Recombinant Salmonella typhimurium Expressing HPV16 E7 Gene Retarded Tumor Growth and Induced Immune Responses in Cervical Tumor-Bearing Mice
- 주제(키워드) Salmonella typhimurium , Cervical cancer , HPV16 E7 , therapeutic vaccine , SipB
- 발행기관 고려대학교 대학원
- 지도교수 박용근
- 발행년도 2012
- 학위수여년월 2012. 2
- 학위구분 석사
- 학과 일반대학원 생명공학과
- 세부전공 의약생명공학전공
- 원문페이지 49 p
- 실제URI http://www.dcollection.net/handler/korea/000000032423
- 본문언어 영어
- 제출원본 000045697061
초록/요약
Human papillomavirus (HPV) infection of the cervical epithelium is causatively combined with the occurence of cervical cancer. The greater part of women will acquire a genital HPV infection one of these days in their lifetime. Approximately 15% of women with a HPV infection cannot induce an effective immune response against the virus. To induce effective immune response, in this study, recombinant Salmonella expressing HPV16 E7 gene was constructed as therapeutic vaccine. An attenuated Salmonella has been developed as an antitumor agent since it can be preferentially amplified within tumors and inhibit their growth without severe pathogenicity in the host. This recombinant Salmonella was expressed the E7 protein that generated a efficacious cytotoxicity and tumor growth retardation in TC-1 cervical cancer cells. Also, in mice bearing TC-1 cells, this recombinant Salmonella expressing HPV16 E7 was treated orally, induced immune response through IFN-g (Interferon-gamma) and TNF-a (Tumor necrosis factor-alpha) cytokines secretion and suppressed tumor growth and prolonged survival as compared with PBS, salmonella control group. These results suggested that Salmonella expressing HPV16 E7 would be used as therapeutic vaccine for cervical cancer.
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ABSTRACT 1
INTRODUCTION 2
MATERIALS AND METHODS
Bacterial strains and Growth conditions 6
Plasimd constructions 6
Mice and tumor cell line 7
Preparation of proteins from whole cell lysates and culture supernatants 8
SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and western blot assay 9
Salmonella invasion 10
Tumor cell cytotoxicity assay 10
Tumor cell challenge and bacterial treatment 11
Cytotoxic T lymphocyte assay 12
Analysis of IFN-g and TNF-a in serum via ELISA 13
RESULTS
Construction of SipB160/HPV16 E7 expressing plasmid vector 14
The confirmation of expression and secretion of SipB160/HPV16 E7 fusion proteins from S. typhimurium 16
Infection of S. typhimurium with and without pN160-16E7 to cervical tumor cell in vitro 18
Generation of the antitumor effect by SipB160/HPV16 E7 fusion protein from S. typhimurium in vitro 21
Induction of Cytotoxic T lymphocyte (CTL) responses in the mice treated with S. typhimurium encoding pN160-16E7 24
Secretion of IFN-g and TNF-a in serum of tumor bearing mice inoculated with S. typhimurium encoding pN160-16E7 26
Retardation of tumor growth in vivo by S. typhimurium with pN160-16E7 29
DISCUSSION 32
REFERENCES 35
ABSTRACT IN KOREAN 37
