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A kind of duf642 protein monoclonal antibody and its application

A DUF642, monoclonal antibody technology, applied in application, immunoglobulin, anti-plant immunoglobulin, etc., can solve the problem of lack of antibody tools, and achieve high specificity and high affinity.

Active Publication Date: 2022-07-22
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no antibody specifically targeting the grape DUF642 gene on the market. Therefore, at the level of protein detection, there is a lack of effective antibody tools for this research field, which can be used for basic research on grape abortion, and at the protein level of commercially available seedless varieties of grapes. detection

Method used

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  • A kind of duf642 protein monoclonal antibody and its application
  • A kind of duf642 protein monoclonal antibody and its application
  • A kind of duf642 protein monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Establishment of hybridoma cell lines and preparation of antibodies

[0026] 1. Experimental materials

[0027] 1.1 Preparation of antigen:

[0028] Monoclonal antibody was prepared by cloning the full-length gene of DUF642, recombinantly expressing the full-length protein of DUF642, and using it as an immunogen to immunize mice to prepare monoclonal antibodies. NPGVEEDPACGPLIDSVAFR ALYPPRPSSK NLLKNGGFEE GPYVFPNTSW GVLIPPNIED DHSPLPGWMV ESLKAVKYIDSDHFSVPQEK RAVELVAGKE SAIAQVARTI PGKTYALSFS VGDASNSCEG SMVVEAFAGR DTIKVPYESKGKGGFKRAVL RFVAVSNRTR IMFLSTFYTM RSDDYASLCG PVLDDVKLLS LRTPPRHI(SEQ ID NO: 1).

[0029] 1.2 Immune mice:

[0030] Three Balb / c mice (8-12 weeks old) were immunized with the antigen and their serum titers were monitored to determine the optimal number of immunizations. Optimized adjuvant and immunization methods produce high-affinity antibodies (IgG subtype) against most antigens. After the initial immunization, 3 to 4 booster immunizations...

Embodiment 2

[0039] Example 2 Validation of anti-DUF642 monoclonal antibody

[0040] The obtained monoclonal antibody 1L3 cell line was verified by immunoenzyme-linking, Western blotting, co-immunoprecipitation plus mass spectrometry, and antibody chip to determine the most effective antibody.

[0041] 2.1 ELISA (immunoenzyme-linked) validation of antibodies and recombinant proteins

[0042] The mouse ascites antibody produced by the monoclonal cell line 1L3 was coated on a 96-well ELISA plate, incubated, washed with skim milk and blocked overnight, washed with PBS, and stored at 4°C until use. Antigen peptides were incubated, washed with PBS, and controls were set. The HRP-labeled detection antibody was added to the ELISA plate incubated as described above. TMB color reaction, microplate reader reading. Antibody titers are shown in Table 1:

[0043] Table 1: Antibody ELISA test data

[0044]

[0045] 2.2 Endogenous Western Blot (WB) Validation of Antibodies

[0046] Using the who...

Embodiment 3

[0048] The hybridoma cell line with the 1L3 clone number anti-DUF642 antibody was cultured, total RNA was extracted, and the mRNA was reverse transcribed into first-strand cDNA; the heavy and light chain genes were amplified by PCR and cloned into a sequencing vector , multiple positive clones were sequenced to obtain the final sequence result.

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Abstract

The present invention provides a DUF642 protein monoclonal antibody and its application. The DUF642 monoclonal antibody of the present invention has high affinity and high specificity for DUF642 protein, can be widely used for DUF642 detection, and can be especially used for detecting the expression of DUF642 protein in various commercially available grape varieties. In addition, the monoclonal antibody of the present invention provides a tool for targeted protein research, so that the regulatory relationship between the expression of DUF642 protein in different abortive grape varieties and the formation of the final grape with or without seed can be effectively studied.

Description

technical field [0001] The present invention relates to a hybridoma cell line and the monoclonal antibody produced therefrom and its application, in particular to a monoclonal antibody against the cell wall synthesis-related protein DUF642 and a hybridoma cell line secreting the antibody and the application of the antibody. Background technique [0002] The cell wall DUF642 protein family is a highly conserved protein in plants that may be related to the structural modification of pectin. The DUF642 family of proteins can be involved in plant cell wall physicochemical modifications, developmental or in response to environmental signals. It plays a role in plant growth and development and also plays an important role in response to plant stress, such as drought, salt, and aluminum oxide. [0003] According to previous research, during the development of grape seeds, there is a clear regulatory relationship between the expression of DUF642 gene and the formation of fertile se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/16C07K14/415C12N15/29G01N33/68G01N33/577
CPCC07K16/16C07K14/415G01N33/68G01N33/577C07K2317/56C07K2317/565G01N2333/415
Inventor 张颖刘锐涛刘崇怀樊秀彩姜建福孙磊
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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