專一性單株抗體對豬內生性逆轉錄病毒的偵測及中和病毒感染之應用

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研究生：	江貞儀 Chen-Yi Chiang
論文名稱：	專一性單株抗體對豬內生性逆轉錄病毒的偵測及中和病毒感染之應用 Application of Monoclonal Antibodies in Detection and Neutralization of Porcine Endogenous Retroviruses
指導教授：	張晃猷 Hwan-You Chang
口試委員:
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生命科學系 Department of Life Sciences
論文出版年：	2005
畢業學年度：	93
語文別：	中文
論文頁數：	95
中文關鍵詞：	豬內生性逆轉錄病毒、單株抗體、中和競爭試驗
外文關鍵詞：	porcine endogenous retrovirus, monoclonal antibody, neutralization, competition assay, epitope mapping
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豬內生性逆轉錄病毒 (PERV) 是普遍存在於豬隻的病毒，由於近年來豬的組織或器官廣泛應用在異種移植上，因此這病毒現在也受到相當程度的重視。研究發現，此病毒具有感染許多哺乳類細胞株的能力，對免疫抑制的移植接受者具有潛在性的危機。目前對於PERVs的感染是否會造成人類相關疾病，尚無定論。因此，發展高敏感高專一性免疫偵測方法，監測臨床受異體移植的病人是否受到此病毒感染是極重要的工作。在本篇論文，我們針對病毒殼及外膜蛋白分別建立單株抗體，稱為A-11，8E10及7C4。由免疫沉澱法及免疫螢光法分析，A-11單株抗體可以辨認全部3種血清型的PERVs，8E10辨認PERV-A及-B，而7C4只辨認PERV-A。這些單株抗體都可以偵測受感染的細胞，尤其是病毒蛋白表現量最高的人類腎臟上皮細胞，而且不會交互辨認其他逆轉錄病毒，例如:老鼠白血病病毒，人類免疫缺失病毒及人類Ｔ細胞白血病病毒的外膜或殼蛋白質。我們也找到單株抗體對抗原的辨認位: A-11辨認殼蛋白第313-332胺基酸;8E10是外膜蛋白第427-434胺基酸;及7C4是外膜蛋白第517-537胺基酸。人工合成胺基酸位於殼蛋白第313-322，外膜蛋白第417-434，重組外膜蛋白397-481及460-539也均可干擾單株抗體結合抗原，尤其以重組外膜蛋白具有中和PERVs感染的能力。另外，配合流式細胞儀分析PERVs對不同人類細胞的感染性，單株抗體也能有效清楚呈現病毒對組織的特異性。PERVs對人類腎臟上皮細胞的感染效率及病毒表現量比其他人類細胞株高，例如: 喉頭上皮，肝臟上皮，及肌肉紡錘細胞。總而言之，此單株抗體除了能應用於在基礎研究上，也有潛力在臨床檢測，辨認病毒的蛋白或應用於病毒感染之中和效力。

