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研究生:曾怡嘉
研究生(外文):Yi-Chia Tseng
論文名稱:淋病雙球菌假設性蛋白質之免疫特性分析
論文名稱(外文):Immunological characterization of Neisseria gonorrhoeae hypothetical proteins
指導教授:楊秋英
指導教授(外文):Chiou-Ying Yang
口試委員:蕭樑基林琦然
口試委員(外文):Leung-Kei SiuChi-Jan Lin
口試日期:2021-10-12
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2022
畢業學年度:110
語文別:中文
論文頁數:69
中文關鍵詞:淋病雙球菌假設性蛋白質
外文關鍵詞:Neisseria gonorrhoeaehypothetical protein
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淋病 (gonorrhea) 是由淋病雙球菌 (Neisseria gonorrhoeae, Ng) 感染所導致之疾病,主要藉由性行為途徑感染,感染後多數患者無明顯癥兆並且可能經由垂直傳染給下一代。除此之外,康復患者體內不具有免疫記憶性,早期只要使用抗生素治療就能達到有效療效,但 Ng 菌多重抗藥性的問題逐漸增加,使得治療變得棘手,開發有效疫苗是目前最佳解決方式。本研究以反向疫苗學 (Reverse vaccinology) 方法作為基礎,從全序列定序完成的參考菌株 FA 1090 的假設性蛋白質 (hypothetical protein) 中挑選標的,首先藉由電腦軟體 SignalP-5.0 Server 預測蛋白質是否為分泌性蛋白質,再以 Gneg-mPLoc 預測蛋白質在菌體位置並以 Blast 比對,選擇高保守性的目標製備重組蛋白質以進行免疫原性 (immunogenicity) 分析。本研究以台灣分離的臨床菌株為模板共分析14個基因,其中有8個基因透過 PCR 成功被增幅出,分別為 NGO1889、NGO1484、NGO0783、NGO2002、NGO2133、NGO0782、NGO0666和NGO2054 。將上述8個基因分別構築至表現載體,送入 E. coli 表現並純化重組蛋白質,純化後的蛋白質進行小鼠免疫試驗,以獲得之抗血清進行西方墨點分析各蛋白質在 Ng 菌的表現情形,證實有5個蛋白質之抗血清具有認回原菌能力且在不同的臨床菌株都有表現。Whole- cell ELISA與免疫螢光染色法分析結果顯示只有 NGO1889、NGO1484、NGO0783和NGO2002 暴露於菌體表面,繼而分析 這些抗血清的殺菌力,結果只有 NGO0783 抗血清對不同臨床菌株具有殺菌活性。進一步進行in vivo 小鼠保護力分析,證實事先接種NGO0783 可增加小鼠對 Ng 菌感染的抵抗力,因此 NGO0783 具有成為開發Ng疫苗之潛力抗原。
Gonorrhea is a sexually transmitted disease caused by infection with the Neisseria gonorrhoeae (Ng) bacterium. Most patients are asymptomatic or signs and may be transmitted to the next generation through vertical transmission. In addition, recovery patients have no immunological memory in their body, and antibiotic treatment is effective for early stage of infection. However, antibiotic resistance is representing a serious problem for treatment. Vaccine development seem to be the best way for treatment. Based on the reverse vaccinology, we select hypothetical protein from the reference strain FA 1090, complete sequence sequencing. First, use SignalP-5.0 Server to predict whether the protein is a secreted protein, and then use Gneg-mPLoc to predict the location of the protein in the bacterial cell. Select highly conserved targets to prepare recombinant proteins for immunogenicity. In this study, the 14 genes template are from Taiwan clinical strains, which 8 genes are successfully amplified by PCR, named NGO1889, NGO1484, NGO0783, NGO2002, NGO2133, NGO0782, NGO0666 and NGO2054. The 8 genes are constructed into expression vectors. Escherichia coli is used expression host for the production of recombinant proteins. After immunogenicity test, the expression of each protein in Ng was analyzed by western blotting with anti-serum, the result showed that only five protein’s antibodies has the ability to recognize the original bacteria. The results of Whole cell ELISA and immunofluorescence staining test showed that only NGO1889, NGO1484, NGO0783 and NGO2002 were surface-exposed protein. Further, we tested serum bactericidal activity against different Ng strains and it showed only NGO0783 anti-serum effect on Ng. Final, we choose NGO0783 test protection assay and then NGO0783’s anti-serum effect protection mouse prevent infection. To sum up, this study showed that NGO0783 may be a potential vaccine antigen.
摘要 i
Abstract ii
目錄 iii
表目錄 v
圖目錄 vi
縮寫字對照表 (Abbreviation) viii
壹、 前言 1
一、 緒論 1
二、 奈瑟氏淋病雙球菌 1
(一) 奈瑟氏淋病雙球菌歷史 1
(二) 奈瑟氏淋病雙球菌流行病學 1
(三) 奈瑟氏淋病雙球菌之抗藥性 2
三、 奈瑟氏淋病雙球菌之疫苗研發 3
(一) Ng 菌疫苗先前發展 3
(二) Ng疫苗研發近況 4
四、 研究目的 5
貳、 材料與方法 6
一、 材料來源 6
(一) 菌株培養條件 6
(二) 動物實驗 6
二、 實驗方法 6
(一) 基因構築 6
(二) 重組蛋白質之誘導表現與純化 9
(三) 蛋白濃度定量 10
(四) 蛋白質膠體電泳分析 (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 10
(五) 免疫小鼠實驗 12
(六) 酵素結合免疫吸附法 (Whole cell enzyme-linked immunosorbent assay, ELISA) 12
(七) 血清殺菌力分析 (Serum bactericidal assay) 13
(八) 免疫螢光染色法 (Immunostaining assay) 14
(九) 致死劑量 (Lethal dose assay) 14
(十) 保護力分析 (protection assay) 15
參、 結果 16
一、 目標抗原之選擇 16
二、 重組蛋白質之製備 16
(一) 基因選殖與表現載體的構築 16
(二) 重組蛋白質表現與純化 17
三、 目標蛋白質在 Ng 菌之表現情形 18
(一) 目標蛋白質在不同 Ng 分離菌株中廣泛表現 18
(二) 以Whole Cell ELISA 及 immunostain 偵測目標蛋白質在 Ng 菌表面蛋白質表現 19
四、 In vitro 體外試驗-抗血清殺菌力分析 (Serum bactericidal assay; SBA) 20
五、 In vivo 主動保護力分析 21
肆、 討論 23
伍、 參考文獻 28
陸、 圖表 34
柒、 附錄 61
1.Oriel JD. The scars of Venus: a history of venereology. Transactions of The Royal Society of Tropical Medicine and Hygiene, Volume 89, Issue 2, 240. (1995).

