Characterization of cathepsin B cysteine protease from Naegleria fowleri excretory-secretory proteins exerting immunomodulatory effects
- 발행기관 아주대학교
- 지도교수 신호준
- 발행년도 2015
- 학위수여년월 2015. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000019230
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
.Naegleria fowleri, a free-living amoeba that is found in diverse environmental habitats, can cause an acute fulminating hemorrhagic meningoencephalitis, called primary amoebic meningoencephalitis (PAM) in experimental animals and in humans. Pathogenicity of N. fowleri may be induced by contact-dependent and contact-independent mechanisms, and both mechanisms lead to death of host cells. Several proteins secreted from N. fowleri are likely to be associated with contactindependent pathogenic mechanism of the amoeba. The excretory and secretory proteins of N. fowleri (Nf-ESPs) include phospholipase, proteases, peroxiredoxins and thrombin receptor. However, the precise mechanism induced by Nf-ESPs in PAM is not fully understood yet. In this study, the cytopathic changes and inflammatory responses induced by Nf-ESPs were analyzed in BV2 microglial cells. Several cytokines, particularly IL-1α and TNF- α were highly up-regulated by Nf-ESPs. Also, activation of P38 and JNK was observed in BV2 cells treated with Nf-ESPs. The results collectively suggest that Nf-ESPs may play an important role in N. fowleri mediated-PAM through induction of inflammatory response in microglial cells and activation of MAPKs signal pathway.
more목차
PART I 1
I. Introduction 2
II. Materials and Methods 13
A. N. fowleri culture and production of excretory-secretory proteins 13
B. Culture of mouse microglial BV2 cell 13
C. MTT assay 14
D. Cytokine array assay 14
E. RNA isolation 15
F. Reverse transcription polymerase chain reaction (RT-PCR) 15
G. Preparation of cell lysate 17
H. Western blotting 17
III. Results 19
A. SDS-PAGE patterns of Nf-ESPs 19
B. Nf-ESPs induces cell death in BV2 cells 20
C. Cytokine expression in BV2 cells treated with Nf-ESPs 22
D. mRNA expression of cytokines genes 24
E. Nf-ESPs induced MAPKs activation in BV2 cells 26
IV. Discussion 28
V. Conclusion 34
References 35
국문요약 48
PART II 50
I. Introduction 51
II. Materials and Methods 58
A. N. fowleri cultures and preparation of Nf-ESPs 58
B. Gene cloning 58
C. Expression, purification and refolding of recombinant NfCPB and NfCPB-L 59
D. Production of anti-NfCPB and anti-NfCPB-L antibodies 61
E. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting 61
F. Semi-quantitative revers transcription PCR 62
G. Enzyme activity assay 63
H. Biochemical properties of rNfCPB and rNfCPB-L protein 64
I. Determination of cysteine protease in rNfCPB and rNfCPB-L protein 64
J. Degradation of host proteins by rNfCPB and rNfCPB-L protein 65
K. MTT assay 66
L. Statistical analysis 66
III. Results 67
A. Cloning and sequence analysis of nfcpb and nfcpb-L 71
B. Differential expression of NfCPB and NfCPB-L in N. fowleri 77
C. Expression, purification and refolding of rNfCPB and rNfCPB-L protein 79
D. Biochemical characterization of rNfCPB and rNfCPB-L protein 81
E. Degradation of host proteins 86
F. rNfCPB and rNfCPB-L induces cell death of BV2 cells 89
IV. Discussion 91
V. Conclusion 96
References 97
국문요약 105
