枯草芽孢杆菌突变株的构建及其对猪肺泡巨噬细胞(MΦ3D4/2)免疫因子表达的影响

Construction of Bacillus Subtilis Mutant Strain and its Effect on Immune Factor Expression of Porcine Alveolar Macrophages(MΦ3D4/2)

试验旨在研究枯草芽孢杆菌(Bacillus subtilis)不萌发芽孢和不生孢营养体对猪肺泡巨噬细胞(MΦ3D4/2)免疫因子表达的影响。利用同源重组敲除B.subtilis PY79芽孢萌发基因clW-gerD和生孢早期基因spo0A,构建芽孢不萌发突变株B.subtilis clW-gerD-和不生孢突变株B.subtilis spo0A-,提取基因组DNA进行PCR检测;结合革兰氏染色和萌发率检测,分别对构建的B.subtilis芽孢不萌发突变株和不生孢突变株进行萌发和生孢能力鉴定;将B.subtilis clW-gerD-和B.subtilis spo0A-分别与猪肺泡巨噬细胞共培养,用ELISA试剂盒检测细胞因子分泌表达水平。结果表明:(1)成功获得不萌发突变株B.subtilis clW-gerD-和不生孢突变株B.subtilis spo0A-;(2)萌发和生孢能力检测结果显示,B.subtilis clWgerD-完全丧失芽孢萌发能力;显微镜观察和生孢率检测表明不生孢突变株B.subtilis spo0A-丧失了生孢能力;(3)B.subtilis clW-gerD-极显著的促进了细胞因子IL-6的表达(P<0.01);B.subtilis clW-gerD-、B.subtilis PY79和B.subtilis spo0A-对细胞因子IL-1β、IL-10和IL-8的表达量无显著影响(P>0.05)。B.subtilis clW-gerD-可刺激巨噬细胞分泌促炎因子IL-6,增强MΦ3D4/2细胞的炎症反应,提高机体的抗感染能力。

To examine the ability of Bacillus.Subtilis spores and vegetative cells to affect the secretion of porcine alveolar macrophages cytokines,we first knocked out the spore germination gene clW-gerD and sporulation gene spo0A from chromosomal DNA of B.subtilis PY79,constructed the deletion mutants B.subtilis clW-gerD-and B.subtilis spo0A-,extracted 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 clW-gerD-and B.subtilis spo0A-respectively cocultured of the porcine alveolar macrophages.We successfully deleted the spore germination gene clW-gerD-and sporulation gene spo0A,constructed the spore germination mutants B.subtilis clW-gerD-and the sporulation mutant B.subtilis spo0A-.The results of the sporulation ability and sporulation ability showed that B.subtilis clW-gerD-of three spore germination mutants completely deprived spore germination ability.Microscope observation and spore production rate test indicated that B.subtilis spo0A-completely lost sporulation ability.In coculture experiment,B.subtilis clW-gerD-significantly increased the expression of cytokine IL-6(P<0.01);Whereas the expression of IL-1β,IL-10 and IL-8 showed a less pronounced increase after B.subtilis clW-gerD-,B.subtilis PY7 and B.subtilis spo0A-interacted with macrophage(P>0.05).In summary,B.subtilis clW-gerD-could stimulate the macrophage secretion of proinflammatory factor IL-6,strengthen inflammatory reaction of MΦ3D4/2 cells and improve the ability of anti-infection.