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Monoclonal antibody of 1-aminocyclopropane-1-carboxylate oxidase and application thereof

A technology of aminocyclopropane and monoclonal antibody, applied in anti-enzyme immunoglobulin, measuring devices, instruments, etc., can solve the problem of lack of antibody tools and achieve high specificity and high affinity

Active Publication Date: 2017-10-20
艾比玛特医药科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, there is no specific antibody against 1-aminocyclopropane-1-carboxylate oxidase for fruit ripening research on the market. Therefore, at the level of proteomics, there is a lack of effective antibody tools for this research field. Study on the expression level of the enzyme in different plant types, different strains or different developmental stages

Method used

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  • Monoclonal antibody of 1-aminocyclopropane-1-carboxylate oxidase and application thereof
  • Monoclonal antibody of 1-aminocyclopropane-1-carboxylate oxidase and application thereof
  • Monoclonal antibody of 1-aminocyclopropane-1-carboxylate oxidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Preparation of a monoclonal antibody to 1-aminocyclopropane-1-carboxylate oxidase hybridoma cells and preparation and purification of monoclonal antibodies against 1-aminocyclopropane-1-carboxylate oxidase

[0018] 1.1 Antigen preparation

[0019] Synthesized four groups of 1-aminocyclopropane-1-carboxylate oxidase polypeptides 'NYPPCPNPEL', 'GGLILLFQDD', 'HSIVINLGDQ' and 'SVEHRVIAQT', and used them as immunogens respectively. The above polypeptides were also coupled to VLP and the traditional KLH system immunogenicity enhancer.

[0020] 1.2 Immunization of mice

[0021] Each group of antigens will be used to immunize 6 Balb / c mice (8-12 weeks old), and their serum titers will be monitored to determine the optimal number of immunizations. Each of the 6 immunogenic peptides is mixed into one set. Optimized adjuvants and immunization methods can produce high-affinity antibodies (IgG subtypes) against most antigenic polypeptides. After the initial immunizatio...

Embodiment 2

[0030] Embodiment 2 Verification of anti-CK18 monoclonal antibody

[0031] The obtained 8 monoclonal antibody cell lines were verified by ELISA, western blotting, co-immunoprecipitation plus mass spectrometry, antibody chips, etc. to determine the most effective antibodies.

[0032] 2.1 Elisa (immunoenzyme-linked) pairing verification of antibody and antigen polypeptide

[0033] Coat the 96-well ELISA plate with ascitic fluid antibody to be paired, incubate, wash and block overnight with skim milk, wash with PBS, and store at 4°C until use. Antigen peptides were incubated, washed with PBS, and controls were set at the same time. HRP-labeled detection antibody was added to the previously incubated ELISA plate. TMB color reaction, microplate reader reading. The titers of the 8 cell lines obtained by screening are shown in Table 1:

[0034] Table I

[0035]

[0036] 2.2 Endogenous western blot (WB) validation of antibodies

[0037] Use apple whole protein lysate, antibod...

Embodiment 3

[0045] Example 3 Application of a monoclonal antibody to 1-aminocyclopropane-1-carboxylate oxidase of the present invention in the detection of fruit development stages

[0046] Because the antibody of the present invention has unique specificity to 1-aminocyclopropane-1-carboxylate oxidase, it can be used as a detection antibody that specifically detects fruit development stages, or it can be further developed as Detection kit.

[0047] First, the antibody of the present invention is subjected to an ELISA experiment with a fixed concentration (1 mg / ml) and a standard product of 1-aminocyclopropane-1-carboxylate oxidase recombinant protein at different concentrations to obtain the corresponding OD value, and draw a standard curve, horizontal Coordinate is the concentration coordinate of 1-aminocyclopropane-1-carboxylate oxidase, and ordinate is the optical density value (OD value) that microplate reader records, as image 3 shown. The standard curve is used to convert the OD...

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Abstract

The invention discloses a monoclonal antibody of 1-aminocyclopropane-1-carboxylate oxidase. A heavy chain variable region sequence of the monoclonal antibody contains an amino acid sequence of CDR shown as SEQ ID NO. 2, 4, and 6; and a light chain variable region sequence of the monoclonal antibody contains an amino acid sequence of CDR shown as SEQ ID NO. 9, 11, and 13. A polypeptide of the enzyme is used as an immunogen to immunize a mouse to obtain lymphocyte, a fusion cell is prepared by a hybridoma cell fusion technique, and a cell strain which can generate high affinity and high specificity and is used for detecting and identifying the enzyme is obtained. The monoclonal antibody of 1-aminocyclopropane-1-carboxylate oxidase can be used for detecting the content change of the 1-aminocyclopropane-1-carboxylate oxidase in different developmental stages of fruits. The monoclonal antibody disclosed by the invention provides a tool for targeted protein study, so that deeper study on protein expression changes and protein network maps of different plant types, different strains or different development stages is realized.

Description

technical field [0001] The invention belongs to the field of monoclonal antibody biotechnology, in particular to a monoclonal antibody of 1-aminocyclopropane-1-carboxylate oxidase and application thereof. Background technique [0002] 1-Aminocyclopropane-1-carboxylate oxidase is a non-heme iron enzyme encoded by the ACO gene (Zhang Z, Schofield CJ, Baldwin JE, Thomas P, John P. Biochem. J. 307:77–85 ), this enzyme belongs to the oxidoreductase family and is widely found in plants, especially in fruits. 1-Aminocyclopropane-1-carboxylate oxidase is a rate-limiting enzyme that catalyzes ethylene synthesis and plays a role in regulating fruit development and ripening (Pirrung MC, Kaiser LM, Chen JBiochemistry.32(29):7445–50) . [0003] Currently, there is no specific antibody against 1-aminocyclopropane-1-carboxylate oxidase for fruit ripening research on the market. Therefore, at the level of proteomics, there is a lack of effective antibody tools for this research field. St...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40G01N33/573G01N33/577
CPCC07K16/40G01N33/573G01N33/577
Inventor 翁炜宁孟逊
Owner 艾比玛特医药科技(上海)有限公司
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