2.Mahoney JF, Ferguson C, Buchholtz M & Van Slyke CI. The use of penicillin sodium in the treatment of sulfonamide resistant gonorrhea in men. A preliminary report. Am J Syphilis, 27, 525–8. (1943).

3.Unemo M, Seifert HS, Hook EW 3rd, Hawkes S, Ndowa F, Dillon JR. Gonorrhoea. Nat Rev Dis Primers, 21, 5(1):79. (2019).

4.Workowski, K.A. Centers for Disease Control and Prevention Sexually Transmitted Diseases Treatment Guidelines. Clin Infect Dis, 61 Suppl 8, S759-762. (2015).

5.Fox, K.K., Thomas, J.C., Weiner, D.H., Davis, R.H., Sparling, P.F., & Cohen, M.S. Longitudinal evaluation of serovar-specific immunity to Neisseria gonorrhoeae. Am J Epidemiol, 149, 353-358. (1999).

6.Jerse, A.E., Bash, M.C., & Russell, M.W. Vaccines against gonorrhea: current status and future challenges. Vaccine, 32, 1579-1587. (2014).

7.Quillin, S.J., & Seifert, H.S. Neisseria gonorrhoeae host adaptation and pathogenesis. Nat Rev Microbiol, 16, 226-240. (2018).

8.Rice, P.A., Shafer, W.M., Ram, S., & Jerse, A.E. Neisseria gonorrhoeae: Drug Resistance, Mouse Models, and Vaccine Development. Annu Rev Microbiol, 71, 665-686. (2017).

9.Cornelisse, V.J., Zhang, L., Law, M., Chen, M.Y., Bradshaw, C.S., Bellhouse, C., Fairley, C.K., & Chow, E.P.F. Concordance of gonorrhoea of the rectum, pharynx and urethra in same-sex male partnerships attending a sexual health service in Melbourne, Australia. BMC Infect Dis, 18, 95. (2018).

10.Hook EW & Hansfield HH. Gonoccoal Infection in the Adult. In: Holmes KK, editor. Sexually transmitted diseases. New York: McGraw-Hill, 627–45. (2008).

11.Palafox SKV, Jasper S, Tauber, Allyson D & Foster SC. Ophthalmia neonatorum. J Clinic Experiment Ophthalmol, 2:119. (2011).

12.Asgeirsson, G., & Wientzen, R.L. Epidemiology and pathophysiology of Neisseria gonorrhoeae infection. Semin Adolesc Med, 2, 99-105. (1986).

