Yeast내에서 탄저병 원인균인 Bacillus anthracis의 치사독소인 Lethal Factor 단백질 발현 = Expression of Anthrax Lethal Factor, a Major Virulence Factor of Anthrax, in Saccharomyces cerevisiae

국문 초록 (Abstract)

Bacillus anthracis는 탄저병의 병원체이다. 탄저병의 독소는 Bacillus anthracis가 가진 세가지 독소로 이루어져 있다. protective antigen (PA), lethal factor (LF)그리고 edema factor (EF)로 구성되어 있다. PA는 세포수용체와 결합하여 활성화 과정을 거친 후 LF 흑은 EF를 세포질 안으로 이동시켜 주는 역할을 한다. LF는 금속이온 $(Zn^{2+})$ 의존적 단백질 가수분해 효소로써 탄저병에 감염된 동물들의 치사독소로 작용하게 된다. 따라서 LF에 대한 특성 분석 및 억제재 개발에 관한 연구는 탄저치료제 개발에 매우 중요한 과정이라 할 수 있다. 본 연구에서는 탄저독소의 치료제 개발을 위해 선행되어야 하는 LF 고처리량 활성검증방법 및 저해제 선별에 더 높은 효율을 가지기 위해 이러한 시스템 방법 등을 이용하여 세포내 검정방법의 기초 자료를 마련하고자 하였다. 이를 위하여 yeast를 숙주로 한 LF 발현 vector의 구축과, 구축한 발현 시스템을 yeast에 형질전환 하여 plasmid의 안정성 및 LF유전자의 발현을 확인하였다. 본 연구는 LF유전자의 발현을 진핵세포 내에서 처음으로 시도했으며, 세포내 검증 시스템 도입의 기초적 자료를 제공하였다. Yeast내에서의 LF의 발현은 탄저병의 저해제 선별이나 활성측정검증을 생체 내에서 용이하게 할 수 있다는 가능성을 나타냈다.

다국어 초록 (Multilingual Abstract)

Anthrax is an infectious disease caused by the gram-positive bacterium, Bacillus anthracis. Anthrax toxin is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF onto the cytosol. LF is a zinc-dependent metalloprotease, which is a critical virulence factor in cytotoxicity of infected animals. Therefore, it is of interest to develop its potent inhibitors for the neutralization of anthrax toxin. The first step to identify the inhibitors is the development of a rapid, sensitive, and simple assay method with a high-throughput ability. Much efforts have been concentrated on the preparation of powerful assays and on the screening of inhibitors using these system. In the present study, we have tried to construct anthrax lethal factor in yeast expression system to prepare cell-based high-throughput assay system. Here, we have shown the results covering the construction of a new vector system, subcloning of LF gene, and the expression of target gene. Our results are first trial to express LF gene in eukaryote and provide the basic steps in design of cell-based assay system.

참고문헌 (Reference)

1 "a bacterial adenylatecyclase that increases cyclic AMP concentrations of eukaryoticcells" 3162-3166, 1982

2 "Toxins of Bacillus anthracis" 1747-1755, 2001

3 "The capsule of bacillus anthacis" 1999

4 "Structural basis forthe activation of anthrax adenylyl cyclase exotoxin by calmodulin" 396-402, 2002

5 "Protolyticinactivation of MAP-Kinase-kinase by anthrax lethal factor" 734-737, 1998

6 "Proteolyticactivation of receptor-bound anthrax protective antigen onmacrophages promotes its internalization" 251-258, 2000

7 "Production and purification of anthrax toxin Methods in Enzymology" Academic press 103-116, 1988

8 "Observations on experimentalanthrax demonstration of a specific lethal factor produced in vivoby Bacillus anthracis" 869-173 870, 1954

9 "Immunological analysis ofcell Associated antigens of Bacillus anthracis" 349-356, 1988

10 "Evidence for plasmid mediated toxin production in Bacillusanthracis" 371-376, 1983

11 "Effect of the lower molecular capsule released from the cellsurface of Bacillus anthracis on the pathogenesis of anthrax" 227-233, 2002

12 "Development of antibodies to protective antigenand lethal factor components of anthrax toxin in humans andguinea pigs and their relevance to protective immunity" 356-363, 1986

13 "Demonstration of a capsule plasmid in Bacillusanthracis" 291-297, 1985

14 "Clinical and epidemiologicalprinciples of Anthrax" 552-555, 1999

15 "Anthrax toxin protective antigen is activated by a cell surface proteasewith the sequence specificity and catalytic properties offurin" 10277-10281, 1992

16 "Anthrax lethal factor cleaves the N-terminusof MAPKKs and induces tyrosine/threonine phosphoryation ofMAPKs in cultured macrophages" 706-711, 1998