A rational engineering strategy to improve the functional half-life of human SerpinE1 for the development of therapeutics against influenza A virus
- 주제(키워드) Virus infection , protein engineering , serine protease , serine protease inhibitor , functional half-life
- 발행기관 아주대학교 일반대학원
- 지도교수 Tae Hyeon Yoo
- 발행년도 2025
- 학위수여년월 2025. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000035120
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Influenza A virus (IAV) infections have emerged as a worldwide public health concern because of their high mortality. Currently available antiviral drugs primarily target viral replication and release, thereby limiting the spread of infection. However, these agents are frequently associated with gastrointestinal side effects such as nausea and vomiting, and the emergence of resistant viral strains particularly those carrying specific mutations can compromise therapeutic efficacy. These limitations highlight the need for alternative therapeutic strategies that act independently of viral genetic variability and instead exploit host-based mechanisms. Host-derived serine proteases play a critical role in the entry of IAV into host cells, by activating hemagglutinin (HA). Thus, inhibiting these serine proteases represents a promising therapeutic approach. This study aims to develop a therapeutic agent that effectively prevents virus entry into the upper respiratory mucosa, by engineering SerpinE1 (Plasminogen Activator Inhibitor-1, PAI-1), one of the host-derived serine protease inhibitors. However, the clinical application of wild-type SerpinE1 is limited by its short functional half-life. To overcome this limitation, we designed SerpinE1 variants fused with natural stabilizing domains and the Fc region of human IgG to enhance stability while retaining inhibitory activity. The fusion protein effectively inhibited serine proteases and retained inhibitory activity at 37℃, whereas the native SerpinE1 lost activity within two hours. Our results suggest that the engineered fusion protein may serve as a new therapeutic approach for preventing IAV infection.
more목차
1. Introduction 1
1.1. Influenza A virus 1
1.2. Host serine protease 5
1.3. Serine protease inhibitor 6
1.4. Somatomedin B domain-mediated stabilization of SerpinE1 9
1.5. Aim of this study 10
2. Methods and Materials 11
2.1. Construction of plasmids 11
2.2. Expression and purification of proteins 28
2.3. Characterization of SerpinE1 vatiants 32
2.4. Size exclusion chromatography 34
3. Results and discussion 35
3.1. Development of a fusion strategy to improve the structural half-life of SerpinE 1 using the SMB domain 35
3.2. Inhibitory activity of SMB-fused SerpinE1 38
3.3. Structural homogeneity of SMB-fused variants confirmed by SEC analysis 40
3.4. Functional half-life analysis of SMB-fused SerpinE1 43
3.5. Human IgG Fc fusion strategy for therapeutic application of SerpinE1 50
3.6. Inhibitory activity and structural assessment of Fc-fused SerpinE1 53
3.7. Extended half-life of Fc-fused SerpinE1 58
3.8. Mutation strategy for Fc domain silencing and functional evaluation of Fc-mutant fused SerpinE1 59
4. Conclusion 62
5. References 64
6. Abstracts in Korean 67