13.Mlisana K., et al. Symptomatic vaginal discharge is a poor predictor of sexually transmitted infections and genital tract inflammation in high-risk women in South Africa. J Infect Dis, 206(1):6-14. (2012).

14.Cohen, M.S., Hoffman, I.F., Royce, R.A., Kazembe, P., Dyer, J.R., Daly, C.C., Zimba, D., Vernazza, P.L., Maida, M., Fiscus, S.A., et al. Reduction of concentration of HIV-1 in semen after treatment of urethritis: implications for prevention of sexual transmission of HIV-1. AIDSCAP Malawi Research Group. Lancet, 349, 1868-1873. (1997).

15.Kelley CF, Haaland RE, Patel P., et al. HIV-1 RNA rectal shedding is reduced in men with low plasma HIV-1 RNA viral loads and is not enhanced by sexually transmitted bacterial infections of the rectum. J Infect Dis, 204(5):761–7. (2011).

16.Bernstein KT, Marcus JL, Nieri G, Philip SS & Klausner JD. Rectal gonorrhea and chlamydia reinfection is associated with increased risk of HIV seroconversion. J Acquir Immune Defic Syndr, 53(4):537–43. (2010).

17.Torrone, E.A., et al. Prevalence of sexually transmitted infections and bacterial vaginosis among women in sub-Saharan Africa: An individual participant data meta-analysis of 18 HIV prevention studies. PLoS Med, 15, e1002511. (2018).

18.Lovett, A., & Duncan, J.A. Human Immune Responses and the Natural History of Neisseria gonorrhoeae Infection. Front Immunol, 9, 3187. (2018).

19.Jerse, A.E. Experimental gonococcal genital tract infection and opacity protein expression in estradiol-treated mice. Infect Immun, 67, 5699-5708. (1999).

20.Song, W. et al. Local and humoral immune responses against primary and repeat Neisseria gonorrhoeae genital tract infections of 17 beta-estradiol-treated mice. Vaccine, 26, 5741-5751. (2008).

21.Feinen, B., Jerse, A.E., Gaffen, S.L., & Russell, M.W. Critical role of Th17 responses in a murine model of Neisseria gonorrhoeae genital infection. Mucosal Immunol, 3, 312-321. (2010).

22.Liu, Y., Islam, E.A., Jarvis, G.A., Gray-Owen, S.D., & Russell, M.W. Neisseria gonorrhoeae selectively suppresses the development of Th1 and Th2 cells, and enhances Th17 cell responses, through TGF-beta-dependent mechanisms. Mucosal Immunol, 5, 320-331. (2012).

23.Unemo, M., & Shafer, W.M. Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future. Clin Microbiol Rev, 27, 587-613. (2014).

24.Cámara J., et al. Molecular characterization of two high-level ceftriaxone-resistant Neisseria gonorrhoeae isolates detected in Catalonia, Spain. J. Antimicrob Chemother, 67(8):1858–1860. (2012).

25.CDC. CDC Update to CDC's Sexually transmitted diseases treatment guidelines, 2010: oral cephalosporins no longer a recommended treatment for gonococcal infections 2012. (2012).

26.Bignell C & Fitzgerald M. Guideline Development Group; British Association for Sexual Health and HIV UK. UK national guideline for the management of gonorrhoea in adults. Int J STD AIDS, 22(10):541-7. (2012).

27.Bignell, C., Unemo, M., & European, S.T.I.G.E.B. 2012 European guideline on the diagnosis and treatment of gonorrhoea in adults. Int J STD AIDS, 24, 85-92. (2013).

28.Whiley, D. M., Jennison, A., Pearson, J. & Lahra, M. M. Genetic characterisation of Neisseria gonorrhoeae resistant to both ceftriaxone and azithromycin. Lancet Infect. Dis, 18, 717–718. (2018).

29.Russell, M.W., Hedges, S.R., Wu, H.Y., Hook, E.W., 3rd, & Mestecky, J. Mucosal immunity in the genital tract: prospects for vaccines against sexually transmitted diseases--a review. Am J Reprod Immunol, 42, 58-63. (1999).

30.Russell, M.W., Jerse, A.E., & Gray-Owen, S.D. Progress Toward a Gonococcal Vaccine: The Way Forward. Front Immunol, 10, 2417. (2019).

31.Ram, S., et al. Binding of C4b-binding protein to porin: a molecular mechanism of serum resistance of Neisseria gonorrhoeae. J Exp Med, 193, 281-295. (2001).

32.Smith, H., Parsons, N.J., & Cole, J.A. Sialylation of neisserial lipopolysaccharide: a major influence on pathogenicity. Microb Pathog, 19, 365-377. (1995).

