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Preparation and application of anti-Staphylococcus aureus eLtaS protein monoclonal neutralizing antibody E4-2

A monoclonal antibody, Staphylococcus technology, applied in the direction of anti-bacterial immunoglobulin, antibody, anti-bacterial drugs, etc., to achieve the effect of broad application prospects

Active Publication Date: 2014-07-30
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant report on the use of specific antibodies against bacterial virulence proteins for anti-bacterial infection treatment

Method used

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  • Preparation and application of anti-Staphylococcus aureus eLtaS protein monoclonal neutralizing antibody E4-2
  • Preparation and application of anti-Staphylococcus aureus eLtaS protein monoclonal neutralizing antibody E4-2
  • Preparation and application of anti-Staphylococcus aureus eLtaS protein monoclonal neutralizing antibody E4-2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 Construction of anti-eLtaS monoclonal antibody hybridoma cell line

[0036] Materials: Freund's complete adjuvant and incomplete adjuvant, TMB is a product of Sigma, 20% fetal bovine serum is a product of Beijing Yuanheng Shengma Biotechnology Research Institute, serum-free RPMI1640 is a product of Gibco, SP2 / 0 cells are from ATCC Imported and kept in our laboratory, Balb / c mice and Kunming mice were purchased from the Experimental Animal Center of Academy of Military Medical Sciences. The rest of the reagents were purchased from the market.

[0037] Method result:

[0038] 1. Expression and purification of eLtaS protein. The eLtaS gene in the genome of Staphylococcus aureus 8325-4 was fished out by using eLtaS coding region-specific primers (the electrophoresis detection of it was as follows: figure 1 shown), and cloned into the pET-28a vector to induce, express, and purify eLtaS protein (the SDS-PAGE results are shown in figure 2 ) shown.

[0039] 2...

Embodiment 2

[0044] Example 2 Screening and Identification of Anti-eLtaS Neutral Monoclonal Antibody Hybridoma Cell Lines

[0045] Material: Protein G Sepharose CL4B column: product of GE healthcare company; Ibid.

[0046] Method result:

[0047] 1. Purification of eLtaS monoclonal antibody. Add 1 ml of pH8.0, 0.1moL / L phosphate buffer to 2 ml of mouse ascites and adjust the pH to 9 with pH9.0, 1moL / L TRIS-HCL. Add the mouse ascites to the Protein G Sepharose CL4B column protein column that has been equilibrated with 0.1moL / L phosphate buffer solution pH8.0, and wash the column with the above buffer solution until no foreign protein can be detected in the effluent. Elute with citric acid buffer of pH 3.0, collect the effluent, and immediately neutralize with 1moL / L TRIS-HCL pH 8.5 buffer, and dialyze with pH 7.2, 0.01M PBS for 72h. Sampling for SDS-PAGE detection ( image 3 ).

[0048] 2. Screening of neutralizing antibodies. Use brain heart infusion medium to culture Staphylococcus ...

Embodiment 3

[0053] Example 3 The protective experiment of monoclonal antibody E4-2 to CD1 mice

[0054] Materials: Same as Examples 1 and 2 above.

[0055] Method result:

[0056] 1. The acute peritonitis model was used to detect the effect of anti-eLtaS monoclonal antibody E4-2 against Staphylococcus aureus infection. 6-week-old female CD1 mice were treated with intraperitoneal injection of PBS, control IgG (100 μg) and E4-2 (100 μg); the single clone of Staphylococcus aureus 8325-4 was picked and cultured in brain heart infusion medium , 37°C, 200rpm for 12hr. After the bacteria were collected, the bacteria were washed once with PBS, and the concentration of the bacteria was adjusted to OD600=1.5, and 500 μl was injected intraperitoneally into the mice, and the survival number of mice at different time points was observed ( Figure 7 ).

[0057] 2. The pneumonia model was used to test the anti-eLtaS monoclonal antibody E4-2 against Staphylococcus aureus infection. 6-week-old female...

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Abstract

The invention discloses an anti-Staphylococcus aureus eLtaS protein monoclonal neutralizing antibody E4-2, belonging to the technical field of preparation of antibodies for immunization. An antibody light / heavy chain variable region gene is cloned from the prepared eLtaS neutralizing monoclonal antibody E4-2, and can code correct mouse antibody variable regions. The light / heavy chain variable region gene based on the monoclonal antibody can be used for constructing and expressing multiple micromolecule gene engineering antibodies; and the polypeptide or protein coded on the basis of the gene can crosslink multiple biological activity molecules for preparing anti-Staphylococcus aureus infection drugs, and has wide application prospects.

Description

technical field [0001] The invention belongs to the technical field of antibody preparation for immunization, and in particular relates to a monoclonal neutralizing antibody E4-2 against Staphylococcus aureus eLtaS protein, its preparation method and its application in the preparation of drugs against Staphylococcus aureus infection . Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) belongs to the genus Staphylococcus and is an important pathogenic bacterium of humans, which can cause various serious diseases such as pneumonia, endocarditis, burn and war wound infection, sepsis, toxic shock, etc. Infection (Lowy FD.New Engl J Med.1998; 339,520-532), it is the main pathogenic bacteria of Gram-positive bacteria sepsis, and also an important pathogenic bacteria of burn wound infection and acute liver failure (Yao Yongming et al. , Some New Developments in Sepsis Research 2000;13, 517-519). It can spread on a large scale through droplets and contact ...

Claims

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Application Information

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IPC IPC(8): C07K16/12A61K39/40A61P31/04
Inventor 杨光刘玉高亚萍董洁冯健男
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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