鮑氏不動桿菌醣蛋白質 A1S_3744 特性分析以及免疫載體蛋白質 P64KN 對保護性抗原致免疫力的影響

楊宗翰

doi:10.6845/NCHU.2015.01087

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鮑氏不動桿菌醣蛋白質 A1S_3744 特性分析以及免疫載體蛋白質 P64KN 對保護性抗原致免疫力的影響

Characterization of the Acinetobacter baumannii glycoprotein A1S_3744 and effects of the immune protein P64KN on the immunogenicity of the identified protective antigens

楊宗翰 , Masters　　Advisor：楊秋英　　

繁體中文 DOI： 10.6845/NCHU.2015.01087

鮑氏不動桿菌；醣蛋白質；融合蛋白質； Acinetobacter baumannii ； glycoprotein ； fusion protein

Abstract │ Reference (77) │ Times Cited (1) │ Altmetrics

Abstract 〈TOP〉

鮑氏不動桿菌 (Acinetobacter baumannii, AB)是一種革蘭氏陰性菌，環境中廣泛存在，是人類體表上常見的菌種之一。 AB 菌感染患者會產生肺炎、腦膜炎、心內膜炎等症狀甚至死亡。AB菌具有多重抗藥性，導致治療困難，也造成病患的高死亡率，因此，尋找能誘導出具殺菌性抗體的保守性抗原以發展有效的疫苗為一對應策略。本研究以 AB 菌之醣蛋白質 A1S_3744 為目標，利用其重組蛋白質的抗血清分析 A1S_3744 在 AB 菌族群中表現情形，發現此蛋白質在各 AB 菌株間皆有表現。為分析 A1S_3744 在 AB 菌體表現位置，本實驗利用抗血清進行 whole cell ELISA、免疫螢光染色法以及細胞分層試驗，結果顯示 AB 菌外膜有 A1S_3744 表現但無法被重組蛋白質的抗血清辨識。進一步利用 biotin 標記AB菌表面蛋白質並將結果與 A1S_3744 抗血清辨認結果進行比對，證實 A1S_3744 在 AB 菌表面表現甚少。此外，將A1S_3744 抗血清進行殺菌力分析，結果顯示其血清缺乏殺菌或是抑菌能力。綜合以上結果得知A1S_3744 蛋白質在 AB 菌中的表現以在內膜及外膜都有，但是僅有其中極小部份表現在細胞表面。然而，以 E. coli 表現重組 A1S_3744 所產生之抗血清卻具無法有效辨識本身經修飾化的醣蛋白質的可能性，因此為了取得更加準確的結果，直接利用 AB 菌本身抗原直接進行實驗會更加理想。

本研究另一主題是利用實驗室前人研究中具有引發強烈免疫反應、可做為免疫載體的 P64K N 端 146 個胺基酸序列的蛋白質 P64KN1-146 (P64KN) 與實驗室中發現具有保護力的三種抗原 Ncsp、Pase1 及 Pas2 一同構築融合蛋白質以提高此三種蛋白質之免疫性為實驗目的。這三個目標抗原在弗氏佐劑的輔助下能夠誘發小鼠產生對抗 AB 菌能力的免疫反應。然而引發的免疫反應不夠強烈再加上弗氏佐劑帶有毒性，因此將 P64KN 與目標抗原結合並觀察是否可以取代弗氏佐劑以協助三種抗原提高免疫性為本研究第二個目標。將三種融合蛋白質抗血清利用西方墨點法及抗血清結合效力分析，結果顯示除了 P64KN-Ncsp 抗血清，其餘融合蛋白質血清皆無法辨識其對應之抗原。進一步針對 anti-P64KN-Ncsp 進行殺菌力分析，結果顯示此抗血清完全失去原本 anti-Ncsp 所具有的抑菌能力。綜合以上結果顯示，本研究所設計之融合蛋白質可能因為融合蛋白質內構型相互影響導致抗原辨識位改變，進一步造成產生之抗血清皆失去其辨識目標抗原或是抑制 AB 菌生長的能力。

