Antibody against alpha-hemolysin and application thereof

An antibody, VH-CDR1 technology, applied in the direction of antibodies, applications, antibody mimics/stents, etc., can solve the problems of limited antibiotics, clinical patient treatment failure and high mortality.

Active Publication Date: 2021-03-23
MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, clinically, MRSA infection is becoming more and more serious, and the choice of antibacterial drugs is very limited, and the treatment failure and mortality rate of clinical patients remain high.

Method used

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  • Antibody against alpha-hemolysin and application thereof
  • Antibody against alpha-hemolysin and application thereof
  • Antibody against alpha-hemolysin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 Recombinant Expression of Staphylococcus aureus α-Hemolysin (α-Toxin) Fused with His Tag

[0094] Taking the amino acid sequence of Staphylococcus aureus α-hemolysin as the target sequence, the corresponding base sequence was artificially synthesized, and cloned into the Pet-21a plasmid containing His tag by using restriction sites NdeI and XhoI. Wherein the Staphylococcus aureus α-hemolysin amino acid sequence is shown in SEQ ID NO: 78, the corresponding base sequence is shown in SEQ ID NO: 79, and the construction of the recombinant plasmid is shown in figure 1 .

[0095] The obtained recombinant plasmid was transformed into competent cells BL21(DE3)pLysS, and a single colony was picked and inoculated into LB liquid medium containing 100 μg / ml ampicillin the next day, and cultured overnight at 37°C with shaking. Inoculate the overnight cultured bacterial solution into LB liquid medium containing 100 μg / ml ampicillin at a volume ratio of 1:100, shake at 20...

Embodiment 2

[0096] Example 2 Recombinant Expression of Staphylococcus aureus α-Hemolysin Mutant (H35Lα-Toxin) Fused with His Tag

[0097] Based on the amino acid sequence of Staphylococcus aureus α-hemolysin, the 35-position histidine (His) was mutated into leucine (Leu) to obtain the mutated amino acid sequence, and the corresponding base sequence was artificially synthesized, and the enzyme cleavage site was used to Click NdeI and XhoI to clone it into the Pet-21a plasmid containing His tag. Wherein the Staphylococcus aureus mutant α-hemolysin (H35Lα-Toxin) amino acid sequence is shown in SEQ ID NO: 80, the corresponding base sequence is shown in SEQ ID NO: 81, and the construction of the recombinant plasmid is shown in figure 2 .

[0098] The obtained recombinant plasmid was transformed into competent cells BL21(DE3)pLysS, and a single colony was picked and inoculated into LB liquid medium containing 100 μg / ml ampicillin the next day, and cultured overnight at 37°C with shaking. I...

Embodiment 3

[0099] Example 3 Purification of Staphylococcus aureus α-hemolysin (α-Toxin) and its mutant (H35Lα-toxin)

[0100] The Escherichia coli induced to express Staphylococcus aureus α-hemolysin and its mutants were disrupted with a sonicator, and worked at 180W for 3s and 3s, with a time of 7-9min; centrifuged at 13,000rpm for 30min, collected the supernatant, and passed through a 0.22μmL filter Filter sterilize.

[0101] The Ni column and the filtered supernatant were mixed on a rotary mixer for 1 h at room temperature, and the Ni column was loaded into the packed column. Use 5 times the column bed volume of BD solution (containing imidazole concentration 30mM) to elute the protein non-specifically bound to the Ni column, wash until the protein chromogenic solution does not change color, and then use 5 times column bed volume BB solution (containing imidazole concentration 300mM ) to elute the target protein. Then use a 10KD concentrator tube to concentrate and replace the elu...

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Abstract

The invention provides an antibody or a fragment thereof combined with staphylococcus aureus alpha-hemolysin, and application of the antibody or the fragment thereof in preventing or treating staphylococcus aureus infection. The antibody is obtained by screening through a strategy of attenuated immunity and virulent screening of the alpha-hemolysin, has high affinity to the alpha-hemolysin, can effectively block the hemolysis of the alpha-hemolysin, proves a significant protective or therapeutic effect in an alpha-hemolysin sepsis model, an MRSA bacteremia model and an MRSA pulmonary infectionmodel, has a synergistic effect with antibiotics, and is a beneficial supplement to the existing antibiotic therapy of staphylococcus aureus.

Description

technical field [0001] The invention relates to the field of antibody medicine, in particular, the invention relates to an anti-alpha-hemolysin antibody and its pharmaceutical application. Background technique [0002] Staphylococcus aureus, belonging to the genus Staphylococcus, is an important Gram-positive pathogen and the most important G + Pathogenic bacteria can cause suppurative infection, pneumonia, pseudomembranous enteritis, pericarditis and other local infections, as well as systemic infections such as sepsis and sepsis. According to data from the China Drug Resistance Surveillance Network, Staphylococcus aureus ranks fourth among pathogenic bacteria detected in hospitals and first among G+ bacteria detected in hospitals. [0003] Staphylococcus aureus releases a large amount of toxins to destroy tissue cells during infection of the human body, and inhibits the body's immune cells from clearing pathogens. At present, β-lactam antibiotics are mainly used clinical...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C07K19/00C12N15/13A61K39/40A61P31/04
CPCC07K16/1271A61P31/04C07K2319/00C07K2317/24C07K2317/56C07K2317/565C07K2317/92A61K2039/505C07K2317/90C07K2317/76
Inventor 安毛毛王骊淳高攀林鉴姜远英慎慧陈思敏郭诗雨方玮
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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