Function epitopes of staphylococcal enterotoxin B (SEB), monoclonal antibodies specifically bound with function epitopes and application of monoclonal antibodies

A Staphylococcus intestinal tract, monoclonal antibody technology, applied in the fields of application, antibody, antibacterial drugs, etc., can solve the problem of SEB cannot be cleared

Inactive Publication Date: 2012-10-31
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has certain limitations due to its inability to remove SEB from the human body

Method used

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  • Function epitopes of staphylococcal enterotoxin B (SEB), monoclonal antibodies specifically bound with function epitopes and application of monoclonal antibodies
  • Function epitopes of staphylococcal enterotoxin B (SEB), monoclonal antibodies specifically bound with function epitopes and application of monoclonal antibodies
  • Function epitopes of staphylococcal enterotoxin B (SEB), monoclonal antibodies specifically bound with function epitopes and application of monoclonal antibodies

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0045] Experimental example 1. Cloning of SEB gene

[0046] Referring to the information and sequence of SEB provided by GENEBANK, synthetic primers: SEB primer sense 4Tsense-1: 5′-ATT T ATGTATAAGCGTCTGTTT-3'; 4Tantisene: 5'-ATT FTACTTTTTTCTTGGTGGTCAG-3'. Amplify the SEB gene fragment, recover the fragment through the gel recovery kit (Shanghai Sangong), digest it with restriction endonucleases Sal I and Not I, recover it by gel electrophoresis, and combine it with the double enzyme Sal I and Not I The cut plasmid vectors were ligated and transformed into Escherichia coli DH5α, and positive clones with inserted fragments were screened. DNA sequence determination confirmed that the nucleotide sequence of the SEB gene is shown in SEQ ID NO.1.

experiment example 2

[0047] Experimental example 2. Prokaryotic cell expression and purification of SEB

[0048] The SEB gene fragment with the correct sequence obtained in Example 1 was digested with corresponding restriction endonucleases and recovered, and loaded into pGEX-4T-2 plasmid. Transform DH5α competent cells and lay LB-Amp plates. Pick a single clone from the plate for Sal I and Not I double enzyme digestion identification, transfer the positive clone into BL21 (DE3) engineering bacteria, do SEB induction expression, and obtain GST-SEB fusion protein after purification by GST affinity chromatography column , digested with EK protease, recovered the flow-through by GST affinity chromatography, removed GST, and confirmed SEB pure protein by SDS-PAGE.

[0049] see attached results figure 1 .

experiment example 3

[0050] Experimental example 3. Screening and preparation of mouse anti-SEB monoclonal antibody

[0051] After 100 μg of purified SEB was emulsified with an equal volume of Freund’s adjuvant, BALB / C mice were immunized by intraperitoneal injection, and boosted immunization once every two weeks. The dose was the same as the first immunization. Spleen lymphocytes were isolated from mice with high SEB antibody titers, and the mouse spleen lymphocytes were fused with NS-1 cells using the classic PEG method. A 96-well plate was coated with 5 μg / ml SEB, and 10 hybridoma cell lines stably expressing anti-SEB antibody were obtained through repeated screening by ELISA. A large number of monoclonal cell lines were expanded, and 5×10 BALB / C mice were injected intraperitoneally 6 / mouse, the ascites of the mice was collected around 10 days, and the monoclonal antibody against SEB was purified by using Protein A column and affinity chromatography. The results of the ELISA test showed that...

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Abstract

The invention belongs to the technical field of biology, and particularly discloses function epitopes 195SFWYDMMP202, 12HKSSKFTGL20, 18TGLMEN23 and 43QFLFYFIY51 of staphylococcal enterotoxin B (SEB), monoclonal antibodies 4A3 and 3E2 specifically bound with the function epitopes and application of the monoclonal antibodies in the preparation of drugs for treating SEB-induced toxic shock syndrome.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention discloses four functional epitopes of SEB proteins, monoclonal antibodies specifically binding to the epitopes and their application in the treatment of SEB-induced toxic shock syndrome . Background technique [0002] The incidence of Gram-positive (G) bacterial sepsis is increasing year by year, among which Staphylococcus aureus is an important pathogen of burn wound infection and acute liver failure. Due to its complex pathogenic factors and increasing drug resistance, especially the emergence of intermediate vancomycin-resistant Staphylococcus aureus, the prevention and treatment of sepsis caused by Staphylococcus aureus infection has become a major challenge in modern trauma surgery and critical care medicine. one of the thorny problems. In recent years, it has been found that more than 80% of penicillin-resistant Staphylococcus aureus that cause hospital in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K16/12C12N15/13A61K39/40A61P31/04
Inventor 郭亚军戴建新王华菁
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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