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Human-derived antibody IgG of anti-anthrax protective antigen PA and application of human-derived antibody IgG

A protective antigen, conservative technology, applied in applications, antibodies, antibacterial drugs, etc., can solve problems such as no effect, and achieve good affinity, good protective effect, and high protective effect.

Active Publication Date: 2015-05-20
中国人民解放军南京军区军事医学研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibiotic treatment mainly refers to high-dose intravenous and oral antibiotics to kill Bacillus anthracis during anthrax infection, such as penicillin, ciprofloxacin, tetracycline, erythromycin, and vancomycin, etc., but for the released Toxins are ineffective and resistant strains have been found

Method used

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  • Human-derived antibody IgG of anti-anthrax protective antigen PA and application of human-derived antibody IgG
  • Human-derived antibody IgG of anti-anthrax protective antigen PA and application of human-derived antibody IgG
  • Human-derived antibody IgG of anti-anthrax protective antigen PA and application of human-derived antibody IgG

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Screening of Human Anti-PA Antibody Fab

[0034] 1) Coat the solid-phase screening ELISA plate with purified recombinant PA83 protein, 2 μg per well, wash, add blocking solution, wash, add natural phage antibody library antibody prepared in our laboratory, and wash to remove unbound phage antibody.

[0035] 2) Add trypsin to elute the specifically bound phage antibody, increase the infection value, and help superinfection with phage VCSM13.

[0036] 3) The above screening steps were repeated, and a total of five rounds of "adsorption-elution-amplification" enrichment screening were performed.

[0037] 4) Dilute the phage obtained from the last round of screening and multiplication, spread on a culture plate with 100 μg / mL of ampicillin and culture overnight, pick 60 single colonies on the cell culture plate, and culture overnight with shaking.

[0038] 5) After overnight, transfer 5 μL of bacterial solution from each well of the first plate to the second plat...

Embodiment 2

[0042] Example 2 Preparation of Human Anti-PA IgG Antibody PA21

[0043] 1) According to the variable region sequence of the obtained antibody, design primers for Infusion PCR

[0044] According to the principle of Infusion PCR, design the heavy and light chain PCR amplification primers of PA21 antibody. The primers need to include 15 bp bases on the expression vector and at least 15 bp bases inserted into the target fragment. The bases inserted into the target fragment are designed according to common primers. Principled design.

[0045] Heavy and light chain PCR amplification primers:

[0046] Primers for heavy chain amplification:

[0047] F: 5'-GGTGTCCACTCGCTAGAGGTGCAGCTGTTGGAGTCTGGGGGAG-3'

[0048] R: 5'-GCCCTTGGTGGATGCTGGGGAGACGGTGACCAGGGT-3'

[0049] Light chain amplification primers:

[0050] F: 5'-ACAGACGCTCGCTGCGAGCTCGTGATGACTCAGTCTCCAGAC-3'

[0051] R: 5'-TGCAGCCACCGTACGTTTGATCTCCAGCTTGG-3'

[0052] 2) Amplify human anti-PA IgG antibody PA21 heavy chain, ligh...

Embodiment 3

[0081] Example 3 Functional Activity Identification of Human Anti-PA IgG Antibody PA21

[0082] 1) ELISA

[0083] Dilute the attenuated strain PA83 protein (gifted by the Plague Department of the Chinese Center for Disease Control and Prevention) with coating solution (0.1M carbonate buffer, pH9.6) to 2 μg / mL coated ELISA 96-well plate, add 100 μL to each well, 4 overnight at ℃; PBST (PBS containing 0.5% Tween20) 5% skimmed milk-washing buffer was blocked, and incubated at 37°C for 2h; after washing 5 times with PBST, 100 μL of PA21 antibody (2 μg / mL initial concentration, 14 Concentration gradient dilution) 37°C for 2h; goat anti-human secondary antibody diluted 1:4000 100μL / well was added to the well, incubated at 37°C for 1h; peroxidase substrate chromogenic solution 100μL / well, after 10 minutes at room temperature Stop the reaction with 2M sulfuric acid, and use dual wavelength 450nm / 690nm for colorimetry on the machine. The result is as figure 2 It was shown that huma...

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Abstract

The invention relates to a total-molecular human-derived monoclonal antibody of an anti-anthrax protective antigen and application of the total-molecular human-derived monoclonal antibody. The amino acid sequence of a heavy-chain variable area of the antibody is shown in SEQ ID NO.2, and the amino acid sequence of a light-chain variable area is shown in SEQ ID NO.4. The total-molecular human-derived antibody is subjected to functional identification and an in vitro cell neutralization experiment, and results show that the total-molecular human-derived antibody can be specifically bound with the anti-anthrax protective antigen PA and has an effect of inhibiting the pathogenic effect of anthrax toxin, so the total-molecular human-derived monoclonal antibody disclosed by the invention can be applied to diagnosis, treatment and prevention of relevant anthraces.

Description

technical field [0001] The invention belongs to the technical field of monoclonal antibody drugs, and relates to a whole-molecular human monoclonal antibody against anthrax protective antigen and its encoded DNA molecule, an expression vector containing the DNA molecule, and a host cell containing the expression vector. And the application of the whole molecular human monoclonal antibody in the preparation of anthrax treatment or preventive medicine. Background technique [0002] Anthrax is an acute zoonotic infectious disease caused by Bacillus anthracis. Herbivorous animals such as cattle and sheep have the highest incidence. People can contact anthrax-affected animals and their livestock products or through the presence of anthrax in the air, anthracis spores in the soil. Bacillus anthracis is highly pathogenic, and its spores have a strong ability to survive in harsh environments. It can be infected through aerosols and is widely distributed around the world. Clinically...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13C12N15/85C12N5/10A61K39/395A61P31/04
Inventor 朱进熊四平冯振卿
Owner 中国人民解放军南京军区军事医学研究所
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