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ADRA1A polypeptide with specific C-terminal and preparation of antibody thereof

A specific, C-terminal technology, applied in the field of biomedicine, can solve the problems of non-specific antibody binding protein position, high antibody price, and low affinity, and achieve the effect of strong affinity, good antibody specificity, and low cost

Inactive Publication Date: 2009-04-22
BEIJING PEOPLE'S POLICE COLLEGE
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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 ADRA1A through immune experiments, and to establish the basic biological products required for corresponding in vitro immune analysis, and provides a synthetic polypeptide antibody that can specifically target the C-terminus of ADRA1A 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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  • ADRA1A polypeptide with specific C-terminal and preparation of antibody thereof
  • ADRA1A polypeptide with specific C-terminal and preparation of antibody thereof
  • ADRA1A polypeptide with specific C-terminal and preparation of antibody thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0015] Example 1: Synthesis of ADRA1A antigen peptide.

[0016] Use DNAstar, OMIGA, UWGCG and other protein analysis software to analyze the amino acid sequence of ADRA1A for hydrophilicity, antigenicity, surface probability and homology, and then Based on our existing experience in the verification of actual antibody preparation, we finally determined that the 440th to 452th positions were the targets, and the amino acid sequence was TPSLDKNHQVPTI. On the automatic peptide synthesizer, the crude product is obtained from the fixed carboxyl group to the amino-terminal decreasing synthesis. After being dissolved by 30% acetonitrile, it was analyzed by high pressure liquid chromatography (HPLC), and the main peak area was calculated and collected. The synthetic peptide was purified after freezing and vacuum extraction and identified by mass spectrometry.

Embodiment 2

[0017] Example 2: Coupling of synthetic polypeptide and carrier.

[0018] The carrier protein is KLH (Keyhole limpet hemocyanin), and the bifunctional reagent maleamide benzoic acid-N-succinate (MBS) connection method is used to couple KLH to the synthetic peptide: take KLH 5mg (0.11μmol, containing lysine) 2.2μmol), dissolved in 0.75mL coupling buffer 1 (50mmol / L borate buffer, pH8.5); 3mg MBS (11μmo) was dissolved in 75μL dimethylformamide (DMF). Add the MBS solution to the KLH solution in 3 times, rotate and mix, and act at room temperature for 30 minutes. After rapid centrifugation, the reaction mixture (about 0.8mL) was added to the pre-coupling buffer 2 (0.1mol / L phosphate, 0.15mol / LNaCL, 0.01mol / LNa 2 EDTA, pH7.0) balanced PD-10 column. Elute with coupling buffer 2 and collect the eluate (ie MBS-KLH solution). Dissolve 1.5 mg of synthetic peptide in 0.15 mL coupling buffer 2, add MBS-KLH buffer 0.56, rotate and mix at room temperature, let the reaction mixture (approximat...

Embodiment 3

[0019] Example 3: Preparation of anti-polypeptide antibodies.

[0020] Take 500 μg of the coupled KLH-polypeptide, dissolve it in 500 μL phosphate buffer, and add an equal volume of complete Freund's adjuvant. For immunization, select New Zealand rabbits (weight standard of 2-2.5 kg) for immunization of the appropriate age, and after adaptive breeding, they are injected into the skin 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. A second booster immunization was performed after 2 weeks, and the method was the same as before. Two weeks later, a small amount of blood was collected from the ear vein, and the enzyme-labeled plate was coated with a synthetic peptide (1 μg / mL), and the immune serum titer was detected by indirect ELISA. Repeatedly boost the immunization, and stop the immunization when the antibody titer reaches 1:160,000, a...

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Abstract

The invention discloses human ADRA1A polypeptide with a special C end and a method for preparing an antibody of the human ADRA1A polypeptide, and belongs to a biological product for in vitro tests with antibodies as characteristics. The amino acid sequence of the ADRA1A polypeptide with the special C end is TPSLDKNHQVPTI. The antibody against the ADRA1A polypeptide is prepared by the following method: (1) the analysis of an antigen epitope of ADRA1A is performed; (2) the polypeptide synthesis of the C end of the ADRA1A is performed; (3) the synthesized polypeptide is crosslinked with carrier protein; (4) a rabbit anti-ADRA1A polypeptide antibody is prepared; and (5) serum containing the antibody is obtained through the collection and separation, and the antibody is purified to obtain the antibody against the ADRA1A polypeptide. The antibody with the special C end against ADRA1A synthetic polypeptide has the advantages that the antibody has high titer, strong affinity, and good specificity, and can perform specificity combining reactions with natural ADRA1A; the preparation cost is low; and the antibody after the purification can be fully used for immunoblotting, enzyme-linked immunosorbent assays, and the establishment of in vitro immunoassay methods. The antibody provides a useful tool for researching in vivo and in vitro biological functions of the ADRA1A.

Description

Technical field [0001] The invention relates to a biological product for in vitro tests characterized by antibodies, in particular to a C-terminal specific ADRA1A polypeptide and a method for preparing antibodies thereof, belonging to the field of biomedical technology. Background technique [0002] ADRA1A is a member of the G protein-coupled receptor superfamily, with 7 transmembrane structures that can bind endogenous catecholamines and epinephrine. The results of pharmacological and molecular cloning studies have confirmed that this receptor family has complex heterogeneity. So far, 9 subtypes have been identified, including 3 alpha 1 subtypes (1A, 1B and 1C), and 3 alpha 2 receptors. Body (2A, 2B and 2C) and 3 beta receptors (1, 2 and 3). ADRA1A is involved in the onset and maintenance of several diseases, such as hypertension, chronic heart failure, diabetes, and glaucoma. The various subtypes of this gene have also been widely found in multiple organs and tissues, and it ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K16/18G01N33/531
Inventor 魏玉芝刘晓维杜宏武梁武斌吴巧雯
Owner BEIJING PEOPLE'S POLICE COLLEGE
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