Construction Of Mutant Strains Of Bacillus Subtilis And Their Effects On The Expression Of Immune Factors In Porcine Alveolar Macrophages(M?3D4/2)

Probiotics improve immune function of animals mainly through cellular and humoral immunity pathways,and promote animals' health growth.Bacillus subtilis is a kind of feed probiotics,which plays an important role in enhancing the immune capacity and activating the immune system.At present,little information is available about the effects of the Bacillus subtilis on the immune system of host.To examine the ability of Bacillus.Subtilis spores and vegetative cells to the effect the secretion of porcine alveolar macrophages cytokines.We first knocked out the spore germination gene cl W,ger D,cl W-ger D and sporulation gene spo0 A from chromosomal DNA of B.subtilis PY79,constructed the deletion mutants B.subtilis cl W-,B.subtilis ger D-,B.subtilis cl W-ger D-and B.subtilis spo0A-,extracted of genomic DNA for PCR detection;The spore germination ability of spore germination mutant strains and the sporulation ability of spore mutant strain were evaluated by Gram staining and germination rate detection;The secretion expression level of cytokines was detected by ELISA test kit after B.subtilis cl W-ger D-and B.subtilis spo0A-respectively cocultured with the porcine alveolar macrophages.The results were shown as follows:(1)we successfully deleted the spore germination gene cl W,ger D,cl W-ger D and sporulation gene spo0 A,constructed the spore germination mutants B.subtilis cl W-,B.subtilis ger D-,B.subtilis cl W-ger D-and the sporulation mutant B.subtilis spo0A-.(2)The results of the sporulation ability and sporulation ability shown that B.subtilis cl W-ger D-completely deprived spore germination ability,B.subtilis cl W-and B.subtilis ger D-still have the ability of germination.Microscope observation and spore production rate test indicated that B.subtilis spo0A-completely lost sporulation ability.(3)In coculture experiment,B.subtilis cl W-ger D-significantly increased the expression of cytokine IL-6and IL-1?(P < 0.01).B.subtilis mutants had no effect on the expression of cytokines after LPS stimulated macrophage.Therefore,Bacillus.Subtilis spores and vegetative cells could regulate the secretion of macrophage cytokines.