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Monoclonal antibody resistant to SasA (staphylococcus aureus surface protein A) antigen and application of monoclonal antibody

A technology of monoclonal antibody and staphylococcus, applied in the fields of application, antibody, antibacterial drug, etc., can solve the problems of limited protection, unsatisfactory protection of monoclonal antibody, and no surface protein antigen can be found, and achieve highly specific binding activity Effect

Active Publication Date: 2014-10-15
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2009, Martha Brown et al. screened multiple mouse monoclonal antibodies against IsdB, but these monoclonal antibodies only showed 10%-50% protection in the mouse lethal challenge model (Martha Brown et al., CLINICAL AND VACCINE Immunology, 2009, 16(8):1095–1104)
In 2012, Hwan Keun Kim et al. reported that multiple mouse monoclonal antibodies against SPA have good protection in the mouse kidney infection model, but the article did not report whether these monoclonal antibodies have protective effects in the mouse lethal challenge model. Protective (Hwan Keun Kim et al., Infection and Immunity, 2012, 80(10):3460–3470)
The current research status of monoclonal antibodies against the surface protein of Staphylococcus aureus is that the protective properties of monoclonal antibodies that have completed clinical trials are not ideal, and there is no FDA-approved monoclonal antibody on the market, and animal experiments have shown that most monoclonal antibodies Protection was also limited in a mouse lethal challenge model
There are two possible reasons: one is that the key surface protein antigen has not been found; the other is that the monoclonal antibody currently studied is mainly aimed at a single domain of the antigen, and its protective effect may be limited in resisting the lethal challenge of Staphylococcus aureus

Method used

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  • Monoclonal antibody resistant to SasA (staphylococcus aureus surface protein A) antigen and application of monoclonal antibody
  • Monoclonal antibody resistant to SasA (staphylococcus aureus surface protein A) antigen and application of monoclonal antibody
  • Monoclonal antibody resistant to SasA (staphylococcus aureus surface protein A) antigen and application of monoclonal antibody

Examples

Experimental program
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Embodiment 1

[0035] Example 1 Preparation of Monoclonal Antibody to SasA Protein

[0036] 1. Division of different structural domains of SasA protein

[0037] The SasA protein has a full length of 2271 amino acids and belongs to the SRRP protein (Serine-rich repeat protein) family of Gram-positive bacteria. According to bioinformatics analysis, the SasA protein can be divided into five main structural domains, which are N-terminal signal peptide, serine-rich repeat region 1 (amino acid sequence 48-229), non-repeat region (amino acid sequence 229-751 amino acid sequence), serine-rich repeat region 2 (amino acid sequence 752-2213) and the cell wall anchor sequence at the C-terminus. where SasA 752-2213 Mainly localized in the capsular polysaccharide of Staphylococcus aureus, while SasA 48-229 and SasA 229-751 localizes on the bacterial surface, thus predicting SasA 48-229 and SasA 229-751 It is the main functional domain of SasA protein.

[0038] 2. SasA 48-229 and SasA 229-751 gene...

Embodiment 2

[0055] Example 2. ELISA detection of binding activity of monoclonal antibody #23 and 2H7 to SasA protein

[0056] The SasA protein fragment was coated with 100 μl per well at a concentration of 2 μg / ml, and left overnight at 4°C. After washing 3 times with ELISA washing solution PBST (PBS+0.1% Tween-20), 100 μl of fixative solution (PBST+2% BSA) was added to each well, and blocked at 37° C. for 1 hour. Wash 3 times with PBST, dilute the monoclonal antibody sample with diluent (PBST+0.2% BSA) and add 100 μl to each well, and incubate at 37° C. for 1 h. Wash 4 times with PBST, add 100 μL of horse anti-mouse HRP secondary antibody (CST, #7076) diluted 1:10,000 times to each well, and incubate at 37°C for 1 hour. Wash 4 times with PBST, add TMB to carry out color reaction, stop reaction with 2mol / L sulfuric acid after color development for 5 minutes. Finally, the absorbance value at 450nm is detected on a microplate reader, and the results are shown in Figure 7 . The monoclon...

Embodiment 3

[0057] Example 3. The protective effect of monoclonal antibodies #23 and 2H7 on the challenge of Staphylococcus aureus USA300 strain in mice

[0058] Use 6-week-old BALB / c female mice and divide them into 5 groups (#23 monoclonal antibody group, 2H7 monoclonal antibody group, #23+2H7 monoclonal antibody group, isotype monoclonal antibody control group, PBS control group), 8 mice in each group . #23 monoclonal antibody group intraperitoneally injected monoclonal antibody #23400ug / body, 2H7 monoclonal antibody group intraperitoneally injected monoclonal antibody 2H7400ug / mouse, #23+2H7 monoclonal antibody group intraperitoneally injected #23 and 2H7 monoclonal antibody 200ug / mouse, same type The monoclonal antibody control group was intraperitoneally injected with IgG1 and IgG2b isotype control monoclonal antibodies at 200ug / mouse, and the PBS control group was injected with the same volume of PBS (200ul). After 24 hours of monoclonal antibody injection, the cultured Staphyloco...

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Abstract

The invention discloses a monoclonal antibody resistant to different structural domains of a SasA (staphylococcus aureus surface protein A) antigen as well as an application of the monoclonal antibody in preparation of a drug for resisting staphylococcus aureus infection. The specific antibody has good protection performance in a mouse model when in combined utilization.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to genes and gene-coded polypeptides of monoclonal antibodies against Staphylococcus aureus (Staphylococcus aureus, S.aureus) surface protein A (Staphylococcus aureus surface protein A, SasA) , and the application of said antibody in resisting Staphylococcus aureus infection. Background technique [0002] Staphylococcus aureus is an important Gram-positive conditional pathogen. In the adult population, approximately 20% carry S. aureus continuously and another 30% carry it intermittently. In healthy individuals, S. aureus usually causes minor infections of the skin and soft tissues; however, in traumatized and immune-compromised patients, S. aureus can cause life-threatening pneumonia, bacteremia, and Severe complications including septic arthritis, osteomyelitis, etc. The exotoxin of Staphylococcus aureus can also cause food poisoning, epidermolysis an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13A61K39/40A61P31/04
Inventor 陈薇杨益隆易绍琼李建民郭强徐俊杰于长明刘树玲
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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