Antibody resistant to staphylococcus aureus and manganese ion binding protein C and application of antibody

A staphylococcus and protein-binding technology, which is applied in the fields of medicine and immunology, can solve the problems of aggravated and prominent bacterial drug resistance

Active Publication Date: 2020-02-28
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The long-term abuse of antibiotics has made the problem of

Method used

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  • Antibody resistant to staphylococcus aureus and manganese ion binding protein C and application of antibody
  • Antibody resistant to staphylococcus aureus and manganese ion binding protein C and application of antibody
  • Antibody resistant to staphylococcus aureus and manganese ion binding protein C and application of antibody

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Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1, expression and purification of Staphylococcus aureus manganese ion-binding protein C (MntC)

[0043] The MntC gene was amplified by PCR from genomic DNA from a S. aureus strain (ATCC accession number BAA-1556). After DNA sequencing proved it, it was expressed in Escherichia coli, cultured overnight at 37°C in LB medium containing ampicillin, and the cells were harvested by centrifugation. The MntC protein was purified by Ni-NTA, and the amino acid sequence of the MntC protein is shown in SEQ ID NO.1.

Embodiment 2

[0044] Embodiment 2, the separation of peripheral blood mononuclear cells (PBMC)

[0045] Recruit healthy volunteers and volunteers who recovered from severe infection of Staphylococcus aureus, collect venous blood samples in anticoagulant tubes containing heparin, and use density centrifugation to separate PBMC cells. Centrifuge at 400×g for 15 minutes; absorb the upper transparent plasma layer and store it at -80°C; after absorbing the supernatant, mix well with the same amount of RPMI1640 (Gibco), and slowly add the same amount of lymphocytes along the inclined tube wall Centrifuge the upper layer of the separation solution at 2000rpm for 20min, absorb the mononuclear cells in the cloud layer into a sterile centrifuge tube, add more than 5 times the volume of RPMI1640, centrifuge at 1000rpm for 5min, wash the cells twice, and resuspend the cells with an appropriate amount of RPMI1640 at 1×10 7 Each tube was frozen and stored in liquid nitrogen for later use.

Embodiment 3

[0046] Example 3, Sorting single plasma cells by flow cytometry

[0047] Using MntC protective antigen protein (HPLC purity > 95%) as the antigen, ELISA was performed on the serum to determine the antibody titer of the sample, and the sample with high antibody titer was selected, and single plasma cells were sorted by flow cytometry, and CD3 / CD14 / CD16 / CD235a-CD19+CD20+ / -CD38hiCD27hi set up gate sorting to separate plasma cell populations at different time points, the results are as follows figure 1 shown. Through serological experiments and B lymphocyte phenotype analysis, we can ensure that we can obtain a large number of single plasma cells from >3% of the plasma cell population, and isolate the gene sequence of the fully human monoclonal antibody against MntC, and reconstruct The nucleotide sequence of the chain is shown in SEQ ID NO.16, and the nucleotide sequence of the light chain is shown in SEQ ID NO.17. A large number of sorted cells are in good condition.

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Abstract

The invention discloses an antibody resistant to a staphylococcus aureus and manganese ion binding protein C and application of the antibody. An epitope peptide recognized by the antibody resistant tothe staphylococcus aureus and manganese ion binding protein C at least comprises MntC126-128aa, and the amino acid sequence is LDN. The monoclonal antibody is generated through cultivation of a hostcell of an expression vector, and the generated monoclonal antibody is secreted into supernatant and then purified through chromatography. The antibody can treat septicopyemia caused by MRSA (methicillin-resistant staphylococcus aureus) infections, and is of important significance in prevention and treatment of staphylococcus aureus infections.

Description

technical field [0001] The invention relates to the fields of medicine and immunology, in particular to an antibody against staphylococcus aureus manganese ion-binding protein C, and also relates to the application of the antibody against staphylococcus aureus manganese ion-binding protein C. Background technique [0002] Staphylococcus aureus (abbreviated as Staphylococcus aureus), a Gram-positive bacterium, is a major human pathogen that is widespread and causes hospital and community-related infections, and can cause various diseases in humans. Infection is mainly acute and suppurative. Systemic infection can lead to severe infection and complications such as osteomyelitis, septic arthritis, endocarditis, pneumonia, and sepsis, and the fatality rate can reach 20%. The long-term abuse of antibiotics has aggravated and highlighted the problem of bacterial drug resistance. Methicillin-resistant Staphylococcus aureus (MRSA) was discovered for the first time in 1961. It is c...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/11A61K39/40A61P31/04
CPCA61K2039/505A61P31/04C07K16/1271C07K2317/34C07K2317/565C07K2317/92
Inventor 曾浩葛爽宋振邹全明刘圆圆敬海明赵莉群张怡
Owner ARMY MEDICAL UNIV
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