학술논문
살모넬라 엔테리티디스와 소 로타바이러스에 대한 다가면역원성 세균외막소포 백신 개발 / DEVELOPMENT OF MULTI-IMMUNOGENIC OUTER MEMBRANE VESICLE VACCINE AGAINST SALMONELLA ENTERITIDIS AND BOVINE ROTAVIRUS
Document Type
Dissertation/ Thesis
Author
이성 / Lee, Seong
Source
Subject
Language
Korean
Abstract
CHAPTER Ⅰ. 저독성 LPS를 생성하는 SALMONELLA ENTERITIDIS 유래 돌연변이주 개발Lipopolysaccharide (LPS)의 독성을 저하시킨 세균외막소포 (OMV) 백신 플랫폼 개발을 위하여, msbB 유전자를 변이시킨 Salmonella enteritidis 변이주를 개발하였고 그 특성을 규명하였다. 실험 결과, S. enteritidis msbB 유전자 변이주의 LPS 구조가 변형되었는데, 특히, 균체내독소의 핵심인 lipid A의 구조가 변화되었음을 확인하였다. 이러한 lipid A의 구조 변화는 lipid A 전구체에 myristic acid의 결합을 촉매 하는 MsbB 효소의 결핍에 의한 것으로 판단되어진다. 전자현미경 관찰 결과에서는, S. enteritidis msbB 유전자 변이주가 정상균주에 비하여 월등히 많은 OMV를 생산하고 있는 것으로 나타났으며, BALB/c 마우스에 접종하여 균체내독소 활성을 측정한 결과, S. enteritidis msbB 유전자 변이주가 정상균주에 비하여 현저히 독성이 낮아져 있음을 확인할 수 있었다. 결론적으로 LPS 독성을 저하시킨 살모넬라 변이주 개발을 통하여 S. enteritidis에 대한 OMV 백신 개발의 기틀을 마련하였다.CHAPTER Ⅱ. 소 로타바이러스(BOVINE ROTAVIRUS) 항원결정기를 포함한 다가면역원성 세균외막소포의 개발OMV를 이용하여 살모넬라와 소 로타바이러스 (BRV)를 동시에 방어할 수 있는 면역원을 개발하기 위하여, LPS의 독성을 저하시킨 S. enteritidis 변이주에서 BRV 항원결정기를 발현시키고 그로부터 OMV를 추출하여 면역원성 시험을 실시하였다. pBAD24 발현 벡터를 이용하여 S. enteritidis 유래 OmpA 단백질에서 BRV VP8 항원 결정기가 발현될 수 있는 체계를 구축하였고 이로부터 유도된 OMV를 SDS-PAGE와 western blot으로 확인한 결과, 50kDa의 OmpA::BRV VP8 chimeric protein이 성공적으로 발현하여 anti-BRV monoclonal antibody와 결합하는 것으로 나타났다. 또한 발현된 OmpA::BRV VP8 재조합 단백질이 세포 외막에 삽입되어 있음을 확인하였다. 그리고 이로부터 얻어진 OMV를 BALB/c 마우스에 면역한 결과, OMV 면역군의 혈청 IgG가 S. enteritidis를 면역한 것과 동일한 수준으로 검출 되었으며, 이 혈청을 이용한 serum bactericidal assay를 통하여 OMV 면역군의 혈청에서 S. enteritidis을 중화할 수 있는 항체가 있는 것을 확인하였다. 또한 면역한 마우스에 challenge test를 실시한 결과, 비면역군에 비하여 OMV를 면역한 군의 비장 및 간 등에서 현저하게 S. enteritidis의 증식이 억제되었다. BRV에 대한 중화항체 생성은 OMV 면역군의 혈청에서 비면역군에 비하여 8배 정도 높은 역가를 나타내었다. 이러한 결과들을 종합하면, LPS의 독성을 저하시킨 S. enteritidis 변이주에서 BRV 항원결정기를 발현시키고 그로부터 추출한 OMV를 이용한 면역원성 시험을 수행하여 S. enteritidis는 물론, BRV를 중화할 수 있는 항체 생성을 유도할 수 있음을 확인함으로서, OMV를 이용한 다가면역원성 백신 개발의 가능성을 확인하였다.
