Molecular studies on antiviral and endospore-inactivating activities of non-thermal atmospheric pressure plasma
- 주제(키워드) plasma , waterborne virus , lentivirus , endospore
- 발행기관 아주대학교
- 지도교수 문은표
- 발행년도 2018
- 학위수여년월 2018. 8
- 학위명 박사
- 학과 및 전공 일반대학원 생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000028020
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
우리 주변에는 많은 미생물이 있고, 그 미생물은 우리의 생명을 위협한다. 따라서 마시는 물과 지하수에 오염되어 있는 바이러스와 사람과 동물에게 감염되어 병을 일으키는 바이러스들을 불활성화 하는 것이 매우 중요하게 되었다. 이번 연구에서는 세 가지의 다른 타겟 미생물을 제 4의 상태인 플라즈마를 이용하여 불활성화 하였다. 세가지 타겟 미생물은 수처리 모델 바이러스인 박테리오파지(핵산의 조성과 구조에 따라 PRD1, ФX174, MS2를 사용)와 HIV의 모델 바이러스인 lentivirus, 마지막으로는 내성이 강한 내생포자이다. 각 실험은 챕터를 나누어 토의 하였다. 이번 실험에서 플라즈마의 가스는 질소가스를 사용하였고, 세가지 처리 방법을 고안하여 이용하였다. 실험 결과를 기반으로 세가지 플라즈마 처리 방법 중에서 직접 발생 처리 방법(direct in suspension method)이 가장 불홯성화 효과가 높았으며, 모든 모델 바이러스가 1분 안에 6 log 이상 불활성화 되었다. PBS / N2 plasma solution 역시 바이러스 불활성화 효과가 좋았으며 실생활에서 사용과 보관이 용이하다. PBS / N2 plasma solution는 4도씨에서 7일동안 보관을 해도 박테리오파지 불활성화 효과가 유지 되었고, -80도씨와 4도씨에서 28일 동안 보관을 해도 lentivirus 불활성화 효과가 유지 되었다. Real-time PCR과 아가로즈젤 전기영동을 통해 박테리오파지의 핵산이 손상 되는 것을 확인 하였고, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)을 통해 박테리오파지 구조 단백질 역시 손상되는 것을 확인하였다. 하지만 지질에는 플라즈마가 거의 영향을 미치지 않았다. Lentivirus에 PBS/N2 plasma 용액을 처리하고 숙주세포에 도입하면, 숙주세포의 세포독성은 없었고 lentivirus만 효과적으로 불활성화 하였다. 이것은 GFP 유전자가 삽입된 lentivirus의 형광단백질 발현이 적어지는 것을 형광현미경을 통해 확인하였다. 그리고 GFP를 순수분리하여 lentivirus가 불활성화 양을 정량적으로 확인하였다. 내생포자는 미생물 중에서 가장 내성이 강한 미생물이다. 그래서 내생포자를 플라즈마로 불활성화 하려면 플라즈마와 반응하는 시간이 필요하다. 플라즈마와 내생포자가 24시간 이상 반응하면 불화성화 효과가 급격하게 올라가고, 특히 직접 발생 처리 방법(direct in solution method )으로는 4log 이상, PBS/N2 plasma 용액 방법은 2 log 이상 불활성화 하였다. 내생포자에 플라즈마를 적절한 처리 조건을 병행하여 처리 한다면, 다른 미생물 불활성화 방법(autoclave, UV)과 비슷한 불활성화 효과를 보였다. 플라즈마를 처리해도 내생포자의 외형변화는 크지 않았고, crack이라는 외형변화가 나타났다.
