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研究生:PRISKILA CHERISCA THENAKA
研究生(外文):PRISKILA CHERISCA THENAKA
論文名稱:雞源抗蛇毒抗體的鑑定瑪家山龜殼花
論文名稱(外文):Characterization of Chicken-Derived Antibodies against Snake Venom Ovophis makazayazaya
指導教授:楊沂淵楊沂淵引用關係
指導教授(外文):YANG, YI-YUAN
口試委員:楊沂淵吳昭容潘玟伃
口試委員(外文):YANG, YI-YUANWU, CHAO-JUNGPAN, WEN-YU
口試日期:2024-06-21
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗暨生物技術學系碩士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2024
畢業學年度:112
語文別:英文
論文頁數:57
中文關鍵詞:免疫球蛋白 Y (IgY)瑪家山龜殼花噬菌體展示技術單鏈抗體 (scFv)蛇毒
外文關鍵詞:Immunoglobulin Y (IgY)Ovophis makazayazayaphage display technologysingle-chain variable fragment (scFv)snake venom
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毒蛇咬傷在全球一直是很重要的醫學議題。Ovophis makazayazaya (OM) 也稱為瑪家山龜殼花,是台灣常見的毒蛇之一,會分泌出血性蛇毒蛋白(OM蛋白)。隨著瑪家山龜殼花咬傷病例數量持續增加,治療方式變得非常重要。然後,目前還沒有針對瑪家山龜殼花咬傷的特異性抗蛇毒血清。此外,傳統利用馬開發特定的抗蛇毒血清除了成本高昂也容易具有像是血清疾病等潛在的副作用。本研究旨針對 OM 蛋白開發生產一種經濟實惠的抗蛇毒血清治療策略。我們以減毒OM蛋白對母雞進行免疫來製備多株抗體IgY。從蛋黃中純化的IgY抗體顯示出對OM蛋白具有特異性結合能力,且雞隻的免疫反應持續至少五週。接著透過噬菌體展示技術我們建構了兩個具有短鏈和長鏈的單鏈抗體(scFv)基因庫,其大小分別為1.6 × 106和5.5 × 105。經過兩次四輪篩選(bio-panning)後,elution噬菌體和噬菌體ELISA 上的訊號都有增加的趨勢。我們隨機挑選52個表現的scFv抗體進行序列分析,第一輪篩選可分為11群短鏈和1群長鏈;第二輪篩選可分為12群短鏈和2群長鏈。在這些抗體當中,四個scFv包含cOML1、cOM2S3、cOM2L1和cOM2L11 的在 ELISA 上對OM蛋白的具有結合力,但在西方墨點法(Western blot)上沒有結合訊號。此外,在ELISA上發現cOM2S3和cOM2L11與其他台灣常見的蛇毒蛋白具有交叉結合反應;而cOML1和cOM2L1只專一性對OM蛋白具有結合力。這四種 scFv 抗體也經過His-tag純化並用於效價(titration)和競爭型 ELISA分析,進而確認它們親和力。此外,IgY和scFv 在血液瓊脂培養基上顯示出對 OM 引起的溶血具有抑製作用。因此,使用雞和噬菌體展示技術生產多株IgY和單株scFv的中和性抗體既符合經濟效應,也可應用於未來的診斷和治療試劑。
Snake-induced envenomation is an important medical issue worldwide. The Ovophis makazayazaya (OM), also known as the Taiwan mountain pitviper, is a hemotoxic venomous snake species commonly found in Taiwan. The availability of treatment is substantial due to the increasing number of snake envenomation. Nonetheless, there is currently no specific antivenom available against OM. Besides, the development of specific antivenom using horses may be costly and challenging with potential side effects such as serum sickness. This study aims to develop an affordable therapeutic strategy for antivenom production against OM proteins. Attenuated OM proteins were used to immunize hens to produce polyclonal IgY antibodies. The purified IgY antibodies from egg yolk, showed specific binding activities to OM and the immune responses in chickens lasted at least for 5 weeks. Two single-chain variable fragment (scFv) antibody libraries with short or long linkers, containing 1.6 × 106 and 5.5 × 105 transformants, were constructed by phage display technology. After four rounds of bio-panning, which was carried out twice, the eluted phage titers and signals on phage-based ELISA were increased. Sequence analysis of 52 randomly selected expressed scFv clones was classified into 11 groups of the short linker and 1 group of the long linker in the first bio-panning experiment, and 12 groups of the short linker and 2 groups of the long linker in the second bio-panning experiment. Among these clones, four groups containing cOML1, cOM2S3, cOM2L1, and cOM2L11 showed binding activity to OM proteins on ELISA but no binding signal on western blot. In contrast with the cOM2S3 and cOM2L11, which showed cross-binding activities to other snake venom proteins that commonly caused snake envenomation in Taiwan, the cOML1 and cOM2L1 had specific binding activities to OM proteins without cross-binding observed on ELISA. These four scFv antibodies were also purified for titration and competitive ELISA to confirm their binding affinity. Additionally, the IgY and scFv showed the inhibition of OM-induced hemolysis on blood agar plates. In conclusion, the production of IgY and neutralizing scFv antibodies using chicken and phage display technology is both sustainable and economically favorable, which can be applied for future diagnostic and therapy agents.
