Isolation And Purification Of Peptidoglycan From Staphylococcus Aureus(ATCC25923), And Theirs Bioactivities.

IntroductionSepsis is systemic inflammatory response syndrome (SIRS) induced by infection,and bacterial infection keeps a high mortality about 95% in all pathogens. Sepsis shock triggered by gram-positive bacterium quickly increases in the past ten years,and the mortality already reaches 52.1% in all sepsis cases. The most common cause of gram-positive bacterial sepsis is peptidoglycan,which is the structural component of the bacterial cellwall.At present, peptidoglycan purchased is short of water solubility,and costly,thereby, this confine the study of gram-positive bacterial sepsis. So, we are going to found a simple and economic method for the soluble peptidoglycan isolation, which can provide subject to study the pathogeny and rivalrous measure of gram-positive bacterial sepsis.ObjectiveTo found a method for the soluble peptidoglycan isolation from Staphylococcus aureus, and identify that biologic activity.Methods1. Isolations of the s.aureus insoluble peptidoglycan:(1) the s.aureus insoluble peptidoglycan was gained by cold, hot and hypersound affection repetat of s.aureus(ATCC25923);(2) the s.aureus insoluble peptidoglycan was gained by 10% trichloracetic acid, 5% trichloracetic acid,NH4HCO3 buffer with trypsin digestion of s.aureus(ATCC25923) in order.2. Isolations of the s.aureus soluble peptidoglycan and quality control:(1) the s.aureus soluble peptidoglycan was released by short dose benzylpenicillin sodium and purified by sephadex G-100. (2) The quality control in the process of isolation: the soluble peptidoglycan was detected by SLP reagent kit; the LPS was detected by kinetic turbidimetric limulus test;and the protein was detected by improved LOWRY reagent kit.(3) The selection of the optimization condition in the course of the soluble peptidoglycan: the dosage of benzylpenicillin sodium, the incubation time of benzylpenicillin sodium addition and the bacterial concentration.3. The biologic activity identification of the s.aureus soluble peptidoglycan:(1) the expression of tumor necrosis factor alpha(TNF-α) in RAW264.7 cell after exposure to the soluble peptidoglycan(250,500,1000,1500ng/ml) were measured by ELISA kit;(2) The effect of the soluble peptidoglycan(40,60,80,100,200mg/kg) attacking mice were observed.Results1. The rate of return was 0.84% in the isolation of the insoluble peptidoglycan with cold,hot and hypersound affection repetat; and the rate of return was 1.02% with 10% trichloracetic acid, 5% trichloracetic acid,NH4HCO3 buffer with trypsin digestion in order.2. The soluble peptidoglycan is gained by short dose benzylpenicillin sodium and sephadex G-100; the SLP reagent kit revealed the prepared soluble peptidoglycan is peptidoglycan; the limulus test showed that the content of LPS in the prepared soluble peptidoglycan(10ng/ml) is lower (0.01ng/ml); the improved LOWRY reagent kit showed the content of protein in the prepared soluble peptidoglycan(1.0mg/ml) was 0.06mg/ml; the optimization condition is 0.75mg/ml of the dosage of benzylpenicillin sodium,1h of the incubation time of benzylpenicillin sodium addition and 5×108CFU/ml of the bacterial concentration.3. TNF-αwas released by RAW264.7 cell after exposure to the prepared soluble peptidoglycans, moreover, it revealed dose-effect relationship with the soluble peptidoglycan, and the effect is 1/15 of LPS; animal experiment showed that the 3-day survival rate of mice from the soluble peptidoglycan(100mg/kg) challenge keeps 40-50%.Conclusion1. The peptidoglycan can be gained by cold, hot and hypersound affection repetat and 10% trichloracetic acid, 5% trichloracetic acid,NH4HCO3 buffer with trypsin digestion in order, but the gained peptidoglycan was water insolublity and lower rate of return.2. The method of the soluble peptidoglycan isolation by short dose benzylpenicillin sodium and sephadex G-100 was founded; through the SLP reagent kit, the limulus test and the improved LOWRY reagent kit detecting, the prepared soluble peptidoglycan include shorter LPS and lower protein, its purity coefficient reach 93%.3. The prepared soluble peptidoglycan can activate RAW264.7 cell to release TNF-αin vitro; the causing death effect of mice from the soluble peptidoglycan(100mg/kg) challenge was obviously.