The Fluorescent Characterization And Analysis Of Macrophages Infected With GFP Transgene B. Melitensis

Object:Relatively stable integration of GFP-expressing recombinant Brucella16M and M5(hereinafter referred to GFP Brucella16M and M5) with normal Brucella16M and M5intracellular survival ability if there are differences. GFP Brucella16M and M5infection the lysosomes, endoplasmic reticulum and Golgi of the host cell and analysis of the fluorecence intensity produced.Ressearch GFP brucella16M and M5into murine macrophage produced GFP+cells and measurement GFP brucella16M and M5infection with intracellular lysosomes, endoplasmic reticulum, Golgi appara-tus combined time. Reproduction studies provide a theoretical basis for the survival of brucella in the cell and molecular mechanisms.Methods:The pMC-221vector was transformed into competent cells of brucella16M and M5,and then obtaining a stable passage GFP brucella16M and M5.By McFarland nephelometry to the desired concentration when GFP brucella16M and M5grown to log phase cells were collected.,according to the bacterial cell ratio of100:1and infection using the plate count method detecting the intracellular survival of capable. Observed GFP brucella16M and M5combined with intracellular lysosomes, Golgi and endoplasmic reticulum by Confocal microscopy,detection positive inclusions fluorescence intensity of GFP brucella16M and M5combined with various organelles.Measuring GFP brucella16M and M5infection into cells and combine with intracellular lysosomes, endoplasmic reticulum, Golgi apparatus of the first time.Results:(1)Successful obtain integration recombinant GFP brucella16M and M5,Compared to survial of normal brucella in murine macrophage has undifferentiated.(2) GFP brucella16M with lysosomal endoplasmic reticulum (ER) and the Golgi apparatus produced the fluorescent intensity of the initial equivalent to Brucella M5.(3) GFP brucella16M and M5infection into macrophages at10min, Brucella reach lysosomes at1.5h, Brucella arrive simultaneously in the endoplasmic reticulum (ER) and the Golgi apparatus at2h.Conclusion:(1)GFP brucella16M and M5in the early infection host cell and intracellular lysosomes, endoplasmic reticulum and Golgi apparatus the same time, both of which may belong to the same type I Brucella strains relevant standards.(2)GFP brucella reach endoplasmic reticulum and Golgi have same time,it may be have a relationship transfer membrane protein.(3)Although brucella16M and M5product the fluorescence intensity of the initial infection and GFP+macrophages were no significant differences, but intracellular survival results to the contrary. These results indicate that Brucella16M and M5into murine macrophages and reach lysosomes, endoplasmic reticulum and Golgi ability is no difference, the difference between the the ability of survive and replicate,this is pathogenic of intracellular the key reason for brucella.