Antibody against staphylococcal enterotoxin B and application thereof

A Staphylococcal Gut, golden yellow technology, applied in the field of immunity, can solve problems such as SEB worry complex

Active Publication Date: 2019-11-26
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Concerns about SEB are compounded by the lack of effective medical countermeasures for mass treatment of affected populations

Method used

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  • Antibody against staphylococcal enterotoxin B and application thereof
  • Antibody against staphylococcal enterotoxin B and application thereof
  • Antibody against staphylococcal enterotoxin B and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, expression and purification of Staphylococcus aureus enterotoxin B (SEB) and mutant Staphylococcus aureus enterotoxin B (mSEB)

[0058] The SEB gene was amplified by PCR from genomic DNA from a S. aureus strain (ATCC accession number BAA-1556). The L45R, Y89A, Y94A variants were then generated by site-directed mutagenesis of the wild-type gene using the QuickChange II XL Site-Directed Mutagenesis Kit. 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 SEB protein and mSEB (L45R, Y89A, Y94A) protein were purified by Ni-NTA, and the amino acid sequence of the SEB protein is shown in SEQ ID NO.1.

Embodiment 2

[0059] Embodiment 2, the separation of peripheral blood mononuclear cells (PBMC)

[0060] 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

[0061] Example 3, Sorting single plasma cells by flow cytometry

[0062] Sorting single plasma cells by flow cytometry: using SEB protective antigen protein (HPLC purity > 95%) as the antigen to perform ELISA detection on the serum to determine the antibody titer of the sample, select the sample with high antibody titer, and pass the flow cytometer Single plasma cells were sorted, and CD3 / CD14 / CD16 / CD235a-CD19+CD20+ / -CD38hi CD27hi were gated and sorted to separate plasma cell populations at different time points. 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 SEB, 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. The number of sorted cells is large and the state is good ...

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Abstract

The invention discloses an antibody against staphylococcal enterotoxin B and application thereof. The heavy chain of the antibody against staphylococcal enterotoxin B has variable regions CDR1, CDR2 and CDR3 of which the amino acid sequences are shown in SEQ ID NO. 5, SEQ ID No. 6 and SEQ ID No. 7, or is composed of CDR variants with equivalent functions; and light chain of the antibody against staphylococcal enterotoxin B has variable regions CDR1, CDR2 and CDR3 of which the amino acid sequences are shown in SEQ ID NO. 9, SEQ ID No. 10 and SEQ ID No. 11, or is composed of CDR variants with equivalent functions. The antibody against staphylococcal enterotoxin B is capable of specifically binding with staphylococcal enterotoxin B or free enterotoxin B; and thus, the antibody can be used fortreating, preventing and diagnosing staphylococcus aureus infection. Therefore, the antibody against staphylococcal enterotoxin B will be an important research direction in the fields of "non-antibiotic" treatment of methicillin-resistant staphylococcus aureus infection and drug resistance development control.

Description

technical field [0001] The invention relates to the field of immunization, in particular to an antibody against Staphylococcus aureus enterotoxin B, and also relates to the application of the antibody. Background technique [0002] Methicillin-resistant Staphylococcus aureus (MRSA) is a typical representative of "super bacteria", which has strong pathogenicity, wide transmission routes, easy outbreaks, and the development of multiple high drug resistance. MRSA is an important pathogen that causes nosocomial infection and community infection. The infection is characterized by acute and suppurative, and the whole body can cause sepsis, acute pneumonia, endocarditis, septic arthritis, osteomyelitis and other serious diseases. Infection and complications, the fatality rate of infection is as high as 20%; it can cause purulent infection of skin and soft tissue locally, which will not heal for a long time. At the same time, the exotoxin of MRSA can also cause systemic fatal infec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13A61K39/40A61P31/04G01N33/569
CPCA61K2039/505A61P31/04C07K16/1271C07K2317/24C07K2317/34C07K2317/92G01N33/56938
Inventor 曾浩刘圆圆邹全明宋振张卫军赵安妮赵莉群
Owner ARMY MEDICAL UNIV
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