1. Introduction 1
1.1. Snake envenomation 1
1.2. Ovophis makazayazaya snake 2
1.3. Antibody immunotherapy 3
1.3.1. Antibody 3
1.3.2. Antivenin for snake envenomation 4
1.3.3. Chicken-derived antibodies 4
1.4. Production of monoclonal antibodies 6
1.5. Phage display technology 6
2. Research aim 8
3. Materials and Methods 9
3.1. Animal models 9
3.2. Preparation and analysis of the snake venom protein 9
3.3. Chicken immunization 9
3.4. IgY purification 9
3.5. Antibody library construction 10
3.6. Bio-panning for selecting specific antibodies 11
3.7. Extraction of total plasmid DNA 13
3.8. Expression and purification of E. coli-derived chicken scFv antibodies 14
3.8.1. Heat-shock procedure for E. coli transformation 14
3.8.2. ScFv antibodies expression 14
3.8.3. Expression test on SDS-PAGE and western blot 15
3.8.4. Binding assay with OM venom protein 15
3.8.5. Western blot of anti-OM scFv antibodies 16
3.9. Enzyme-linked immunosorbent assay (ELISA) 16
3.9.1. Polyclonal IgY Titration 16
3.9.2. Eluted Phage Titration 17
3.9.3. Binding Test of Randomly Selected scFv Clones against OM Protein 17
3.9.4. Binding Specificity of Anti-OM scFv Antibodies against Different Snake Venoms 17
3.9.5. Titration Assay of Anti-OM scFv Antibodies 18
3.9.6. Competitive ELISA of Anti-OM scFv Antibodies 18
3.10. Sequence analysis of E. coli-derived chicken scFv antibodies 18
3.11. Neutralization test of anti-OM scFv antibodies on blood agar plate 19
4. Results 20
4.1. Characterization of OM snake venom protein 20
4.2. Analysis of purified chicken polyclonal IgY antibodies 20
4.3. Binding assay of polyclonal antibodies against OM snake venom protein 20
4.4. Construction of anti-OM venom proteins scFv antibodies libraries 21
4.5. Bio-panning of recombinant phage display of anti-OM scFv libraries 21
4.6. Protein expression and sequence analysis of selected anti-OM scFv antibodies 22
4.7. Binding assay of selected scFv antibodies against different snake venom proteins 23
4.8. Titration assay of anti-OM scFv antibodies 23
4.9. Competitive ELISA of anti-OM scFv antibodies 24
4.10. Neutralization test of scFv antibodies against OM venom on blood agar plate 24
5. Discussion 25
6. Conclusion and Future Prospects 32
7. Table 33
Table 1. Chicken Immunization Schedule 33
Table 2. List of Primers used in Library Construction 33
Table 3. Anti-OM scFv Library Size and Eluted Titer after Each Round of Bio-panning 34
Table 4. Classification of anti-OM scFv Clones According to the Heavy- and Light-chain Variable Regions 35
Table 5. Amino Acid Mutation Rates of anti-OM scFv Clones in Comparison with Chicken Germline 36
Table 6. IC50 of anti-OM scFv Antibodies 36
8. Figures 37
Figure 1. Analysis of OM snake venom protein on SDS-PAGE. 37
Figure 2. Analysis of purified chicken anti-OM polyclonal IgY after each immunization. 38
Figure 3. Binding assay of chicken anti-OM IgY antibody titer after immunization on ELISA. 40
Figure 4. Monitoring the immune response in chicken on ELISA. 41
Figure 5. Construction of anti-OM scFv antibody libraries. 42
Figure 6. Determination of eluted phage titers after four rounds of bio-panning. 43
Figure 7. Binding assays of total amplified phage after each round of bio-panning. 43
Figure 8. Expression test of the randomly selected scFv clones after the 4th round of bio-panning. 44
Figure 9. Binding assay of the randomly selected scFv antibodies against OM proteins on ELISA. 45
Figure 10. Protein expression and purification of anti-OM scFv antibodies. 46
Figure 11. Analysis binding activity of anti-OM scFv antibodies on western blot. 46
Figure 12. Binding specificity of anti-OM scFv antibodies to different snake proteins on ELISA. 47
Figure 13. Titration assay of anti-OM scFv antibodies. 48
Figure 14. Inhibition assay of anti-OM scFv antibodies by competitive ELISA. 49
Figure 15. Neutralization assay of anti-OM antibodies on blood agar plate. 50
9. References 51
10. Appendix 57
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