Parallel Abstract 〈TOP〉

Acinetobacter baumannii is a Gram-negative, opportunistic nosocomial pathogen that infects immunocompromised or burn patient and causes infections. Because of its high mortality and multiple antibiotic resistances, A. baumannii has been taken as extremely dangerous pathogens. Base on the fact that the effects of antibiotic have been limited, one of the alternative strategies to take place the antibiotics is to develop effective vaccines. Glycoproteins play an important role in providing physiological function of A. baumannii. At the same time, we found that glycoprotein A1S_3744 got a signal peptide amino acid sequence of lipobox motif that may allow it to set in the membranes. Because A1S_3744 may express on the surface of A. baumannii, which fits the important characteristic of being an effective antigen, we focus on this protein for testing its potential to be a vaccine candidate in this study. First, mice were immunized with purified recombinant protein A1S_3744 (rLS3744) and non-leader sequence A1S_3744 (rNL3744) for producing of the antiserum. By Western blot analysis, we found that A1S_3744 were detected in 100 % of the tested AB isolates suggested that this antigen is highly conserved in AB. To figure out the location of this protein, whole cell ELISA was used in this study. The result showed two possibilities, this protein may not express on the surface of AB or the antiserum of rA1S_3744 is not an effective antiserum. Immunostaining and cell fractionation were used to detect the actual protein location. Results suggested that this protein mainly expressed at the inner membrane of AB, just few of them were found at the outer membrane. Biotinylation were used to labeling the whole surface proteins of AB, we can indirectly proved that there were just a few of A1S_3744 express on the surface. We also tested the bactericidal actives of the antiserum, and it showed no effect on AB. The above results demonstrated that A1S_3744 may not be an effective antigen because of the low surface expression and non-effect on inhibit AB activity.

The other subject of this study is to construct the fusion protein between immune-carrier P64KN and the other protective antigen Ncsp, Pase1 and Pase2 with restriction enzyme EcoRI splicing sequence trying to enhance the immunogenicity of these three antigens. For testing the different of immunogenicity between fusion protein and their target antigens, Western blot analysis and antiserum titration test were used in the study. Results show that only P64KN-Ncsp antiserum can recognize rNcsp and showed the same antiserum efficiency of Ncsp antiserum. During the bactericidal assay, we found that the antiserum of P64KN-Ncsp lost its ability to inhibit the activity of AB. The above results demonstrate that those fusion proteins may lost their original structure and change their epitopes cause of the ineffective linker design. Using the linkers that can truly separate each parts of the fusion protein might be a better way to construct P64KN related fusion proteins.

Reference ( 77 ) 〈TOP〉

1. Peleg AY, Seifert H, Paterson DL (2008) Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 21: 538-582.
連結：
2. Howard A, O'Donoghue M, Feeney A, Sleator RD (2012) Acinetobacter baumannii: an emerging opportunistic pathogen. Virulence 3: 243-250.
連結：
3. Dell A, Galadari A, Sastre F, Hitchen P (2010) Similarities and differences in the glycosylation mechanisms in prokaryotes and eukaryotes. Int J Microbiol 2010: 148178.
連結：
4. Iwashkiw JA, Seper A, Weber BS, Scott NE, Vinogradov E, et al. (2012) Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation. PLoS Pathog 8: e1002758.
連結：
5. Nothaft H, Szymanski CM (2010) Protein glycosylation in bacteria: sweeter than ever. Nat Rev Microbiol 8: 765-778.
連結：

Times Cited (1) 〈TOP〉

李鴻毅(2016)。腦膜炎雙球菌蛋白質 P64K 及 Ag473 對鮑氏不動桿菌抗原免疫性之影響。中興大學分子生物學研究所學位論文。2016。1-71。

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