| In this study,three strains of BCG-pasteur,△BCG 3112 and △BCG 3113 were cultured in the early stage,and the growth rate and uniform concentration of the bacteria were determined.Extraction of non-polar lipids from three strains by optimized lipid extraction.Then,the lipid components of the three strains were analyzed by thin layer chromatography,and then an adjuvant was prepared by comparing non-polar lipids of BCG-pasteur and △BCG 3112 strains.To study the effect of non-polar lipid adjuvants on the potency of tuberculosis vaccines,mice were immunized with BCG,BCG+BCG non-polar lipids and BCG+△BCG 3112 non-polar lipids respectively.The number of spleen lymphocytes producing IFN-γ in each group was detected by flow cytometry and ELISpot method..The concentration of Ig G and Ig M antibody was detected by ELISA.Finally,mice were immunized with H37 Rv strain.After 4 weeks,pathological sections of mice were observed to study the role of non-polar lipid adjuvants.The results of thin-layer chromatography showed that the non-polar lipid components of the three strains may include triacylglycerol,methylthiol di-imide,family protein PAT,diglyceride,free fatty acid,free mycolic acid,Glycosphingolipids,sulphur lipids I,sulphur lipids III,glyceryl myristate and trehalose dimycolate.By comparing the relative values of non-polar lipid shadows,it was found that the content of triacylglycerol in △BCG 3112 strain was significantly higher than that in BCG-pasteur(P<0.05).Therefore,the non-polar lipid of △BCG 3112 strain was used as adjuvant to study.At the same time compare the effect of BCG non-polar lipid adjuvant.After vaccination of mice,the number of T lymphocytes producing IFN-γ CD4 and CD8 was detected by flow cytometry.The results showed that there were no significant differences in CD4,CD8,and CD4/CD8 ratios between the blank control group,the BCG group,the BCG+BCG non-polar lipid group,and the BCG+△BCG 3112 non-polar lipid group(P>0.05).By PMA stimulation,the CD8 and CD4 T lymphocyte numbers of BCG group,BCG+BCG non-polar lipid group and BCG+△BCG 3112 non-polar lipid group produced IFN-γ were significantly higher than the blank control group(P<0.05),There were no significant differences in the number of CD8 and CD4 T lymphocytes between the BCG+BCG non-polar lipid group and the BCG+△BCG 3112 non-polar lipid group compared with the BCG group(P>0.05).By PPD stimulation,the number of CD8 and CD4 T lymphocytes producing IFN-γ in BCG+BCG non-polar lipid group and BCG+△BCG 3112 non-polar lipid group was significantly higher than BCG group and blank control group(P<0.05).There was no significant difference between the BCG group and the blank control group(P>0.05).The ELISpot experiment showed that the number of spots in the BCG+BCG non-polar lipid group and the BCG+ △ BCG 3112 non-polar lipid group was significantly increased compared with the BCG group(P<0.05).There was no significant difference between the BCG+BCG non-polar lipid group and BCG+△BCG 3112 non-polar lipid group,but the BCG+△BCG 3112 non-polar lipid group produced IFN-γ producing T lymphocytes higher than the BCG+BCG non-polar lipid group.The results suggest that both non-polar lipids of BCG and △BCG 3112 strains have the effect of improving the immune effect of BCG vaccine,and the effect of non-polar lipids of △BCG 3112 strain is better.The serum Ig G antibody level showed that compared with the blank control group,the BCG group,the BCG+BCG non-polar lipid group and the BCG+△BCG 3112 non-polar lipid group were significantly increased(P<0.05).In addition,the Ig G antibody levels in the BCG+BCG non-polar lipid group and the BCG+△BCG 3112 non-polar lipid group were significantly higher than BCG group(P<0.05),however,there was no significant difference in Ig G antibody concentration between the BCG non-polar lipid group and the BCG+△BCG 3112 non-polar lipid group.The results of Ig M antibody detection showed that the BCG group,BCG+BCG non-polar lipid group and BCG+△BCG 3112 non-polar lipid group were significantly higher than the blank control group(P<0.05),there were no significant differences in Ig M antibody levels between the BCG group,the BCG+BCG non-polar lipid group,and the BCG+△BCG 3112 non-polar lipid group(P>0.05).The above results suggest that the non-polar lipids of BCG and △BCG 3112 strain can increase the antibody titer of BCG vaccine,and then improve the humoral immune function of animals,while the non-polar lipid of △BCG 3112 strain has a better effect.The results of pathological section of mice showed that the BCG group,BCG+BCG non-polar lipid group and BCG+△BCG 3112 non-polar lipid group had significantly reduced lung hemorrhage and less red blood cells in the tissue compared with the attack group.There was a large amount of lymphocyte infiltration in the lung tissue of BCG group and BCG+BCG non-polar lipid group,and the lesions of alveolar space and epithelioid nodules did not improve.In the BCG+△BCG 3112 non-polar lipid group,the lymphocyte infiltration decreased and the alveolar space narrowed in the lung tissue,and no epithelioid nodules were formed in the lung.The results suggest that the BCG+△ BCG 3112 non-polar lipid group has the best protective effect on the lung of challenged mice. |