more목차
Table of contents
Abstract i
Table of contents iii
List of figures viii
List of table xii
CHAPTER I: Inactivation of surrogate bacteriophages for waterborne virus
I. Introduction 2
II. Material & Methods
A. Comparison of bacteriophage inactivation effect by gas source 5
B. Inactivation of bacteriophage using N2 plasma with direct method 5
C. Plasma treatment of host and bacteriophage plaques 6
D. Inactivation of bacteriophage using N2 plasma with direct in phage suspension method 6
E. Inactivation of bacteriophage using N2 plasma with PBS/N2 plasma solution method 7
F. Effect of antioxidant on bacteriophage during direct in phage suspension method 7
G. Effect of inactivation by diluting PBS/N2 plasma solution 8
H. Inactivation effect of storage duration and storage temperature on the PBS/N2 plasma solution 9
I. Effects of plasma treatment on genomic DNA of PRD1 9
J. Effect of plasma treatment on genomic DNA of ФX174 and genomic RNA of MS2 10
K. Effect of plasma treatment on bacteriophage structure proteins 10
L. Effect of plasma treatment on lipid peroxidation 11
M. Anti-viral effects of underwater plasma devices equipped with bubblers 11
III. Results
A. Comparison of bacteriophage inactivation effect by gas source 13
B. Inactivation of bacteriophage using N2 plasma with direct method 13
C. Plasma treatment of host and bacteriophage plaques 13
D. Inactivation of bacteriophage using N2 plasma with direct in phage suspension method 14
E. Inactivation of bacteriophage using N2 plasma with PBS/N2 plasma solution method 14
F. Effect of antioxidant on bacteriophage during direct in phage suspension method 15
G. Effect of inactivation by diluting PBS/N2 plasma solution 16
H. Duration of antiviral activity with PBS/N2 plasma solution 16
I. Effects of plasma treatment on genomic DNA of PRD1 17
J. Effect of plasma treatment on genomic DNA of ФX174 and genomic RNA of MS2 17
K. Effect of plasma treatment on bacteriophage structure proteins 18
L. Effect of plasma treatment on lipid peroxidation 18
M. Anti-viral effects of underwater plasma devices equipped with bubblers 19
IV. Discussion 20
V. List of figure 24
CHAPTER II: Inactivation of HIV based lentivirus
I. Introduction 63
II. Material & Methods
A. Lentivirus infection 64
B. Lentivirus GFP quantification 64
C. MTT assay 64
D. Cell counting using Image-J program 65
E. Plasma treat using PBS/N2 plasma solution method 65
III. Results
A. Inactivation of lentivirus using PBS/N2 plasma solution treatment 67
B. Cytotoxic effect of pretreated PBS on U87 MG cell 67
C. Comparison of bacteriophage inactivation effect and lentivirus inactivation efficiency 68
D. Inactivation effect of PBS/N2 plasma solution storage time and temperature 68
IV. Discussion 70
V. List of figure 72
CHAPTER III: Inactivation of Bacillus endospore resistant to other inactivation method
I. Introduction 84
II. Material & Methods
A. Bacillus atrophaeus endospore preparation 86
B. Inactivation of B.atrophaeus endospore using direct in suspension PBS/N2 plasma solution method 86
C. Comparison of endospore inactivation effects between plasma and other inactivation methods 87
D. Effects of plasma treatment on genomic DNA of B.atrophaeus 87
E. SEM image of B.atrophaeus endospore and vegetative cell after plasma treatment 88
III. Results
A. Inactivation of B.atrophaeus endospore using direct in suspension PBS/N2 plasma solution method 89
B Comparison of endospore inactivation effects between plasma and other inactivation methods 89
C. SEM image of B.atrophaeus endospore and vegetative cell after plasma treatment 89
IV. Discussion 91
V. List of figure 93
References 100
국문요약 106
List of figures
Chapter I
Fig 1. Schematic illustration of the atmospheric pressure cold plasma system. 24
Fig 2. Comparison of bacteriophage inactivation effect by gas source. 25
Fig 3. Direct in bacteriophage method. 26
Fig 4. Direct in bacteriophage solution method. 27
Fig 5. PBS/N2 plasma solution method. 28
Fig 6. Plasma treatment using direct in phage method. 29
Fig 7. Plasma treatment of host and bacteriophage plaques. 30
