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Monoclonal antibody of S-adenosylmethionine synthetase and application thereof

An adenosylmethionine, monoclonal antibody technology, applied in the field of monoclonal antibody biology, can solve problems such as lack of antibody tools

Active Publication Date: 2017-11-24
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

[0004] Currently, there is no specific targeting antibody against S-adenosylmethionine synthase on the market. Therefore, at the proteomics level, there is a lack of effective antibody tools for this research field, which can be used in different plant types, different strains or Study on the Expression Ability Level of the Enzyme at Different Developmental Stages

Method used

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  • Monoclonal antibody of S-adenosylmethionine synthetase and application thereof
  • Monoclonal antibody of S-adenosylmethionine synthetase and application thereof
  • Monoclonal antibody of S-adenosylmethionine synthetase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Preparation of a monoclonal antibody against S-adenosylmethionine synthase hybridoma cells and preparation and purification of monoclonal antibodies against S-adenosylmethionine synthetase

[0020] 1.1 Antigen preparation

[0021] Synthesized four sets of S-adenosylmethionine synthetase polypeptides 'FTKCPEEIGA', 'TDETPELMPL', 'QVTVEYYNDK' and 'VLISTQHDET', and used them as immunogens respectively. The above polypeptides were also coupled to VLP and enhanced the immunogenicity of the traditional KLH system factor.

[0022] 1.2 Immunization of mice

[0023] 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 immunization, there will b...

Embodiment 2

[0032] Embodiment 2 Verification of anti-CK18 monoclonal antibody

[0033] 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.

[0034] 2.1 Elisa (immunoenzyme-linked) pairing verification of antibody and antigen polypeptide

[0035] 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:

[0036] Table I

[0037]

[0038] 2.2 Endogenous western blot (WB) validation of antibodies

[0039] Use cherry whole protein lysate, antibo...

Embodiment 3

[0047] Example 3 Application of a monoclonal antibody to S-adenosylmethionine synthetase of the present invention in detecting the content of S-adenosylmethionine synthetase in different animals

[0048] Since the antibody of the present invention has unique specificity to S-adenosylmethionine synthetase, it can be used as a detection antibody for specifically detecting the content of S-adenosylmethionine synthetase in different animals and plants, or it can also be used according to conventional technical means in the field. Further developed as a detection kit.

[0049] First, the antibody of the present invention was subjected to an ELISA experiment with a fixed concentration (1 mg / ml) and a standard product of S-adenosylmethionine synthetase recombinant protein at different concentrations to obtain the corresponding OD value, and draw a standard curve. The concentration coordinate of glycoside methionine synthase, the ordinate is the optical density value (OD value) that m...

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Abstract

The invention discloses a monoclonal antibody of S-adenosylmethionine synthetase. A heavy chain variable region sequence of the monoclonal antibody contains CDR amino acid sequences shown by SEQ ID NO.2, 4 and 6; a light chain variable region sequence contains CDR amino acid sequences shown by SEQ ID NO.9, 11 and 13. The polypeptide of the enzyme is used as an immunogen for immunizing a mouse to obtain lymphocyte; a hybridoma fusion technology is used for preparing fusion cells; a cell strain capable of detecting and identifying the S-adenosylmethionine synthetase at high affinity and high specificity is obtained. The monoclonal antibody can be used for detecting the content of S-adenosylmethionine synthetase in bodies of animals and plants. The monoclonal antibody provides a targeted protein study tool, so that the deep level study of protein network atlas and the protein expression change on different plant types, different strains or different development periods can be realized.

Description

technical field [0001] The invention belongs to the field of monoclonal antibody biotechnology, in particular to a monoclonal antibody of S-adenosylmethionine synthetase and application thereof. Background technique [0002] S-adenosylmethionine (SAM), the English name is S-adenosyl-methionine, is an important metabolic intermediate substance in animals, plants and other living bodies, and is also an important physiologically active substance in the human body, participating in various biochemical reactions (Jingpei , Adenosylmethionine, Chemistry of Life, 1995,5:49), such as participating in transmethylation, transaminopropylation and transsulfuration in vivo (LU S C, Regulation of hepatic glutathione synthesis Sem Liv Dis, 1998, 18:331-343). Most of the existing technologies start with the preparation method of SAM to study it. [0003] SAM is enzymatically synthesized by the substrate L-methionine and ATP by S-adenosylmethionine synthetase, therefore, S-adenosylmethioni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40G01N33/577G01N33/573
CPCC07K16/40C07K2317/565G01N33/573G01N33/577G01N2333/91171
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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