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Antibody against alpha-hemolysin and stable formulations thereof

A technology of antibodies and antigens, applied in the direction of antibodies, antibody medical components, medical preparations of non-active ingredients, etc., to prevent or alleviate sepsis, shorten the course of treatment, and improve immunity

Pending Publication Date: 2021-11-26
MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no suitable stable formulation on the market

Method used

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  • Antibody against alpha-hemolysin and stable formulations thereof
  • Antibody against alpha-hemolysin and stable formulations thereof
  • Antibody against alpha-hemolysin and stable formulations thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1 Recombinant Expression of Staphylococcus aureus α-Hemolysin (α-Toxin) Fused with His Tag

[0122] 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 .

[0123] 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

[0124] Example 2 Recombinant Expression of Staphylococcus aureus α-Hemolysin Mutant (H35Lα-Toxin) Fused with His Tag

[0125] 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 .

[0126] 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

[0127] Example 3 Purification of Staphylococcus aureus α-hemolysin (α-Toxin) and its mutant (H35Lα-toxin)

[0128] 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.

[0129] 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 combined with staphylococcus aureus alpha-hemolysin or a fragment thereof, and a composition containing the antibody combined with the staphylococcus aureus alpha-hemolysin or the fragment thereof, wherein the composition is preferably a hydro-acupuncture preparation. The antibody disclosed by the invention is obtained by screening through strategies of attenuated immunization and virulent screening of alpha-hemolysin, has high affinity to the alpha-hemolysin, can effectively block the hemolysis of the alpha-hemolysin, and proves a remarkable protection or treatment effect in an alpha-hemolysin sepsis model, an MRSA bacteremia model and an MRSA pulmonary infection model. On the basis of the antibody against the staphylococcus aureus alpha-hemolysin or the fragment thereof, the freeze-thaw stability, the oscillation stability and the illumination stability of the antibody are improved by an antibody stabilizing preparation provided by further component selection and concentration optimization, and the storage life of the antibody preparation, especially a hydro-acupuncture preparation, is prolonged.

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 pathogens detected in hospitals, and G + Bacteria number 1. [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 clinically to treat Staphylococcus aureu...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/13A61K39/395A61K47/18A61K47/26A61P31/04
CPCC07K16/1271A61K39/39591A61K47/183A61K47/26A61P31/04C07K2317/565C07K2317/56C07K2317/76C07K2317/94A61K39/395A61K47/18C07K16/00C07K16/12A61K47/06A61K39/40
Inventor 章燕珍欧阳子均汤沛霈李纲季霜仪叶烨安毛毛王骊淳
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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