to immunize mice. Among the five proteins, it is known that Opc is only expressed in certain strains. To evaluate the specificities of the resultant antisera, bacterial lysates of seven NM strains were subjected to Western blotting. In contrast to the RplJ, Opa, H.8 antigen, and Laz, which were detected in all seven strains, Opc was only detected in one strain confirming the specificities of the antibodies towards the denatured proteins. Whether the antisera can be used in other immunological methods remain to be determined.中文摘要………………………………………………………………………………3 英文摘要…………………………………………………………….……...…………4 前言…………………………………………………………...……….………………5 實驗方法……………………………………………………………….…………… 10 結果與討論…………………….……………………………………….……………23 結論……….…………………………………………………………….……………33 圖表 …………………………………………………………………………………34 參考文獻 ……………………………………………………………………………6