CHAPTER I. DEVELOPMENT OF LOW ENDOTOXIC SALMONELLA ENTERITIDIS MUTANTFor development of outer membrane vesicle (OMV) vaccine platform showing a low endotoxicity of its lipopolysaccharide (LPS) component, an msbB mutant of S. enteritidis was constructed and characterized. In the results, LPS structure of the msbB mutant of S. enteritidis was changed, especially in the lipid A portion, according to lack of a myristate addition in the lipid A molecules, which catalyzed by MsbB activity. In electron microscopic observations, the mutant S. enteritidis produced more OMVs than wild type. As expected, the msbB mutant of S. enteritidis expressed low endotoxic LPS in a lethality test. In conclusion, creation of msbB mutation caused a significant reduction of LPS endotoxicity in the Salmonella. Therefore, these results would provide a concrete basis for the development OMV vaccine against S. enteritidis.CHAPTER II. DEVELOPMENT OF OUTER MEMBRANE VESICLES CONTAINED WITH BOVINE ROTAVIRUS EPITOPEFor development of multi-immunogenic OMVs derived from the low endotoxic S. enteritidis mutant, an expression vector carrying the gene for OmpA::BRV VP8 epitope fusion protein was constructed and introduced into the Salmonella msbB mutant. To express the BRV epitope in the outer membrane of the Salmonella, the expression vector was genetically engineered for the fusion of OmpA with the BRV epitope so that the chimeric protein tagged into the OmpA was intended to be expressed under the control of an arabinose-inducible pBAD24 promoter. The expected expression of the chimeric protein in the outer membrane was verified with the OMV and outer membrane fraction extracted from the S. enteritidis carrying the expression vector. The mice were immunized with the OMVs tagged with the BRV epitope, and the immune responses were monitored by measuring specific antibodies raised against the BRV epitope as well as Salmonella antigens in the OMV-immune sera.In the results, OMVs derived from the msbB mutant of S. enteritidis that transformed with the OmpA::BRV VP2 epitope-expressing vector were shown to be contained with about 50 kDa chimeric protein and the chimeric protein band was reacted with anti-BRV monoclonal antibody in western blot analysis. Sera from the mice immunized with either the msbB mutant of S. enteritidis or the OMVs showed strong antibody responses against S. enteritidis and recombinant BRV VP8 protein. Also, the OMV- and the mutant Salmonella-immunized mice showed a discrete bacterial clearance in the spleen and liver after the oral challenge with wild type S. enteritidis. In accordance, the immune-serum showed a significantly higher bactericidal activity than the non-immune control. In hemagglutination inhibition (HI) assay, there is an 8-fold increase of HI titer in OMV-immunized group than that of control against BRV. These results suggested that specific neutralizing antibody response was raised against BRV in the OMV-immunized mice, as well as the protective immune response against challenge of S. enteritidis.
CHAPTER I. DEVELOPMENT OF LOW ENDOTOXIC SALMONELLA ENTERITIDIS MUTANTFor development of outer membrane vesicle (OMV) vaccine platform showing a low endotoxicity of its lipopolysaccharide (LPS) component, an msbB mutant of S. enteritidis was constructed and characterized. In the results, LPS structure of the msbB mutant of S. enteritidis was changed, especially in the lipid A portion, according to lack of a myristate addition in the lipid A molecules, which catalyzed by MsbB activity. In electron microscopic observations, the mutant S. enteritidis produced more OMVs than wild type. As expected, the msbB mutant of S. enteritidis expressed low endotoxic LPS in a lethality test. In conclusion, creation of msbB mutation caused a significant reduction of LPS endotoxicity in the Salmonella. Therefore, these results would provide a concrete basis for the development OMV vaccine against S. enteritidis.CHAPTER II. DEVELOPMENT OF OUTER MEMBRANE VESICLES CONTAINED WITH BOVINE ROTAVIRUS EPITOPEFor development of multi-immunogenic OMVs derived from the low endotoxic S. enteritidis mutant, an expression vector carrying the gene for OmpA::BRV VP8 epitope fusion protein was constructed and introduced into the Salmonella msbB mutant. To express the BRV epitope in the outer membrane of the Salmonella, the expression vector was genetically engineered for the fusion of OmpA with the BRV epitope so that the chimeric protein tagged into the OmpA was intended to be expressed under the control of an arabinose-inducible pBAD24 promoter. The expected expression of the chimeric protein in the outer membrane was verified with the OMV and outer membrane fraction extracted from the S. enteritidis carrying the expression vector. The mice were immunized with the OMVs tagged with the BRV epitope, and the immune responses were monitored by measuring specific antibodies raised against the BRV epitope as well as Salmonella antigens in the OMV-immune sera.In the results, OMVs derived from the msbB mutant of S. enteritidis that transformed with the OmpA::BRV VP2 epitope-expressing vector were shown to be contained with about 50 kDa chimeric protein and the chimeric protein band was reacted with anti-BRV monoclonal antibody in western blot analysis. Sera from the mice immunized with either the msbB mutant of S. enteritidis or the OMVs showed strong antibody responses against S. enteritidis and recombinant BRV VP8 protein. Also, the OMV- and the mutant Salmonella-immunized mice showed a discrete bacterial clearance in the spleen and liver after the oral challenge with wild type S. enteritidis. In accordance, the immune-serum showed a significantly higher bactericidal activity than the non-immune control. In hemagglutination inhibition (HI) assay, there is an 8-fold increase of HI titer in OMV-immunized group than that of control against BRV. These results suggested that specific neutralizing antibody response was raised against BRV in the OMV-immunized mice, as well as the protective immune response against challenge of S. enteritidis.