| Objective:In order to observe the difference parasitism and activity of Brucella in the same host cells and host individuals more intuitively,the aim of this test is to reconstruct the eukaryotic expression vector p MC-221-RFP of Brucella by using overlapping extension PCR technology to reconstruct the eukaryotic expression vector of Brucella,which is red fluorescent protein,p MC-221-GFP,and can be obtained by electrical transformation.The Brucella strain M5 line(hereinafter referred to as M5-p MC-221-GFP)and the 16 M strain(hereinafter referred to as 16M)that can stabilize the expression of red fluorescent protein(hereinafter referred to as16M-p MC-221-RFP),which can stabilize the expression of red fluorescent protein(hereinafter referred to as 16M-p MC-221-RFP),can be used to identify the murine macrophages by combining two Brucella species with M5-p MC-221-GFP and 16M-p MC-221-RFP,and we can identify Brucella under laser confocal microscopy.The ability of bacteria 16 M and Brucella M5 to infect the same host cell is different from each other.At the same time,this experiment provides a visible tool for subsequent studies of Brucella and provides a solid foundation for visual tools.Methods:1.In this experiment,the ribosome binding site,red fluorescent protein fragment(hereinafter referred to as RBS-RFP fragment)and the specific DNA fragment of Brucella(hereinafter referred to as BDNA fragment)were obtained by PCR,and RBS-RFP-BDNA overlapped fragments were obtained by optimizing the reaction conditions of overlapping extension PCR,and RBS-RFP-BDNA overlapped fragments were connected to p LB vector and Sm was used in Sm.A I and Sac II have double enzyme cut on the recombinant vector,which connect the product of the enzyme cut glue to the fragment obtained by the same two enzymes and double enzyme digestion,and convert the Escherichia coli to obtain the p MC-221-RFP plasmid.2.In this experiment,the p MC-221-GFP plasmid preserved in our laboratory and the p MC-221-RFP plasmid obtained in this experiment were respectively transformed into M5 strains of Brucella and 16 M strain of Brucella,respectively.The resistance screening and the identification of bacterial liquid PCR were used to obtain the M5-p MC-221-GFP and16M-p MC-221-RFP positive strains.3.The infection experiment of mice was carried out by continuous culture of M5-p MC-221-GFP and 16M-p MC-221-RFP in F10 generation.The results showed that the two kinds of Brucella could express the corresponding fluorescence in the eukaryotic environment of mice.4.Using two Brucella of M5-p MC-221-GFP and 16M-p MC-221-RFP to infect mouse macrophages in equal proportion,only red fluorescence was found in the 28% cells of laser confocal image,69% cells only appeared green fluorescence,red fluorescence and green fluorescence appeared at the same time in 3% cells,and red and green overlapping orange yellow region appeared.5.The mouse macrophages were infused by M5-p MC-221-GFP first,then 16M-p MC-221-RFP was added to the infection.The laser confocal images showed that all the cells appeared green fluorescence,but 11% of the cells appeared red and green at the same time,and the red and green overlapping orange area appeared.6.The mouse macrophages were infused by 16M-p MC-221-RFP first,then M5-p MC-221-GFP was added to the infection.The laser confocal pictures showed that all the pictures were only red fluorescence.Results:1.Using the overlap extension PCR technology after optimized conditions,we successfully obtained the red fluorescent protein Brucella eukaryotic expression vector p MC-221-RFP.2.By using electric conversion technology,the M5 strain M5-p MC-221-GFP,which can express green fluorescent protein under eukaryotic conditions,and 16M-p MC-221-RFP of Brucella16 M,which can express red fluorescent protein,have been successfully obtained.3.The infection experiment of mice was carried out by continuous culture of M5-p MC-221-GFP and 16M-p MC-221-RFP in F10 generation.The results showed that the two kinds of Brucella could express the corresponding fluorescence in the eukaryotic environment of mice.4.Using two Brucella of M5-p MC-221-GFP and 16M-p MC-221-RFP to infect mouse macrophages in equal proportion,only red fluorescence was found in the 28% cells of laser confocal image,69% cells only appeared green fluorescence,red fluorescence and green fluorescence appeared at the same time in 3% cells,and red and green overlapping orange yellow region appeared.5.The mouse macrophages were infused by M5-p MC-221-GFP first,then 16M-p MC-221-RFP was added to the infection.The laser confocal images showed that all the cells appeared green fluorescence,but 11% of the cells appeared red and green at the same time,and the red and green overlapping orange area appeared.6.The mouse macrophages were infused by 16M-p MC-221-RFP first,then M5-p MC-221-GFP was added to the infection.The laser confocal pictures showed that all the pictures were only red fluorescence.Conclusion:1.In this experiment,a p MC-221-RFP vector was successfully constructed and combined with p MC-221-GFP.By electric transfer,a M5-p MC-221-GFP strain expressing a stable point of green fluorescent protein and a 16M-p MC-221-RFP strain capable of expressing red fluorescent protein were obtained.2.We found that when Brucella M5 strain and 16 M strain infected multiple mouse macrophages,97% of the cells were infected only by a single type of Brucella,and the infection ability of Brucella 16 M strain was stronger than that of Brucella,but at the same time,3% murine macrophages could be successfully infected by two Brucella.3.The study found that when Brucella M5 strain and 16 M strain infected mouse macrophages successively,all of the cells were infected successfully by Brucella M5 strain,and then 11% of the macrophages could continue to be infected by Brucella 16 M strain after being successfully infected by Brucella strain M5 strain.4.The experimental study found that when Brucella 16 M strain and M5 strain infected mouse macrophages successively,all the cells were infected successfully by Brucella 16 M strain,but all the infected cells could not continue to be infected successfully by Brucella M5 strain. |
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