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Anti-anthrax lethal factor chimeric monoclonal antibody, and preparation method and application thereof

A monoclonal antibody, anti-anthrax technology, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of human injury and limit the clinical application of LF8, and achieve high affinity and good safety effects

Inactive Publication Date: 2010-11-24
曹伯良 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present inventor once prepared a cell line that can secrete a high-affinity mouse monoclonal antibody that neutralizes LF, and named it LF8 cell, and the high-affinity mouse monoclonal antibody that it secreted neutralizes LF was named as LF8 antibody (Zhao , P., X.Liang, J.Kalbfleisch, H.-M.Koo, and B.Cao. 2003.Neutralizing monoclonal antibody against anthrax lethal factor inhibitintoxication in a mouse model.Human Antib.12(4):129-135. ), but the mouse antibody is immunogenic to the human body, that is, the human anti-mouse antibody is produced, causing damage to the human body, which greatly limits the clinical application of LF8

Method used

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  • Anti-anthrax lethal factor chimeric monoclonal antibody, and preparation method and application thereof
  • Anti-anthrax lethal factor chimeric monoclonal antibody, and preparation method and application thereof
  • Anti-anthrax lethal factor chimeric monoclonal antibody, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Preparation of HcLF8 Antibody

[0044] 1.1 Amplification of antibody variable region genes

[0045] method:

[0046] Hybridoma cells (LF8 cells) expressing LF8 antibody were cultured, total RNA was extracted with TRIzol reagent (Invitrogen, USA), and the OD was determined 260 and OD 280 . Using RNA as a template, reverse transcription was performed using SuperScript II reverse transcriptase (Invitrogen, USA) to obtain cDNA. Using cDNA as a template, specific primers were used to amplify the heavy chain variable region (variable region of heavy chain, V H ) and light chain variable region (variable region of light chain, V L ), the response looks like this:

[0047] (a) Primers:

[0048] V H Upstream: 5'-GGGGATATCCACCATGGRATGSAGCTGKGTMATSCTCTT-3'

[0049] Downstream: 5'-GACHGATGGGGSTGTYGTGCTAGCTGMRGAGACDGTGA-3'

[0050] V L Upstream: 5'-GGGGATATCCACCATGGATTTTCAAGTGCAGATTTTCAG-3'

[0051] Downstream: 5'-GGATACAGTTGGTGCAGTCGACTTACGTTTKATTTCCARCTT-3' ...

Embodiment 2

[0135] Example 2 HcLF8 Antibody Affinity Determination

[0136] method:

[0137] The affinity of HcLF8 antibody was determined by non-competitive immunosorbent assay (non-competitive ELISA).

[0138] 1) Coat a 96-well plate with two concentrations of recombinant LF protein (4 μg / ml and 2 μg / ml), overnight at 4°C;

[0139] 2) The next day, wash the plate twice with PBS solution (PBST) containing 0.1% Tween-20;

[0140] 3) Add 200 μl of 1% BSA solution to each well, and block for 1 hour at room temperature;

[0141] 4) Wash the plate twice with 0.1% PBST;

[0142] 5) Add HcLF8 antibody in gradient dilution, 50 μl per well, let stand at room temperature for 2 hours, and make 4 duplicate wells for each concentration; untransfected Sp2 / 0 cell supernatant, selective medium, 1% BSA, PBS are negative controls;

[0143] 6) Wash the plate 4 times with 0.1% PBST;

[0144] 7) Add alkaline phosphatase-labeled goat anti-human IgG (Fc specific), dilute 1:2000, 50 μl per well, and let st...

Embodiment 3

[0155] Example 3 HcLF8 Antibody Neutralization Test of Anthrax Lethal Toxin in Vitro—Neutralizing Effect of Different Concentration Antibodies on Lethal Toxin

[0156] 1. Methods and steps

[0157] (1) Take out J774A.1 cells from liquid nitrogen, and culture them in DMEM medium containing 10% FBS.

[0158] (2) When J774A.1 cells (ATCC) are in the logarithmic growth phase, adjust the concentration to 5×10 5 pieces / ml. The cells were transferred to a 96-well cell culture plate, 100 μl per well (5×10 4 cells).

[0159] (3) 12 hours after plating, 100 μl of HcLF8 antibody and lethal toxin mixture of different concentrations were added to each well. The lethal toxin concentration was fixed, and the final concentration of PA was set to 0.5 μg / ml and the final concentration of LF was set to 0.1 μg / ml according to the literature. According to the pre-experimental results, the initial concentration of HcLF8 antibody was set at 66nM (10μg / ml), diluted with culture medium and then mix...

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Abstract

The invention discloses an anti-anthrax lethal factor human-rat chimeric neutral monoclonal antibody, which can, as proved by in vitro experiment and animal experiment, effectively neutralize anthrax lethal toxin and be used for the preparation of antibody medicine for the treatment of human anthrax infection.

Description

Technical field: [0001] The invention relates to an anti-anthrax lethal factor human-mouse chimeric monoclonal antibody; the invention also relates to a preparation method of the monoclonal antibody; the invention also relates to the application of the monoclonal antibody in preparing medicine for treating human anthrax infection. Background technique: [0002] Anthrax is an acute infectious disease caused by Bacillus anthracis, and the population is generally susceptible. [0003] Anthrax toxin is the key pathogenic factor of anthrax, which consists of three protein components: protective antigen (protectiveantigen, PA), lethal factor (lethal factor, LF) and edema factor (edema factor, EF). LF or EF alone has no toxic effect, PA and LF combine to form PA-LF complex, which is called lethal toxin (Lethal Toxin, LeTx). When PA binds to cell surface receptors, heptamer pores are formed, and LF or EF is brought into cells to form lethal toxin or edema toxin to play a toxic role...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K16/46C07K16/12C12N15/13A61K39/40A61P31/04C12R1/91
Inventor 曹伯良丁贵鹏
Owner 曹伯良
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