Research On Isolation, Purification,Immunological Activity Of Glycolipid Components From Bacille Calmette Guerin Cell Wall

Aim:To study the immunological activity of glycolipids on Mycobacterium tuberculosis, we systematically investigate immunological activity of glycolipids on Mycobacterium bovis Bacille Calmette Guerin (BCG) cell wall, separately from two areas of adjuvant and Antigen activity analysis. Antigenicity are mainly researched by two ways of cellular immunity and humoral immunity; Then we analyze that the glycolipids on BCG cell wall can be used as adjuvant to improve immunogenicity and protective effects of protein vaccine, compared with the existing adjuvant DDA or glycolipid adjuvant TDM.Method:Total glycolipids were extracted from BCG cell wall with chloroform: methnol (2:1), then glycolipid Ga and Gb were isolated and purificated repeatedly from total glycolipids by silica gel column chromatography and TLC. We immunized C57BL/6mice with glycolipids Ga and Gb, and DDA as adjuvant of Mycobacterial antigen Ag85B. Five weeks after final immunization, we detected the level of specific IFN-γ secreted by lymphocytes in spleen,and IgG1and IgG2b antibody to test the adjuvant effect of glycolipid Ga and Gb. Based on the previous total lipids and different purification methods, eight glycolipids were isolated and purificated repeatedly from total glycolipids by the methods including silica gel column chromatography, sephadex column chromatography and TLC. Then to test the antigenicity of these eight glycolipids, we detected the level of IFN-γ by ELISPOT in spleen lymphocyte which were from C57BL/6administrated BCG and the level of IgG by improved ELISA method. In addition, we also examined the level of IgG against the eight glycolipids in TB patients and healthy control to analyze the humoral immune response in TB patients.Further, we investigated the chemical structure characteristics of glycolipids on BCG cell wall. The chemical structure characteristics of Glycolipid Ga was focus analyzed by high-resolution mass spectrometry, infrared spectrometry(IR), nuclear magnetic resonance spectroscopy(NMR), and then according to our data and the previous report, we confirmed the structure and group.Result:Firstly, in DDA+Ga group IFN-γ secretion and the level of IgG1was lower than DDA adjuvant (P=0.071), and IgG2b was high; and the level of IFN-γ in DDA+Gb group was higher than BCG group(P=0.054). Secondly, five glycolipids (G1, G2, G4, G7, G8) obtained from the the BCG surface were potently induced a certain secretion level of IFN-γby spleen lymphocytes, and the IFN-γ secretion was similar to the control of Ag85B protein antigen. Finally, in TB patients the level of IgG against G2or G3was the highest in eight stimuli (P<0.05), and significantly higher than healthy control group (P<0.001). According to related reports and our date, Ga was chaining aliphatic ester.Conclusion:Glycolipids isolated from BCG cell wall in the research all have immune activity. Ga and Gb have immunomodulatory effects. G2, G3had the significant difference in specific IgG antibody in TB patients and healthy controls groups, and were expected to be the active TB diagnostic biomarkers.