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N terminal specific human Eme1 polypeptide and antibody preparation method

A kind of specific and specific technology, applied in the preparation of human Eme1 polypeptide and its antibody, the field of biological products, can solve the problems of non-specific antibody binding protein position, high antibody price, poor antigenicity, etc., to achieve strong affinity, good antibody specificity, cost low effect

Inactive Publication Date: 2008-12-10
BEIJING IMMUNOHUNT CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The present invention is to solve the problem of specifically detecting the expression and natural existence of Eme1 through immune experiments, and to establish the basic biological products required for corresponding in vitro immune analysis, and provides a kind of N-terminal synthetic polypeptide antibody that can specifically target Eme1 and its Preparation
The present invention can also solve the problems of high price, low affinity, non-specific position of antibody-binding protein and poor antigenicity of similar antibodies currently used.

Method used

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  • N terminal specific human Eme1 polypeptide and antibody preparation method
  • N terminal specific human Eme1 polypeptide and antibody preparation method
  • N terminal specific human Eme1 polypeptide and antibody preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Example 1: Synthesis of Emel antigenic peptide.

[0017] DNAstar, OMIGA, UWGCG and other protein analysis software were used to analyze the hydrophilic structure (hydrophilicity), antigenicity (antigenicity), surface accessibility (surface probability) and homology analysis (homology) of the amino acid sequence of Eme1, and then Combined with our existing verification experience of actually preparing antibodies, the 26th to 40th positions were finally determined as the target, and its amino acid sequence was LKKEPSSTKRRQPER. The crude product was obtained after decremental synthesis from the fixed carboxyl group to the amino terminal on an automatic peptide synthesizer. After being dissolved in 30% acetonitrile, it was analyzed by high pressure liquid chromatography (HPLC), the area of ​​the main peak was calculated and collected, and the synthesized peptide was purified and identified by mass spectrometry after refrigerated vacuum drying.

Embodiment 2

[0018] Example 2: Coupling of synthetic polypeptides to carriers.

[0019] Select KLH (Keyhole limpet hemocyanin) as the carrier protein, and use the bifunctional reagent maleamide benzoic acid-N-succinate (MBS) to couple KLH with the synthetic peptide: Take 5 mg (0.11 μmol of KLH, containing lysine Acid 2.2μmol), dissolved in 0.75mL coupling buffer 1 (50mmol / L borate buffer, pH8.5); 3mg MBS (11μmol) was dissolved in 75μL dimethylformamide (DMF). Add the MBS solution to the KLH solution in 3 times, rotate and mix well, and let it act at room temperature for 30 minutes. After rapid centrifugation, the reaction mixture (about 0.8 mL) was added to the coupling buffer 2 (0.1 mol / L phosphate, 0.15 mol / L NaCl, 0.01 mol / L Na 2 EDTA, pH 7.0) equilibrated PD-10 column. Elute with coupling buffer 2, and collect the eluate (ie MBS-KLH solution). Dissolve 1.5mg of synthetic peptide in 0.15mL of coupling buffer 2, add MBS-KLH buffer 0.56, rotate and mix at room temperature, let it rea...

Embodiment 3

[0020] Example 3: Preparation of anti-polypeptide antibody.

[0021] Take 500 μg of coupled KLH-polypeptide, dissolve in 500 μL phosphate buffer, add an equal volume of complete Freund’s adjuvant. New Zealand rabbits (weight standard: 2-2.5 kg) were selected for immunization at the right age, and after adaptive feeding, they were injected intradermally at no less than 15 points on the back. After 2 weeks, the amount of antigen was halved (250μg), and an equal volume of incomplete Freund's adjuvant was added for the first booster immunization. The second booster immunization was carried out 2 weeks later, and the method was the same as before. Another 2 weeks later, a small amount of blood was collected from the ear vein, and the enzyme plate was coated with synthetic polypeptide (1 μg / mL), and the titer of the immune serum was detected by indirect ELISA. The immunization was repeatedly strengthened, and the immunization was stopped when the antibody titer of the blood test...

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Abstract

The invention discloses N-terminal specific human Eme1 polypeptide, as well as an antibody and a preparation method thereof, belonging to the biomedicine technical field. The amino acid sequence of the N-terminal specific human Eme1 polypeptide is LKKEPSSTKRRQPER. Antihuman EME1 polypeptide antibody is prepared according to the following method that: (1) human EME1 epitope is analyzed; (2) human EME1 N-terminal polypeptide is synthesized; (3) synthesized polypeptide is crosslinked with carrier protein; (4) rabbit anti-human EME1 polypeptide antibody is prepared; (5) serum containing the antibody is obtained through collection and separation, and the antibody is purified, and then the antihuman EME1 polypeptide antibody can be obtained. The N-terminal specific antihuman BTRC synthesized polypeptide antibody is high in potency, strong in affinity, good in specificity, capable of having specific conjugation with natural human Eme1; purified antibody can be completely used for immunoblot and enzyme linked immunosorbent assay, as well as the establishment of in vitro immunization analytical method. The antibody provides a useful tool for the research on the biological effects of Eme1 in DNA damage and genomic stability.

Description

technical field [0001] The invention relates to biological products for in vitro tests characterized by antibodies, specifically an N-terminal specific human Eme1 polypeptide and a method for preparing antibodies thereof, belonging to the technical field of biomedicine. Background technique [0002] The combination of Eme1 (essential meiotic endonuclease 1homolog 1) protein and Mus81 constitutes an exonuclease capable of cutting branched DNA structures. Studies have shown that embryonic stem cells knocked out of the Eme1 gene are highly sensitive to DNA cross-linking agents such as mitomycin C, and slightly less sensitive to ionizing radiation, UV radiation, and hydroxyurea treatment. In addition, the findings also show that Eme1 plays a critical role in the maintenance of genome integrity and DNA repair in mammals, and its deficiency can lead to spontaneous genome instability. Contents of the invention [0003] The present invention is to solve the problem of specificall...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K16/18
Inventor 陈光宇杜宏武王德仙陈吾奇
Owner BEIJING IMMUNOHUNT CORP
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