Fundamental studies on the molecular therapy for botulisms

Research Project

Project/Area Number	08456146
Research Category	Grant-in-Aid for Scientific Research (B)
Allocation Type	Single-year Grants
Section	一般
Research Field	Basic veterinary science/Basic zootechnical science
Research Institution	Iwate University
Principal Investigator	SYUTO Bunei Iwate University, Veterinary Mdicine, Professor, 農学部, 教授 (60001533)
Co-Investigator(Kenkyū-buntansha)	INANAMI Osamu Hokkaido University, Veterinary Medicine, Associate Professor, 大学院・獣医学研究科, 助教授 (10193559)
Project Period (FY)	1996 – 1997
Project Status	Completed (Fiscal Year 1997)
Budget Amount *help	¥7,300,000 (Direct Cost: ¥7,300,000) Fiscal Year 1997: ¥2,200,000 (Direct Cost: ¥2,200,000) Fiscal Year 1996: ¥5,100,000 (Direct Cost: ¥5,100,000)
Keywords	botulisms / neurotoxin / chimera / antitoxin / Fab / neutralization / subunit / binding fragment / ボツリヌス神経毒素 / キメラ分子 / 神経細胞内指向性抗体 / キチン結合性タンパク質 / 分子治療法 / ボツリヌス毒素
Research Abstract	Botulisms is a very serious disease with high mortality. In many cases antitoxin therapy is applied, but it is not always effective after intoxication, because toxin molecules invade into inside of nerve terminals, where antitoxin molecules are inaccessible. To neutralize the toxin invaded into inside of the cell, it is necessary to develope the novel antitoxin molecules penetrable into inside of the cell like toxin molecules. Botulinum type C toxin (strain Stockholm) molecule consists of a heavy chain and a light chain with a disulfide linkage between them. The C-terminal region of the heavy chain binds to nerve ending, and N-terminal region forms a chanel for the translocation of the light chain accross the cell membrane. Based on these results, we attempted to prepare the chimera molecules of toxin heavy chain and anti-light chain Fab or IgG molecule. In both cases chimera molecules, toxin heavy chain (Hc) with anti-toxin light chain Fab and Hc with anti-light chain IgG, were effective 2.5-fold comparing with the mixture of Hc and anti-light chain F(ab')2 or Hc and anti-light chain IgG.The recoveries of chimera molecules were about 5 96 in polyclonal antibodies and 10 % in monoclonal antibody, respectively, Application of the binding fragment instead of Hc reduced a neutoralization activity. The use of other type of toxin heavy chain was also investigated in order to avoid the aitibody production by the repeted application by using type D toxin from strain South African (DSA). DSA toxin heavy chain-used chimera molecules neutralized type C toxin at the same level. A candidate molecule of the toxin carrier present in the serum was isolated and characterized. The purified protein is a glycoprotein containing mannose, and has a molecular size of 1800kDa consisting of two subunits with the molecular weights of 85,000 and 30,000, respectivly. Preinjection of this protein raises the lethality of toxin to 1.2-fold.