| Objective:In this study, peptidoglycan(PG) was isolated from Bacillus subtilis PabO2 and analyzed its chemical composition.In order to provide theoretical basis for the development and application of microbial ecological agents and immunopotentiators, the effect on immune function of mice with different concentrations of PG was examined. Method: (1)Prepared Bacillus subtilis PG samples through TCA extracting and analyzed their amino acid composition; determined protein contents by using Bradford assay; determined polysaccharide contents by using phenol-sulfuric acid method; and qualified fat contents by vanillin assay.(2)Investigate the effect of PG on immune function of mice.Animal grouping:208 five week-old healthy Kunming Mice were randomly divided into four groups.Control group:0.5m10.9% asepsis normal saline intraperitoneal injection per day for 7days.Low dose group:0.5 ml 0.5 mg/ml Bacillus subtilis PG intraperitoneal injection per day for 7days.Middle dose group:0.5 ml 1mg/ml Bacillus subtilis PG intraperitoneal injection per day for 7days.High dose group:0.5 ml 2mg/ml Bacillus subtilis PG intraperitoneal injection per day for 7days. Several Immune Indexes were detected:①the immune organs quality,index and delayed type hypersensitivity were detected.②The level of serum lysozyme,serum hemolysin,murine spleen cell and antibacterial activity were detected by the spectrophotometry.③Peripheral blood and spleen T lymphocyte subsets were detected by flow cytometry.④The carbon dearance test for macrophage phagocytic function.⑤Spleen lymphocyte proliferation index were detected by MTT. Results:(1) PG in Bacillus subtilis cell wall was successfully extracted and purified by trichloroacetic method. Yield rate was 11.26%. The composition of amino acid in peptidoglycan samples was similarity with Bacillus subtilis. Chemical analysis indicates that PG was composed of 53.55% protein,32.98% sugar and 2.91% adipose. (2) Different concentrations of Bacillus subtilis PG were increased the Immune Indexes in mice:①PG group is absolutely increased immune organ weight and index,and showed significant dose-effect relationship.The foot-pad of delayed type hypersensitivity in PG group were Thicker than Control group, indicates that PG can promote the growth and development of immune organs, improve the body's immunity against specific antigens.②Different concentrations of Bacillus subtilis PG were increased the level of serum lysozyme,serum hemolysin,Murine Spleen Cell and antibacterial activity. It indicates that can improve the body PG specific and non-specific immunity.③Peripheral blood and spleen T lymphocyte showed the level of CD3+CD4+,CD3+CD8+,CD3+CD4+/CD3+CD8+ in PG group was higher than control group, clewed PG can improve the content of T lymphocyte subsets, thus maintaining a higher immunity.④The carbon dearance test showed PG can improve the macrophage phagocytic function.⑤PG can increased spleen lymphocyte proliferation index, so it can promote the growth of immune cells, thus contributing to the development of the immune system, improving immune function. |
