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Specific histidine methylation modified S100A9 protein immunogen, polyclonal antibody and preparation method of polyclonal antibody

A technology of S100A9, histidine methyl, applied in the field of polyclonal antibody and its preparation, can solve the problems of complex platform, no specific antibody, long sample processing time, etc.

Pending Publication Date: 2022-02-18
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the specific function of the S100A9 protein targeting the above-mentioned specific histidine methylation modification has not been studied, nor is there any specific antibody for its specific histidine methylation modification site.
At present, the method of identifying histidine methylation on S100A9 can only be identified by mass spectrometry, but the platform required by mass spectrometry technology is relatively complicated, and general laboratories do not have such conditions; in addition, the entire process of mass spectrometry requires a long time to process samples , the results obtained cannot be presented intuitively.

Method used

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  • Specific histidine methylation modified S100A9 protein immunogen, polyclonal antibody and preparation method of polyclonal antibody
  • Specific histidine methylation modified S100A9 protein immunogen, polyclonal antibody and preparation method of polyclonal antibody
  • Specific histidine methylation modified S100A9 protein immunogen, polyclonal antibody and preparation method of polyclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Preparation of Polypeptide

[0062] Entrust Hangzhou Huaan Biotechnology Co., Ltd. to synthesize the following peptides:

[0063] The polypeptide whose amino acid sequence is shown in SEQ ID No.1 is named as polypeptide 1;

[0064] The polypeptide whose amino acid sequence is shown as SEQ ID No.2 is named as polypeptide 2;

[0065] The polypeptide whose amino acid sequence is shown as DEGPGHHH(methyl)KPGLGEC is named as polypeptide 3;

[0066] The polypeptide whose amino acid sequence is shown as PRGHGHSH(methyl)GKGCGKC is named as polypeptide 4.

Embodiment 2

[0067] Example 2 Preparation of human S100A9 protein immunogen modified by histidine methylation at position 105

[0068] By protein coupling technology, the polypeptide 3 in Example 1 was coupled with hemocyanin (KLH) to obtain the human S100A9 protein immunogen modified by the methylation of the 105th histidine, and the specific steps were as follows:

[0069] 1. Peptide 3 coupled to KLH

[0070] (1) Dissolve 20mg KLH in 2mL, 5mM EDTA aqueous solution;

[0071] (2) Weigh 8 mg Sulfo-SMCC and dissolve it completely in 50 μL DMSO, then add 150 μL 1×PBS and mix well;

[0072] (3) Add the Sulfo-SMCC solution dropwise to the KLH solution, shake gently while adding, and place at room temperature for 1 hour;

[0073] (4) Put the above-mentioned activated KLH solution into a dialysis bag, clamp the dialysis clamp, and dialyze in 2L of 1×PBS under magnetic stirring in a refrigerator at 4°C for 1 hour;

[0074] (5) Replace with new 1×PBS and dialyze for 2 hours, repeat once; put the...

Embodiment 3

[0086] Example 3 Preparation of polyclonal antibody specifically recognizing human S100A9 protein modified by methylation of histidine at position 105

[0087] Animal immunity:

[0088]Take the human S100A9 protein immunogen modified by histidine methylation at position 105 in Example 2 out of the -20°C refrigerator, dissolve at room temperature, avoid repeated freezing and thawing, and mark the syringe; according to Table 1 According to the configuration in , extract the adjuvant according to the volume ratio of the adjuvant and the human S100A9 immunogen modified by histidine methylation at the 105th position of 1:1, fully emulsify, and the emulsification standard is: the emulsified immunogen is dropped into 37 ℃ non-dispersible in water is qualified.

[0089] Before the first immunization, number all New Zealand white rabbits, the first immunization antigen concentration is 1mg / mL, the rabbit is 0.5mL / rat, the second immunization - the fourth immunization antigen amount ...

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Abstract

The invention discloses a specific histidine methylation modified S100A9 protein immunogen, a polyclonal antibody and a preparation method of the polyclonal antibody. The specific histidine methylation modified S100A9 protein immunogen comprises a polypeptide which has an amino acid sequence as shown in SEQ ID No.1 or SEQ ID No.2 and has methylated N at a specific histidine pi site. By immunizing animals with the specific histidine methylation modified S100A9 protein immunogen, the polyclonal antibody capable of specifically recognizing the specific histidine methylation modified S100A9 protein can be obtained, so that the change of specific histidine methylation of S1000A9 in a biological sample can be rapidly and simply detected. The research on the functional influence of the specific histidine methylation S100A9 protein (or calprotectin) and the development of the specific histidine methylation dynamic change of the S100A9 (or calprotectin) in the specific life activity or inflammation generation process are promoted.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an immunogen of S100A9 protein modified by specific histidine methylation, and also relates to a polyclonal antibody capable of specifically recognizing S100A9 protein modified by specific histidine methylation and its Preparation. Background technique [0002] S100A9 protein is a member of the calcium-binding protein S100 protein family. It is highly expressed in neutrophils and monocytes. It usually forms a heterodimer through chemical combination with S100A8. Its main function is to inhibit casein kinase Ⅰ and Ⅱ activity, so S100A8 / S100A9 is collectively called Calprotectin (CP, calprotectin). [0003] Calprotectin is widely distributed in human cells, tissues, and body fluids. It is the main protein of granulocytes, monocytes, and keratinocytes. Its expression is tissue- or cell-specific and can be used as a marker of acute inflammatory cell activation. Specifically...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K1/107C07K16/24C07K1/22G01N33/68
CPCC07K14/4728C07K16/24G01N33/68G01N2469/10G01N2333/4727
Inventor 潘文曹丹
Owner UNIV OF SCI & TECH OF CHINA
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