2026-09-18T18:12:10Zhttps://repository.ajou.ac.kr/oai/request

oai:repository.ajou.ac.kr:201003/117982024-10-11T02:21:23Zcom_201003_14372com_201003_14368col_201003_14374

Characterization of cathepsin B cysteine protease from Naegleria fowleri excretory-secretory proteins exerting immunomodulatory effects 이, 진영 신, 호준 대학원 의생명과학과 200824385 이, 진영 Naegleria fowleri excretory/secretory proteins cytokine MAPK microglial cell 파울러자유아메바 시스테인 단백분해효소 배설-분비 단백질 Cathepsin B 단백질 Cathepsin B-like 단백질 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. 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 Doctor 2015-10-22T23:42:21Z 2015-10-22T23:42:21Z 2015 2015 Thesis Theses http://repository.ajou.ac.kr/handle/201003/11798 http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000019230 000000019230 en