Anti-Staphylococcus aureus alpha hemolysin monoclonal antibody and applications thereof

A staphylococcus, golden yellow technology, applied in the direction of antibodies, applications, antibacterial drugs, etc., can solve the problem of reducing antibiotic sensitivity, achieve high affinity, and improve the effect of survival

Active Publication Date: 2019-02-26
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

With the extensive use of antibiotics, most clinically isolated Staphylococcus aureus are less sensitive to o...

Method used

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  • Anti-Staphylococcus aureus alpha hemolysin monoclonal antibody and applications thereof
  • Anti-Staphylococcus aureus alpha hemolysin monoclonal antibody and applications thereof
  • Anti-Staphylococcus aureus alpha hemolysin monoclonal antibody and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, preparation and functional research of alpha hemolysin (Hla protein)

[0041] 1. Preparation of Hla protein

[0042] (1) The small fragment between the recognition sequences of the restriction enzymes BamHI and HindIII of the vector pET28a was replaced with the DNA molecule (gene encoding the Hla protein) shown in sequence 5 in the sequence listing to obtain the recombinant plasmid pET28a-Hla.

[0043] In the recombinant plasmid pET28a-Hla, the DNA molecule shown in sequence 5 of the sequence table is fused with the coding sequence of the His-tag tag (composed of 6 histidine residues) on the vector backbone to form a fusion gene, expressing a His- The Hla protein of tag label (ie fusion protein). The amino acid sequence of the Hla protein is shown in sequence 6 in the sequence listing.

[0044] (2) The recombinant plasmid pET28a-Hla was introduced into Escherichia coli BL21 to obtain a recombinant bacterium, which was named E. coli BL21(pET28a-Hla).

[...

Embodiment 2

[0058] Embodiment 2, phage antibody library screening anti-Hla monoclonal antibody

[0059] 1. Biopanning of anti-Hla monoclonal antibodies

[0060] A. Hla group

[0061] 1. The first round of affinity panning

[0062] (1) Coat the immunotube with 500 μL of Hla protein solution (obtained by adding sterile PBS buffer solution to Hla protein) with a concentration of 10 μg / mL, overnight at 4°C.

[0063] (2) Take the immunotube, add 4% (v / v) Milk-PBST buffer, and block at room temperature for 1 hour.

[0064] (3) Take the phage antibody library, add 4% (v / v) Milk-PBST buffer, and block at room temperature for 1 hour.

[0065] (4) Take the immune tube that completed step (2), first add sterile PBS buffer to wash 3 times, and then add the phage antibody library that completed step (3) (the input amount of phage is about 1.2×10 12 ), let stand at room temperature for 1h.

[0066] (5) After completing step (4), take the immunotube, add an appropriate amount of sterile PBS buffer ...

Embodiment 3

[0086] Example 3, Functional Identification of AAH-1 Antibody

[0087] 1. Preparation of AAH-1 Antibody

[0088] Vector pCDNA3.1 is a product of Invitrogen.

[0089] 1. Construction of recombinant plasmids

[0090] The small fragment between the recognition sequences of restriction endonuclease XhoI and HindIII of the vector pCDNA3.1 was replaced with the DNA molecule shown in sequence 1 in the sequence listing to obtain the heavy chain expression vector.

[0091] The small fragment between the restriction endonuclease XhoI and HindIII recognition sequences of the vector pCDNA3.1 was replaced with the DNA molecule shown in sequence 3 in the sequence listing to obtain the light chain expression vector.

[0092] 2. Construction of recombinant cells

[0093] The heavy chain expression vector and the light chain expression vector were co-transfected into 293T cells to obtain recombinant cells.

[0094] 3. Antibody preparation

[0095] (1) Take the recombinant cells obtained i...

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Abstract

The invention discloses an anti-Staphylococcus aureus alpha hemolysin monoclonal antibody and applications thereof, wherein the monoclonal antibody comprises a heavy chain and a light chain, the aminoacid sequence of the variable region of the heavy chain is represented by the sequence 2 in the sequence listing, and the amino acid sequence of the variable region of the light chain is representedby the sequence 4 in the sequence listing. According to the present invention, the full human source anti-alpha hemolysin monoclonal antibody is rapidly screened from the phage antibody library by using the alpha hemolysin as the target antigen; and the obtained monoclonal antibody is the completely-new antibody, has high affinity, can highly inhibit the hemolytic activity of alpha-hemolysin, canimprove the survival rate of mice infected with Staphylococcus aureus, and further has important application value in the prevention and control of Staphylococcus aureus.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an anti-Staphylococcus aureus α-hemolysin monoclonal antibody and its application, in particular to the use of the monoclonal antibody in the preparation of drugs for the prevention and / or treatment of diseases caused by Staphylococcus aureus Applications. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is a Gram-positive pathogenic bacteria widely distributed in nature. It is estimated that 20-30% of the population carries the pathogen, which mainly exists in the mucous membranes, skin, and especially the nasopharynx of the human body. Staphylococcus aureus can cause a range of diseases, from minor skin infections, to scalded skin syndrome, to abscesses, to life-threatening pneumonia, meningitis, endocarditis, osteomyelitis, toxic shock syndrome, etc. . Staphylococcus aureus infection affects a wide range of skin, soft tissue, respiratory tract, bone m...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/13A61K39/40A61P31/04
CPCC07K16/1271C07K2317/76C07K2317/92
Inventor 杨光刘方杰刘玉刘成华高亚萍冯健男沈倍奋
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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