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YycG-PAS monoclonal antibody and application thereof

A technology of monoclonal antibody and application, applied in the direction of antibody, antibacterial drug, anti-enzyme immunoglobulin, etc.

Inactive Publication Date: 2016-12-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether blocking the YycG (histidine kinase) extracellular signal sensing region (PAS) of the YycG / F two-component signal transduction system can affect the formation of bacterial biofilms has attracted the attention of researchers in this field, but it is still unclear There is no literature report that antibodies to YycG / F two-component signal transduction system can inhibit staphylococcal biofilm formation

Method used

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  • YycG-PAS monoclonal antibody and application thereof
  • YycG-PAS monoclonal antibody and application thereof
  • YycG-PAS monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of YycG-PAS monoclonal antibody:

[0030] Amplify and amplify the pas fragment (nt 100 to 552 bp) of the yycG extracellular signal sensing region from the genomic DNA of Staphylococcus epidermidis ATCC 35984 strain, connect with the prokaryotic expression vector pET28a plasmid and express the DNA in Escherichia coli BL21 Fragment encoded YycG-PAS protein.

[0031] Using Ni 2+ The YycG-PAS protein was purified by affinity chromatography, TEV protease hydrolyzed protein tag, molecular sieve and other methods to prepare the recombinant YycG-PAS protein for immunized mice.

[0032]The present invention uses the PAS region (the 34th to 184th amino acid) of Staphylococcus epidermidis YycG protein as an antigen, and immunizes Balb / c mice to prepare a YycG-PAS monoclonal antibody. The measurement results show that the monoclonal antibody is small Mouse IgG.

Embodiment 2

[0033] Example 2 The ELISA method was used to detect the binding force of the YycG-PAS monoclonal antibody to the recombinant protein, and to detect its specificity in recognizing bacterium protein by immunospot method (Dot blot) and Western blot method (Western blot)

[0034] The ELISA detection method for the binding ability of YycG-PAS monoclonal antibody to YycG-PAS protein is as follows:

[0035] The monoclonal antibody with a stock solution concentration of 5 mg / mL was serially diluted 2-fold, and then added to the 96-well ELISA plate coated with YycG-PAS protein, and incubated at 37°C for 1 hour. After washing the plate repeatedly, add HRP-labeled goat anti-mouse secondary antibody and incubate at 37°C for 1 hour. After washing the plate repeatedly, add the chromogenic substrate to incubate the color development and terminate the reaction, and read the absorbance values ​​of each well at 450nm and 630nm.

[0036] Table 1 shows the ELISA detection results of YycG-PAS mo...

Embodiment 3

[0045] Example 3 YycG-PAS monoclonal antibody inhibits the formation of Staphylococcus epidermidis biofilm

[0046] Using trace bacterial biofilm detection test (96-well plate biofilm formation test), YycG-PAS monoclonal antibody was mixed with Staphylococcus epidermidis biofilm-forming positive strain ATCC 35984 strain (final concentration 80 μg / mL), and inoculated in 96-well plate on, 37°C for 6 or 24 hours. The bacterial biofilm was detected by the crystal violet staining method, and after washing with crystal violet staining, the strength of the bacterial biofilm formation was judged according to the A570 reading. The experimental method is as follows:

[0047] 1. Staphylococcus epidermidis was inoculated in fresh TSB medium and cultured overnight at 37°C.

[0048] 2. Inoculate the overnight cultured ATCC 35984 strain bacterium solution (3 μL) in a 96-well plate containing 80 μg / mL YycG-PAS monoclonal antibody (200 μl per well); normal mouse IgG (80 μg / mL) at the same co...

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Abstract

The invention belongs to the field of biotechnology, and relates to a monoclonal antibody capable of specifically being bound with extracellular region PAS region of YycG histidine kinases, and an application of the monoclonal antibody for preparing preparations for restraining formation of staphylococcus biomembranes. The monoclonal antibody adopts the PAS region of the staphylococcus epidermidis YycG histidine kinases as an antigen and is prepared from immunized mice. The monoclonal antibody can recognize protein located at YycG, and can restrain formation of the staphylococcus epidermidis. The monoclonal antibody disclosed by the invention can be used for preparing medicines for treating biomembrane-related diseases caused by staphylococcus, and YycG-PAS polypeptides of the monoclonal antibody can be used for preparing vaccines of resisting formation of staphylococcus biomembranes.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a monoclonal antibody for inhibiting staphylococcal biofilm formation, in particular to a monoclonal antibody capable of specifically binding to the PAS region of the extracellular region of YycG histidine kinase, and its preparation for inhibiting Use of a preparation for staphylococcal biofilm formation. Background technique [0002] Staphylococcus (including Staphylococcus epidermidis and Staphylococcus aureus) is an important pathogen that causes human biofilm infectious diseases. Implanted into the patient's body to chronicize and persist. Staphylococci that form biofilms are resistant to multiple antibiotics and clearance by the host's immune system. At present, there is no effective clinical treatment for bacterial biofilm disease. Once staphylococcal biofilm infection occurs, the only way to completely treat it is surgical removal or replacement of implanted medical m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40A61K39/40A61P31/04
Inventor 瞿涤吴旸
Owner FUDAN UNIV
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