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Human anti-anthrax protective antigen pa antibody IgG and its application

A technology of protective antigen and cloned antibody, applied in application, antibody, antibacterial drug, etc., can solve the problem of no effect, and achieve the effect of good affinity, high specificity and high protection

Active Publication Date: 2017-10-10
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 anti-anthrax protective antigen pa antibody IgG and its application
  • Human anti-anthrax protective antigen pa antibody IgG and its application
  • Human anti-anthrax protective antigen pa antibody IgG and its application

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, incubated at 37°C for 2 hours; after washing with PBST for 5 times, 100 μL of PA21 antibody (2 μg / mL initial concentration, 14 concentrations) was added to each well gradient dilution) at 37°C for 2 hours; 100 μL / well of goat anti-human secondary antibody diluted at 1:4000 was added to the well, and incubated at 37°C for 1 hour; 100 μL / well of peroxidase substrate chromogenic solution was used after 10 minutes at room temperature 2M sulfuric acid was used to stop the reaction, and the colorimetry on the machine was detected...

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Abstract

The present invention relates to a full-molecular human monoclonal antibody against anthrax protective antigen and its application. The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is As shown in SEQ ID NO.4. The functional identification and in vitro cell neutralization experiments of the whole molecule human antibody showed that it can specifically bind to the anthrax protective antigen PA and effectively block the pathogenic effect of anthrax toxin. Therefore, the whole molecule human antibody of the present invention The original monoclonal antibody can be used in the diagnosis, treatment and prevention of anthrax.

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 Patents(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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