Structurally constrained peptides prevent SARS-CoV-2 spike – hACE2 interaction and neutralize pseudovirus in vitro.
- 주제(키워드) SARS-CoV-2
- 주제(DDC) 570
- 발행기관 아주대학교
- 지도교수 Woo Hyun Goo
- 발행년도 2023
- 학위수여년월 2023. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000033107
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
COVID-19 became a worldwide pandemic shortly after the first emergence of SARS-CoV-2 in December 2019. The virus possesses a high dynamic of evolution, with several mutations have been occurred and many variations of concern (VOCs). The infection is initiated by the interaction of SARS-CoV-2 receptor binding domain (RBD) and hACE2. ACE2 converts angiotensin I to angiotensin 1-9, and hACE2 is the receptor for the spike of SARS-CoV-2. The RBD is immunodominant, this region is the target of vaccine, antibodies, and peptides design. RBD is the poorly conserved region of S protein, the mutations in RBD resulted in many immune escapes, and the approved antibodies lost their susceptibility. In this study, the neutralizing effect of neutralizing peptides CSNP1, CSNP4 and Pep1 was measured in vitro, using the model of HEK293 cells that highly expresses hACE2 as the host cell, and lentiviruses that carry the SARS- CoV-2 spike proteins on their surface as the pseudovirus particles. The results showed that all CSNP1, CSNP4 and Pep1 strongly inhibit the infection of SARS- CoV-2 and have neutralizing effects on all VOCs. The peptides work in dose- dependent manner and do show cytotoxicity even in high concentrations. Taken together, neutralizing peptides based of the structure of hACE2 universally deter the SARS-CoV-2 infections, and the three neutralizing peptides CSNP1, CSNP4, and pep1 are promising candidates in treatment of the new emerging SARS-CoV-2 variants and ready to go in clinical tests.
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BACKGROUND 1
1. The pandemic 1
2. SARS-CoV-2 1
a. Biology of SARS-CoV-2 1
b. Cell entry of SARS-CoV-2 2
c. Evolution of SARS-CoV-2 and Spike mutation 4
3. SARS-CoV-2 spike neutralization. 5
MATERIALS AND METHODS 7
Neutralizing peptides designing process and synthesis. 7
Surface plasmon resonance (SPR) analysis 7
Cell culturing 7
Transient transfection 8
Pseudovirus particles production 8
Fluorescence microscope 9
qPCR 9
Pseudovirus-based neutralizing assay (Luciferase assay) 10
Cell viability assay 10
Proteinase K assay 10
RESULTS 12
1. Candidates for neutralizing peptides 12
2. The neutralizing peptides are not toxic to the cells. 15
3. Human ACE2 expressing cell line construction. 16
4. Neutralize effects of peptides on different variants 18
5. The peptides work in dose-dependent manners. 20
6. Stability of neutralizing peptides 22
DISCUSSION 23
CONCLUSION 25
REFERENCES 26
