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Monoclonal antibody for detecting tobacco apyrases and application of monoclonal antibody

An ATPase and tissue ATPase technology, which is applied in the field of monoclonal antibodies for detecting tobacco ATPase, can solve problems such as lack of antibody tools, and achieve the effect of high specificity and high affinity

Pending Publication Date: 2020-06-19
YUNNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no targeted antibody specifically targeting tobacco adenosine triphosphatase (APY) on the market. Therefore, at the protein level, there is a lack of effective antibody tools for this research field to quickly and sensitively detect adenosine triphosphatase Expression changes at the protein level for functional studies of APY in cell growth and reproductive pollination in tobacco and other plants

Method used

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  • Monoclonal antibody for detecting tobacco apyrases and application of monoclonal antibody
  • Monoclonal antibody for detecting tobacco apyrases and application of monoclonal antibody
  • Monoclonal antibody for detecting tobacco apyrases and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Preparation of a monoclonal antibody to adenosine triphosphatase hybridoma cells and preparation and purification of an anti-adenosine triphosphatase monoclonal antibody

[0031] 1.1 Antigen preparation

[0032] Prokaryotic expression of whole adenosine triphosphatase protein as an immunogen, total protein-coupled VLPs, and immunogenicity enhancers of conventional KLH systems.

[0033] 1.2 Immunization of mice

[0034] Six 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 adjuvants and immunization methods can produce high-affinity antibodies (IgG subtypes) against most antigenic polypeptides. After the initial immunization, there will be 3 to 4 times of boosting. After the boosting, the mouse serum will be taken to detect the titer (the recombinant protein of adenosine triphosphatase is used as an anti-antigen coating). Mice with qualified titers ...

Embodiment 2

[0043] Example 2: Verification of Anti-ATPase Monoclonal Antibody

[0044] The obtained monoclonal antibody cell lines were verified by enzyme-linked immunosorbent immunoassay, western blotting, co-immunoprecipitation plus mass spectrometry, antibody chip, etc. to determine the effectiveness of the antibody.

[0045] 2.1 ELISA (enzyme-linked immunosorbent) verification of antibodies and antigenic peptides

[0046] 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. Antibody titers are shown in Table 1:

[0047] Table 1 Antibody ELISA detection data

[0048]

[0049] 2.2 Endogenous protein immunoblotting (WB) validation of antibodies

[0050] Us...

Embodiment 3

[0054] Example 3: Sequencing of Anti-ATPase Monoclonal Antibodies

[0055] The hybridoma cell line of anti-APY antibody was cultured and the total RNA was extracted, and the mRNA was reverse transcribed into the first-strand cDNA; the heavy chain and light chain genes were amplified by PCR and the amplified genes were cloned into the sequencing vector for multiple The final sequence results were obtained by sequencing the positive clones.

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Abstract

The invention discloses a monoclonal antibody for detecting tobacco apyrases. Sequences of a heavy chain variable region of the monoclonal antibody comprise an amino acid sequence GYSITSGYY of CDR1 shown as SEQ ID NO.2, an amino acid sequence ISYDGSN of CDR2 shown as SEQ ID NO.4, and an amino acid sequence FGKGY of CDR3 shown as SEQ ID NO.6; and sequences of a light chain variable region comprisean amino acid sequence QSIVHSNGNTY of CDR1 as shown in SEQ ID NO.9, an amino acid sequence KVS of CDR2 as shown in SEQ ID NO.11, and an amino acid sequence FQGSHVPWT of CDR3 as shown in SEQ ID NO.13.The invention provides the monoclonal antibody for detecting tobacco apyrases (APY) and application of the monoclonal antibody, and aims to overcome the defects in the prior art. By providing a targeted protein research tool, the detection of APY protein expression change of different tissue of tobaccos and other plants can be realized under different physiological conditions.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody for detecting tobacco adenosine triphosphatase and its application. Background technique [0002] Apyrase (Apyrases, APY, EC 3.6.1.5), responsible for catalyzing the hydrolysis of phosphate anhydride bonds in nucleoside triphosphates (NTP) and nucleoside diphosphates (NDP) to form nucleoside monophosphate (NMP) The enzyme is widely found in bacteria, fungi, animals and plants, and its function is the most widely studied in mammals, and in plants, it is found in potatoes (Solanum tuberosum), Arabidopsis (Arabidopsis), cotton (Gossypium hirsutum), pea (Pisum sativum), soybean (Glycinesoja) and alfalfa (Medicago truncatula) were reported. The research results in the above plants show that: Apyrases play multiple roles in regulating plant growth and development, such as regulating fiber growth, stomatal opening and closing, cold resistance and pollen ge...

Claims

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Application Information

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IPC IPC(8): C07K16/40G01N33/573
CPCC07K16/40G01N33/573C12Y306/01005C07K2317/56C07K2317/565
Inventor 陈穗云廖菊够陶柯良魏雪梅
Owner YUNNAN UNIV
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