| Dental pulpitis, periapical diseases, and periodontitis are all caused mainly by anaerobic grame-negative bacteria. Endotoxins are primary virulance factors of these bacteria. So it would be beneficial for prephelexation and treatment of oral diseases to develop endotoxins-neutralizing agents. Endotoxin, or lipopolysaccharide, is a major component of the outer membrane of gram-negative bacteria. It mainly consists of three domains, namely O-polysaccharide, core oligosaccharide and lipid A. The lipid moiry is the molecular"endotoxic principle", and highly conserved among all genus. Lipid A is the ideal target for developing endotoxin neutralizer. Sequestrating and neutraling endotoxin activity by small molecules, especially peptides, is a prosperous field.1 Selection of lipid A-binding peptide from phage displayed peptide libraryLipid A was dissolved in chloroform:methanol(4:l) (Imgç©–L-1) and then diluted by 10-fold with methanol. The organic solution was applied to wells,sealed the wells with tape, and incubated with lipid A at room temperature for 3h, then they were evaporated to dryness. After three rounds of biopanning and amplification, selected clones were further analyzed by ELISA, compete ELISA and sequencing.The results of ELISA showed most of the third eluted phage clones bound lipidA specifically, as confirmed binding of lipid A to wells. Polymyxin B sulfate (20ng/well) was used to compete with phage clones binding lipid A, PMB could competely inhibit the binding of phage clones to lipid A, as further confirmed lipid A, coated with this method, was recognized by PMB, and the binding site of eluted phage clones was near that of PMB. Sequencing results showed one displayed peptide, named Al(HLFNVSHWLKKS), andPMB(mo-K'TK'-cyclo-[K'K'fLK'K'T]) had some similar amino acid combination(underlined charachaters). Searching most fitness short sequences in BLASTP at NCBI website and found similar short sequences in some proteins, which could interact with lipid A. Querying American patent datebase with all or part of Al amino acid sequences, no patent applied to protect this sequence, some patents applied to protect some amino acid sequence, which included LKK(leucine-lysine-lysine) combination, just like Al. Some important parameters were predicted with softwares.2 Lipid A-binding peptide's synthesized and its role in endotoxin-neutralizing processPeptide was solid-phase synthesized, Crude peptide was purified on a Qg colume. Purity and composition of the peptide was verified by HPLC and MS. The activity of the synthetic peptide during endotoxin-neutralizing proccess was evaluated using THP-1 cells and murine endotoxin shock model.The purity of the synthetic peptide was 98.56%. Molecular weight measured with mass spectrocopy was 1495.7, consist with theoritical MW, 1495.77.Under this culture condition, THP-1 cells didn't secrete TNF-a without additional added LPS, as confirmed endotoxin-free procedure was successful. TNF-a secretion from THP-1 cells increased in accordance with additional added LPS(?coli O111:B4, sigtna) concentration in cultural fluid. Secreted TNF-a amount was a linar function of LPS concemtration range from 8ng/ml to 200ng/ml, so 50ng/ml was pointed as stimulating concemtration of LPS. 20% FBS,PMB reduced endotoxin stimulated TNF-a secretion from THP-1 cells, 4ug/ml PMB5could reduce about 40% TNF-a secretion. THP-1 cell viability measuring with MTT method was lowed under high concerntration of PMB, but wasn't influenced when PMB concerntration not higher than 4u.g/ml. The synthetic peptide opsonized the capability of endotoxin to stimulate THP-1 cells under FBS-free condition, but this opsonic effect wasn't significant under 20% FBS. 20% FBS, the synthetic peptideenhanced the capability of polymyxin B to neutralize endotoxin.Pre-experimental results showed, sensitivity varied among mice speaces, C57BL/6J mice were more sensitive to endotoxin than Balb/c mice, Balb/c mice were more sensitive than Kunming mice. After sensitized with i.p. injec...
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