Porcine endogenous retrovirus (PERV) is ubiquitous in pigs. The virus has drawn much attention recently because of the widespread use of pig organs and tissues in xenotransplantion. The virus has been found to be capable of infecting a bread range of cells including those of human origins in vitro and thus possesses a potential threat to xeno-transplant recipients who are generally immunosuppressed. Nevertheless, it remains unclear whether PERVs infection can cause human diseases. Therefore, the development of a highly sensitive and specific immunoassay for clinical surveillance is imperative in patients receiving xenotransplantation. We describe here the generation of monoclonal antibodies (mAbs) named A-11, 8E10, and 7C4 that specifically recognize either Gag or Env protein of PERV. In immunoblotting assay, the A-11 mAb was found to be able to detect all three classes of PERV: 8E10 mAb recognizes PERV-A and -B, whereas 7C4 mAb recognized only PERV-A. A-11 mAb can be used in detection of PERV in cultured cells, especially human kidney epithelial cells, larynx epithelial cells, hepatoma epithelial cells, and muscle spindle cells by immunochemistry. No cross-reaction with Gag and Env proteins of murine leukemia virus and human immunodeficiency virus-1,2 was observed, indicating that it was highly specific to PERV. In addition, the region recognized by mAb A-11, 8E10, and 7C4 was localized to amino acid positions 313 to 322 on the Gag protein, 427 to 434 and 517 to 537 on the Env protein, respectively. The synthetic peptides Gag313-322, Env417-434, recombinant Env397-481, and Env460-539 proteins effectively competed the binding of the mAb with recombinant Gag and Env proteins, respectively. The recombinant protein of Env was able to neutralize PERV infection. Furthermore, A-11 mAb was used in tissue tropism of PERV infection for different human cell lines by flow cytometry. Higher efficiency of PERV infection and higher expression of viral proteins were observed in human kidney cell line than others human cell lines. Therefore, these mAbs can be used as tools for the identification of viral proteins in basic research as well as clinical studies and application in neutralization of virus infection.

中文摘要    i
Abstract    iii
Table of contents    v
Acknowlegement    vii
Abbreviation    viii
Chapter I: Background and Significance    1
1.1. Characterization of retroviruses    2
1.2. The life cycle of retroviruses    2
1.3. Characterization of endogenous retroviruses    3
1.4. Porcine endogenous retrovirus    4
1.5. Specific aims    6
Chapter II: Characterization of a monoclonal antibody specific to the Gag protein of porcine endogenous retrovirus and its application in detecting the virus infection    9
Abstract    10
Introduction    12
Materials and Methods    14
2.1. Nucleic acid isolation, Polymerase Chain Reaction (PCR), Reverse Transcription reaction (RT), and Reverse Transcription-PCR (RT-PCR).    14
2.2. Overexpression and purification of recombinant Gag proteins.    14
2.3. Cell lines and viruses.    15
2.4. Production, purification, isotyping of antibody.    16
2.5. ELISA and competitive binding assay.    17
2.6. Western analysis and dot immunoblotting.    18
2.7. Immunocytochemistry assay.    19
2.8. Flow cytometry analysis.    20
Results    21
3.1. Generation and isotyping of mAbs specific to PERV Gag protein.    21
3.2. Specificity and sensitivity of the Anti-Gag mAbs.    21
3.3. Mapping of epitopes on Gag234-524 recognized by A-11 mAbs.    23
3.4. Detection of PERV infected cells by immunocytochemistry.    24
3.5. Detection of Gag of PERV in different infected human cell lines.    25
Discussion    27
Chapter III: Identification of the epitopes on PERV Env protein recognized by neutralizing monoclonal antibodies    43
Abstract    44
Introduction    46
Materials and Methods    49
2.1. Cell lines and Viruses.    49
2.2. Expression and purification of recombinant envelope protein.    50
2.3. Production, purification, isolation of monoclonal antibody.    50
2.4. ELISA and competition assay.    52
2.5. Western analysis and dot immunoblotting.    53
2.6. Immunocytochemistry assay of PERV.    54
2.7. Immunofluorescence staining.    55
2.8. PERV neutralization and competition binding assay.    56
2.9. PERV immunoprecipitation assay.    57
2.10. Preparation of plasmids    57
Results    59
3.1. Generation and isotyping of mAbs specific to PERV Env protein.    59
3.2. Specificity and sensitivity of the anti-Env mAb.    60
3.3. Detection of PERV infected cells by immunocytochemistry.    61
3.4. Neutralization of PERV by anti-Env mAb.    62
3.5. Epitope mapping of anti-Env mAb.    62
3.6. Identification of the mAb binding sites by competition assay.    63
3.7. Nonneutralizing specific mAbs can also bind PERV virions.    64
3.8. Determinination of the sequence of the variable region of mAb.    65
Discussion    66
References    85

                                

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