The Fluorescent Characterization And Analysis Of Macrophagocytes Infected With Two Vaccine Strains Of Brucella

Object:We had constructed the stable expression GFP of conformity Brucella M5(GFP-M5) and S19(GFP-S19); compared the survival ability of GFP-M5 and GFP-S19 with normal Brucella M5 and S19 in macrophage, to prove that the insertion of GFP gene will not infect the further step of experiment. To give the experiment basis of survival, reproduction and pathogenic mechanism of Brucella in mouse and cells, we had analyzeed the quantity of positive yellow fluorescent protein of GFP-M5 and GFP-S19 in lysosome, ER and golgi complex after the combination; we had analyzed the quantity of GFP-M5 and GFP-S19 infect the murine macrophage successfully in the early stage; we had analyzed the lost of green fluorescent gene expression of GFP-M5 subculture in specific time; we observed the fluorescent expression of mouse spleen section after GFP-M5 infection. Method:1. pMC-221 carrier entered the competent cell of Brucella M5 and Brucella S19 by electrotransformation. We had obtained the GFP-M5 and GFP-S19 after successful detection.2. Normal Brucella M5,S19 and GFP-M5,GFP-S19 were cultrured to log phage, collected the bacteria. Cells were infected by Mc FarLand with bacteria in the ratio of 1:100. To compare the survival ability of normal Brucella and GFP-Brucella by plate count method.3. The combination of GFP-M5 and GFP-S19 with lysosome, ER and golgi complex were observed by laser scanning confocal microscope and analyzed the quantity of positive yellow fluorescent protein.4. The percentage of GFP+ macrophage in different stage after GFP-M5 and GFP-S19 infecting the murine macrophage respectively were detected and analyzed by using flow cytometer.5. GFP-M5 were subcultrued in the liquid Brucella culture medium with chlorampenicol resistant and without chlorampenicol resistant respectively. After infecting murine macrophage by the bacteria of different subculture stage, the lost of gene of green fluorescent protein were detected and analyzed by using flow cytometer.6. After GFP-M5 and GFP-S19 infecting mouse, the spleen were removed for photography, analyzed and sectioned for observation under fluorescence microscope. Result:1. The PCR test of electrotransformational bacteria indicated that pMC-221 was transformed into two kinds of brucella are successful.2. Though the result of CFU, in different time phage GFP-M5, GFP-S19 and normal M5, normal S19 had’t show significant difference, in terms of quantity of bacteria. In addition, the ability of bacteria infect macrophage had not relate with the existence of pMC-221 carrier.3. After the infection of macrophage by GFP-M5 and GFP-S19, we had observed, the result reveal two bacteria have the same ability to combine with mice organelle in the macrophage by laser scanning confocal microscope in 1h, 2h, 3h and 4h. This result had proved that two bacteria have same infection, replication and reproduction ability in macrophage in 4h.4. Through the flow cytometer, two bacteria already entered into the cell in 30 min. In the infection process form 30 mins to 6h, the infection ability of two bacteria hadn’t show significant difference which indicate that two bacteria had same infection and reproduction ability in macrophage in 6h.5. Through the fluorescent inverted microscope, murine macrophage hadn’t express the green fluorescent protein after infected by normal M5 bacteria. However, the green fluorescent protein could be found in macrophage which infected by GFP-M5, means the pMC-221 carrier express successfully. Through macrophage infected by GFP-M5 in different time, using flow cytometer to detected the percentage of GFP+ macrophage, the result showed the existence of chlorampenicol resistant in culture medium would not affect the lost of green fluorescent protein gene. Moreover, the GFP-M5 hadn’t found any green fluorescent protein gene lost expression within 33 days subculture, indicated that, in this time period the expression and observation of fluorescent protein hadn’ affected. Thus, given a good basis of study of GFP- Brucella infected mice which can directly observe the GFP- Brucella by the mice organ.6. From the photography of mice spleen, mice which infected by GFP-M5 had darken spleen comparing with normal mice. Through the observation of spleen section from infected mice, the Brucella which had expressed the green fluorescent protein could be found in the GFP-M5 and GFP-S19 infected mice spleen. It means that Brucella already infect the macrophage in mice spleen. Conclusion:This result proved that two bacteria had same infection, replication and reproduction ability in macrophage in early days; In 30 days, the expression of GFP gene fragment was not be lost, this study that Brucella infected vivo animals which can express green fluorescent provides a the experiment basis; after Brucella infecting mice a week, spleen of mice under fluorescent microscope could be observe Brucella which can express green fluorescence, which laid the foundations for study in Brucella infected vivo animal on fluorescence Characterization level.