2026-09-19T02:14:11Zhttps://repository.ajou.ac.kr/oai/request

oai:repository.ajou.ac.kr:201003/14332024-10-11T02:17:30Zcom_201003_14369com_201003_14368col_201003_14371

Roles of Interferon Lambda on Hepatitis B Virus Replication B형 간염 바이러스의 감염과 관련된 인터페론 람다 시스템의 역할과 조절 홍, 승호 박, 선 대학원 의학과 104772 홍, 승호 Hepatitis B Virus IFN-lambda-1 protein Virus replications "Background/Aims: Chronic hepatitis B virus (HBV) infection is a major cause of liver disease. Only interferon-α (IFN-α) and the nucleosidic inhibitors of the viral polymerase, lamivudine and adefovir, are approved for therapy. However, these therapies are limited by the side effects of interferon and by the substantial resistance of the virus to nucleosidic inhibitors. New antiviral molecules suitable for monotherapy or combination therapy are highly desired. Recently described IFN-λ family utilizing its distinct cellular receptor has been reported to exert inhibitory effect on replication of several viruses. In this study, I wanted to know whether IFN-λ1 inhibits HBV replication in human hepatoma cell lines and whether HBV modulates expression of CRF2-12, a subunit of IFN-λ1 receptor. In addition, I wanted to establish ELISA for IFN-λ1 to examine HBV mediated modulation of IFN-λ1 production. Methods: I produced IFN-λ1 using E.coli expression system. For antiviral activity of IFN-λ1, I used a functional form of MBP-IFN-λ1 and for generation of antibody against IFN-λ1, I used His-IFN-λ1. I analyzed HBV replication in WT10 and PEB8 human hepatoma cell lines supporting HBV replication, after treatment of these cells with IFN-λ1 by real-time PCR and Southern blotting. After treatment of PEB8 with IFN-λ1, I examined the transcription level of HBV by Northern blotting and the amount of secretory viral Ag by ELISA. I performed the RT-PCR to investigate the induction of antiviral proteins, MxA and 2’5’-OAS, by IFN-λ1. I investigated the expression of CRF2-12 by RT-PCR and Western blotting. I generated monoclonal Abs against IFN-λ1 using splenocytes from immunized mice and determined the detection limit of ELISA using these Abs. Results: HBV replication in PEB8 but not in WT10 was suppressed by IFN-λ1 treatment. In both cell lines, similar amount of CRF2-12 to that in their parental cell line was expressed, and similar amount of transcripts of MxA as well as 2’5’-OAS were induced by IFN-λ1. In PEB8, neither HBV transcripts nor secretory Ag was affected by IFN-λ1 treatment. IFN-λ1 was detected by ELISA using monoclonal Abs and polyclonal Ab generated in this study with the detection limit of 40 ng/ml. Conclusions: Antiviral activity of IFN-λ1 on HBV was demonstrated in one of two human hepatoma cell lines suggesting that the effect of IFN-λ1 may be dependent on the cellular factors, and/or viral factors. My results showed that CRF2-12 expression was not regulated by HBV replication. Finally, an assay for IFN-λ1 production was established. " "ABSTRACT i TABLE OF CONTENTS iii LIST OF FIGURES vi LIST OF TABLES vii ABBREVIATION viii I. INTRODUCTION 1 A. Background 1 1. Hepatitis B virus 1 2. Interferon alpha 5 3. Interferon lambda 8 B. Aims 12 II. MATERIALS AND METHODS 13 A. Expression vectors for IFN-λ1 and its receptor subunit CRF2-12 13 B. Purification of the recombinant proteins 14 1. Expression and purification of MBP-IFN-λ1 in E. coli 14 2. Expression and purification of His-IFN-λ1 and His-CRF2-12 in E. coli 16 3. Expression and purification of CRF2-12-mIg in COSM6 17 C. SDS-PAGE 17 D. Western blotting 17 E. RNA preparation 19 F. Reverse transcription-polymerase chain reaction 19 G. Isolation of HBV core DNA 22 H. Real-time PCR 23 I. Southern blotting 24 J. Northern blotting 24 K. Enzyme-linked immunosorbent assay 25 1. Secretion of HBsAg and HBeAg 25 2. Screening of monoclonal anti-IFN-λ1 Ab 25 3. Sensitivity of anti-IFN-λ1 Abs against IFN-λ1 25 L. Cell fusion 26 M. Limiting dilution 27 N. Isotyping of monoclonal Abs 28 O. Large production of monoclonal anti-IFN-λ1 Ab 28 P. The production of polyclonal anti-IFN-λ1 Ab and anti-CRF2-12 Ab 29 III. RESULTS 30 A. Expression and purification of IFN-λ1 30 B. Functional activity of IFN-λ1 32 C. Expression of CRF2-12 in HBV replicating human hepatoma cell lines 34 D. Induction of antiviral transcripts in HBV replicating human hepatoma cell lines by IFN-λ1 38 E. The effect of IFN-λ1 on HBV replication 40 F. The effect of IFN-λ1 on HBV transcription 43 G. The effect of IFN-λ1 on the secretion of HBsAg and HBeAg 43 H. Generation of the polyclonal and the monoclonal Abs against IFN-λ1 46 I. Establishment of ELISA for IFN-λ1 48 IV. DISCUSSION 50 V. CONCLUSION 54 REFERENCES 56 국문요약 64 " Doctor 2011-02-08T05:40:59Z 2011-02-08T05:40:59Z 2006 2006 Thesis Theses http://repository.ajou.ac.kr/handle/201003/1433 http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000001230 000000001230 en