| 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. 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 , but the time phase of antibody application is hard to master. Therefore, establishment of active immune against LPS which means to develop a vaccine is very necessary.There are two major difficulties in preparing effective LPS vaccine with broad spectrum protection: (1) LPS is a kind of Thymus-independent antigen (TI antigen), which can not elicit secondary antibody response and high affinity antibodies with strong opsonization; (2) there are too many kinds of specificities of strains and serotypes in Gram negative bacteria with LPS. In this work , peptides multi-mimotopes of LPS displayed on phage were screened from phage display peptide library by using monoclonal and polyclonal anti-LPS antibodies with cross protection against G" bacteria infection, and the peptides mimicry to LPS epitope were synthesized with Fmac amino acide and expressed in E. coli ,which were estimated for a possibility to induce production of anti-LPS antibody. Our objectives are to change LPS as TI antigen to peptide mimotope of LPS as TD antigen, and induce the cross protection against LPS.This research can be divided into the following four parts.Part I Preparation and identification of monoclonal and polyclonal antibodies with cross protection against LPS from different bacteriaIn this part, rabbits were immunized with S. typhi. treated specially . By this way, we got polyclonal antisera with cross reactions with LPS from different strainsof bacteria, such as S. typhi. LPS(L7261) and E.coli. LPS(L2630), especially LPS with conservative structure, e.g. LPS from strain S. minne. Re595 (L9764) and LipidA(L0744&L5399).In protection test in mice attacked with bacteria, the result showed that the antiserum can protect mice from infection (LD90) either by E. coli. (P<0.005 vs control) or by P. pyocyanea (PO.05 vs control). Monoclonal and polyclonal antibodies with high activity were prepared by affinity chromatography , and can be used as targets for screening.Part II Screening and identification of the mimotopes for LPS multi-epitopes from phage displayed peptide librariesPeptide motopes were screened from The cyclic 7mer phage displayed peptide library was screened with purified mAb to S. Typhi. LPS. 34 of selected positive showed excellent binding to mAb 1B6 by identified using ELISA. The binding of positive clones to 1B6 can be inhibited by S. typhi. LPS at a concentration of 0.5ug/ml by competitive inhibition assay. The amino acid sequences of peptide deduced from the corresponding DNA sequences of 10 of the positive clones were PSWASFW(3/10), PEWASSW(3/10), PAWASMW(1/10), MSWFPPY(1/10), QSWFLPY(l/10)and PPGWPGF(1/10).In order to obtain peptides mimetic to multi- epitopes of LPS, the same peptide library was used for screening of mimotopes with purified polyclonal antibody to S. Typhi. LPS. 20 of positive clones were identified as positive clones. The binding of all positive phage clones to polyclonal antibody can be inhibited by S. typhi. LPS at a concentration of 0.5fJ,g/ml by competitive inhibition assay. The amino acid sequences of peptide deduced from the corresponding DNA sequences of 10 of the positive clones were FQFYPAA(4/10) , LQFYPGA(1/10) , LFTFAHY(3/10) YQYYPAA(2/10).Mice were immunized with four phage clones(PSWASF\V\ QSWFLPY, FQFYPAA and YQYYPAA) respectively and also with mixed phage clones of the above four. Only a part of mice immunized with clones QSWFLP Y , FQFYPAA and mixed clones produced anti-LPS with titer of 1:100-1:800 in serum. Mice of immunized with other antigens and control group did not produce anti-LPS. These results suggested that peptides QSWFLPY and FQFYPAA dis...
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