Fig 8. Inactivation of bacteriophage using N2 plasma with direct in phage suspension method. 31
Fig 9. Inactivation of bacteriophage using N2 plasma with PBS/N2 plasma solution method. 32
Fig 10. Effect of antioxidant on PRD1 during N2 PBS with direct in phage suspension method treatment. 33
Fig 11. Effect of antioxidant on ФX174 during N2 PBS with direct in phage suspension method treatment. 34
Fig 12. Effect of antioxidant on MS2 during N2 PBS with direct in phage solution suspension treatment. 35
Fig 13. Effect of inactivation by diluting PBS/N2 plasma solution. 36
Fig 14. Inactivation effect of storage duration and storage temperature on the PBS/N2 plasma solution. 37
Fig 15. Inactivation effect of storage duration and storage temperature on the PBS/N2 plasma solution. 38
Fig 16. Inactivation effect of storage duration and storage temperature on the PBS/N2 plasma solution. 39
Fig 17. Agarose gel electrophoresis of PRD1 DNA treat with plasma. 40
Fig 18. PRD1 gene mapping for qPCR. 41
Fig 19. Agarose gel electrophoresis of PRD1 DNA PCR product. 43
Fig 20. ФX174 gene mapping for qPCR. 45
Fig 21. Agarose gel electrophoresis of ФX174 DNA PCR product. 47
Fig 22. MS2 gene mapping for qPCR. 49
Fig 23. Agarose gel electrophoresis of MS2 DNA PCR product. 51
Fig 24. Inactivation of PRD1 and change of bacteriophage PRD1 DNA ratio using N2 plasma with direct in phage suspension method. 53
Fig 25. Inactivation of ФX174 and change of bacteriophage ФX174 DNA ratio using N2 plasma with direct in phage suspension method. 54
Fig 26. Inactivation of MS2 and change of bacteriophage MS2 RNA ratio using N2 plasma with direct in phage suspension method. 55
Fig 27. SDS-PAGE analysis of PRD1 structure proteins. 56
Fig 28. SDS-PAGE analysis of ФX174 structure proteins. 57
Fig 29. SDS-PAGE analysis of MS2 structure proteins. 58
Fig 30. Linoleic acid peroxidation of N2/PBS plasma solution. 59
Fig 31. Anti-viral effects of underwater plasma devices equipped with bubblers. 60
Chapter II
Fig 1. Schematic illustration of plasma treatment method. 72
Fig 2. Transduction mechanism of lenti-GFP virus. 73
Fig 3. GFP expression in lentivirus-transduced cells after PBS/N2 plasma solution treatment. 74
Fig 4. Quantification of green fluorescent protein extracted from U89MG cells infected with lenti-GFP. 75
Fig 5. Cytotoxic effect of pretreated PBS on U87 MG cell line, cell viability after exposure to various concentrations of PBS/N2 plasma solution. 76
Fig 6. Comparison of green fluorescent protein quantitation and cell quantity. 77
Fig 7. Inactivation effect of PBS/N2 plasma solution storage time and temperature. 78
Fig 8. Inactivation effect of PBS/N2 plasma solution storage time and temperature. 79
Fig 9. Inactivation effect of PBS/N2 plasma solution treatment on lentivirus and bacteriophage according to storage duration. 80
Fig 10. Inactivation effect of PBS/N2 plasma solution treatment on lentivirus and bacteriophage according to storage duration. 81
Fig 11. Inactivation effect of PBS/N2 plasma solution treatment on lentivirus and bacteriophage according to storage duration. 82
Chapter III
Fig 1. Inactivation of B.atrophaeus endospore using N2 plasma with direct in suspension method and PBS/N2 plasma solution method. 93
Fig 2. B.atrophaeus dnak gene mapping for qPCR. 94
Fig 3. Agarose gel electrophoresis of B. atrophaeus DNA PCR product. 96
Fig 4. SEM images were obtained after treatment of inactivation substances in B.atrophaeus endospore and vegetative cells. 98
Fig 5. SEM images were obtained after treatment of plasma in B.atrophaeus endospore and vegetative cells. 99
List of tables
Chapter I
Table 1. PRD1 Primer sequence and PCR product size 42
Table 2. PRD1 DNA ratio using qPCR assay 44
Table 3. ФX174 Primer sequence and PCR product size 46
Table 4. ФX174 DNA ratio using qPCR assay 48
Table 5. MS2 Primer sequence and PCR product size 50
Table 6. MS2 DNA ratio using qPCR assay 52
Chapter III
Table 1. B.atrophaeus primer sequence and PCR product size 95
Table 2. Comparison of endospore inactivation effects between plasma and other inactivation methods 97