33.Arko, R.J., Duncan, W.P., Brown, W.J., Peacock, W.L., & Tomizawa, T. Immunity in infection with Neisseria gonorrhoeae: duration and serological response in the chimpanzee. J Infect Dis, 133, 441-447. (1976).

34.Eyre JWH & Stewart BH. The treatment of gonococcus infections by vaccines. Lancet, 2:76–81. (1909).

35.Boslego, J.W., et al. Efficacy trial of a parenteral gonococcal pilus vaccine in men. Vaccine, 9, 154-162. (1991).

36.Rotman, E., & Seifert, H.S. The genetics of Neisseria species. Annu Rev Genet, 48, 405-431. (2014).

37.Lytton, E.J., & Blake, M.S. Isolation and partial characterization of the reduction-modifiable protein of Neisseria gonorrhoeae. J Exp Med, 164, 1749-1759. (1986).

38.Joiner, K.A., Scales, R., Warren, K.A., Frank, M.M., & Rice, P.A. Mechanism of action of blocking immunoglobulin G for Neisseria gonorrhoeae. J Clin Invest, 76, 1765-1772. (1985).

39.Plummer, F.A., Chubb, H., Simonsen, J.N., Bosire, M., Slaney, L., Maclean, I., Ndinya-Achola, J.O., Waiyaki, P., & Brunham, R.C. Antibody to Rmp (outer membrane protein 3) increases susceptibility to gonococcal infection. J Clin Invest, 91, 339-343. (1993).

40.Li, G., et al. Antibodies with higher bactericidal activity induced by a Neisseria gonorrhoeae Rmp deletion mutant strain. PloS one, 9, e90525. (2014).

41.Gulati, S., McQuillen, D.P., Mandrell, R.E., Jani, D.B., & Rice, P.A. Immunogenicity of Neisseria gonorrhoeae lipooligosaccharide epitope 2C7, widely expressed in vivo with no immunochemical similarity to human glycosphingolipids. J Infect Dis, 174, 1223-1237. (1996).

42.Gulati, S., Zheng, B., Reed, G.W., Su, X., Cox, A.D., St Michael, F., Stupak, J., Lewis, L.A., Ram, S., & Rice, P.A. Immunization against a saccharide epitope accelerates clearance of experimental gonococcal infection. PLoS Pathog, 9, e1003559. (2013).

43.Gulati, S., Beurskens, F.J., de Kreuk, B.J., Roza, M., Zheng, B., DeOliveira, R.B., Shaughnessy, J., Nowak, N.A., Taylor, R.P., & Botto, M., et al. Complement alone drives efficacy of a chimeric antigonococcal monoclonal antibody. PLoS Biol, 17, e3000323. (2019).

44.Semchenko, E.A., Tan, A., Borrow, R., & Seib, K.L. The Serogroup B Meningococcal Vaccine Bexsero Elicits Antibodies to Neisseria gonorrhoeae. Clin Infect Dis, 69, 1101-1111. (2019).

45.Petousis-Harris H, Paynter J, & Morgan J. Effectiveness of a Group B OMV meningococcal vaccine against gonorrhoea in New Zealand – a case control study. Lancet, 390:1603–1610. (2017).

46.José Juan Almagro Armenteros, Konstantinos D. Tsirigos, Casper Kaae Sønderby, Thomas Nordahl Petersen, Ole Winther, Søren Brunak, Gunnar von Heijne & Henrik Nielsen. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nature Biotechnology, 37, 420-423. (2019).

47.50 Shen, H.B., & Chou, K.C.. Gneg-mPLoc: a top-down strategy to enhance the quality of predicting subcellular localization of Gram-negative bacterial proteins. J Theor Biol, 264, 326-333. (2010)

48.Marshall L. Snyder, Herman C. Lichstein. Sodium Azide As An Inhibiting Substance for Gram-Negative Bacteria. The Journal of Infectious Diseases, 113–115. (1940).

49.Appelmelk, B.J., Verwey-van Vught, A.M., Maaskant, J.J., Schouten, W.F., Thijs, L.G., & Maclaren, D.M. Use of mucin and hemoglobin in experimental murine gram-negative bacteremia enhances the immunoprotective action of antibodies reactive with the lipopolysaccharide core region. Antonie Van Leeuwenhoek, 52, 537-542. (1986).

50.Semchenko, E. A., Tan, A., Borrow, R., & Seib, K. L. The Serogroup B Meningococcal Vaccine Bexsero Elicits Antibodies to Neisseria gonorrhoeae. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 69(7), 1101–1111. (2019).
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