Attenuated Salmonella enterica serovar Typhimurium Expressing PspA Fused to the N-terminal Domain of SipB Induces Protective Responses against Streptococcus pneumoniae WU2 in Mice
- 주제(키워드) Salmonella , Straptococcus pneumoniae , Pneumococcal surface protein A , Salmonella invasion protein B
- 발행기관 고려대학교 대학원
- 지도교수 박용근
- 발행년도 2013
- 학위수여년월 2013. 2
- 학위구분 석사
- 학과 일반대학원 생명공학과
- 세부전공 분자생물학 전공
- 원문페이지 53 p
- 실제URI http://www.dcollection.net/handler/korea/000000037683
- 본문언어 영어
- 제출원본 000045746059
초록/요약
Live attenuated bacteria can be used as a carrier for the delivery of foreign antigens to a host’s immune system. Attenuated Salmonella strains can reach deep tissues, including the liver, spleen and Peyer’s patches, via the circulatory system. The N-terminal domain of SipB, a translocon protein of the type III secretion system of Salmonella enterica serovar Typhimurium, is required for secretion and outer membrane localization. In this study, vaccine plasmids for antigen delivery in which the pneumococcal surface protein A (PspA), which contains a protection-eliciting epitope, is fused to the N-terminal 160 amino acids of SipB were developed. Western blotting experiment showed that recombinant proteins are secreted into the culture media. And the fusion proteins localized to the bacterial surface. PspA-specific antibody responses are significantly increased in mice orally immunized with attenuated S. typhimurium strains BRD509 carrying PspA delivery plasmids. The vaccination using SipB1-160-PspA fusion protein considerably promoted protective antibody responses, resulting that it increased longevity of the mice. These results suggest that expression and delivery of foreign antigens fused to the N-terminus of SipB enhance the induction of antigen specific immune responses and protective immunity against S. pneumoniae, and that the N-terminal domain of SipB can be used as a versatile delivery system for foreign antigens.
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CONTENTS
ABSTRACT 1
INTRODUCTION 2
MATERIALS AND METHODS 6
Bacterial strains, media and growth conditions 6
General Techniques 6
Salmonella mutant construction 6
Construction of the recombinant plasmid and transformation to S. typhimurium 7
Preparation of proteins from whole-cell lysates and culture supernatants 8
Membrane fractionation 9
SDS-PAGE and immunoblot analysis 10
Surface Immunofluorescence assay 10
Purification of PspA 11
Salmonella invasion assay 12
Analysis of serum cross-reactivity by ELISA 12
Immunization of mice 13
Pneumococcal challenge 14
RESULTS 17
Construction of a series of plasmids expressing PspA fusion proteins 17
SipB N-terminal region mediates the expression and secretion of PspA in the attenuated S. typhimurium BRD509 strain 20
SipB N-terminal region and PspA fusion proteins are localized on the bacterial outer-membrane 24
INT407 cell invasion of S. typhimurium with a series of plasmids expressing PspA fusion proteins 29
Antibody responses in mice immunized with S. typhimurium BRD509 expressing PspA delivery plasmid 31
Oral immunization with S. typhimurium BRD509 strain expressing SipB-PspA fusion proteins protects BALB/c mice against S. pneumoniae WU2 challenge 33
DISCUSSION 36
REFERENCES 40
ABSTRACT IN KOREAN 46
ACKNOWLEDGEMENT 47
