Marker for diagnosing drug resistance of streptococcus pneumoniae to vancomycin and application of marker
A technology of Streptococcus pneumoniae and vancomycin, applied in the direction of microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as time-consuming, limited space, and large volume
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Embodiment 1
[0039] In the present invention, vancomycin-sensitive Streptococcus pneumoniae VSSP (or wild-type Streptococcus pneumoniae) is domesticated and treated with vancomycin to obtain the vancomycin-resistant Streptococcus pneumoniae VRSP, and then iTRAQ differential proteomics is used to compare and analyze VSSP and VRSP A total of 260 differentially expressed proteins (DEPs) with a differential fold greater than or equal to 2.0 and p<0.01 were identified in three independent replicates, of which 129 DEPs were up-regulated in VRSP and 131 DEPs were up-regulated in VRSP. expression decreased.
[0040] In order to further screen out the proteins potentially regulating VRSP formation, we performed KEGG pathway enrichment analysis on DEPs, and used cytoscape software to construct the interaction map between these DEPs and pathways, such as figure 1 As shown, the two-component system regulatory protein CiaR was significantly overexpressed in VRSP, while the two-component system ComD and...
Embodiment 2
[0042] To further verify the reliability of mass spectrometry data, we used RT-qPCR to detect the expression of ciaR, comD and comE in VRSP. like figure 2 Panel A shows that ciaR was significantly up-regulated in VRSP compared with VSSP, while comD and comE were significantly down-regulated in VRSP, consistent with the proteomic results.
[0043]It is known that the two-component regulatory systems CiaRH and ComDE are the key proteins regulating the competence formation of Streptococcus pneumoniae, and CiaRH is negatively correlated with competence, and ComDE is positively correlated with competence. Since the ability of Streptococcus pneumoniae to take up exogenous DNA (ie, transformation efficiency) is positively correlated with the formation of competence, the ability of VSSP and VRSP to take up exogenous DNA was compared and analyzed. like figure 2 As shown in panel B, whether it is plasmid DNA or PCR fragment DNA, the ability of VRSP to take up exogenous DNA is signif...
Embodiment 3
[0045] In order to further study the relationship between the two-component system response regulatory protein CiaR and Streptococcus pneumoniae resistance to vancomycin, the present invention constructed a ciaR overexpression strain (ciaR OE) and a ciaR deletion mutant (ciaR mutant), and used the time sterilization curve. The vancomycin tolerance of wild-type strains, ciaR overexpression strains and ciaR deletion mutants was compared and analyzed.
[0046] The result is as image 3 As shown in A, the present invention has successfully constructed a ciaR overexpression strain and a ciaR deletion mutant strain. like image 3 As shown in middle B, the same amount of wild-type strains, ciaR overexpression strains and ciaR deletion mutant strains were then taken and treated with 10×MIC vancomycin respectively, and the plates were sampled and counted at different time points. The survival rate of the expression strain was about 20 times higher than that of the wild type strain, w...
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