Study Of Peptides Mimetic To The Cross Reacitve Epitope Of Lipopolysaccharide

Sepsis caused by gram negative bacteria infections is one of the most dangerous causes leading to death in the clinic, and so far the mortality from it has not declined despite the use of supportive therapy and many kinds of antibiotics. Lipopolysaccharide(LPS), ie, endotoxin, which is the component of the cell wall of gram negative bacteria, has been considered as an very important factor starting the pathologic cascade leading to multi- organ failure and death. Lipid A and the inner core(KDO)are the conservative structures and toxic sites of LPS,which may be cross reactive between different bacteria. Up to now, there is no ideal antagonists against LPS or lipid A for clinical prophylaxis and therapy .Although there were some evidences that the antibodies against LPS are protective and the amount of antibody against core glycolipid of LPS in vivo is correlated with the survival in sepsis patients, the time phase of antibody application is hard to master and repeated use of antibody may lead to the occurrence of allergy. Therefore,establishment of active immune against LPS is very necessary. But the major difficulties of preparing LPS vaccine come from the facts that (1) LPS is a kind of I cell independent antigen(TI antigen) ,which can not elicit effective immune response and immunological memory ;(2) there are so many kinds of the serotypes in LPS that makes it hard to get broad spectrum vaccines to all serotypes of LPS. However,ideal vaccines should be able to elicit the effective immune response,especially the secondary immuneresponse and to provide broad spectrm protection. Our objective is to screen the peptide mimotopes of the conservative epitopes of LPS. The significance of it lies in the following aspect: First, it will change the properties of lipopolysaccharide antigen into peptide epitope,ie, change the T cell independent antigen (TI antigen) into T cell dependent antigen(TD antigen), so that it will be able to induce effective immune response and immunological memery. Second,it is possible for the mimotopes of LPS conservative epitopes to elicit broad spectrum protections against different strains of bacteria or different serotypes of LPS. Third, by screening peptide library with an antibody specifically against the conservative epitopes, it is possible to get novel LPS antaganists, or at least, to get lead compounds which can mimick the epitopes binding to LPS recepters and as a result can inhibite the binding between LPS and LPS recepter. In this work ,we first prepared a monoclonal antibody specifically against LPS conservative epitope, which can cross react with either S.typhimurium T8-61 LPS or E.coli. O111:B4 LPS and can cross react with either P. Aeruginosa or Brucella bacteria ,also which can provide cross protection against the attacks challenged by deadly amount of E.coli.O 111 B4. Then we screened the mimotopes of the conservative epitopes of LPS with this antibody from two bacteriophage displayed peptide libraries, 1 5mer and l2mer peptide library. This work would be expected to be the basis for developing such kinds of mimotope peptide as novel vaccines or antaganists and to get some experiences for the investigations in peptide mimotopes for non-peptides epitope. This reserch can be divided into the following four parts. Part I: Preparation and identification of LPS and antibody against the conservative epitope of LPS. In this part, we prepared E.coli.O1 11 B4 LPS and S.Typhi.T8-61 LPS with high...