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Complete antigen of AG15 polypeptide as well as preparation method and antibody thereof

A complete antigen, AG15 technology, applied in the field of immunology, can solve the problems of low immunogenicity of AG15 polypeptide and inability to use complete antigen

Inactive Publication Date: 2014-02-05
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the AG15 polypeptide itself has low immunogenicity and cannot be used as a complete antigen

Method used

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  • Complete antigen of AG15 polypeptide as well as preparation method and antibody thereof
  • Complete antigen of AG15 polypeptide as well as preparation method and antibody thereof
  • Complete antigen of AG15 polypeptide as well as preparation method and antibody thereof

Examples

Experimental program
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preparation example Construction

[0039] Such as figure 1 The method for preparing the complete antigen of the above-mentioned AG15 polypeptide includes the following steps:

[0040] S10. Providing keyhole limpet hemocyanin activated by a crosslinking agent, and reconstituting the keyhole limpet hemocyanin activated by a crosslinking agent with sodium phosphate buffer to obtain a reconstituted keyhole limpet hemocyanin solution activated by a crosslinking agent .

[0041] The structure of keyhole limpet hemocyanin activated by cross-linking agent is shown as figure 2 shown.

[0042] The sodium phosphate buffer used in this embodiment has a pH of 6.6, and 1 mL of the sodium phosphate buffer contains 20 mM sodium phosphate, 230 mM NaCl, 2 mM EDTA and 80 mM sucrose.

[0043] In this embodiment, the concentration of the keyhole limpet hemocyanin solution activated by the cross-linking agent is 5 mg / mL.

[0044] In this embodiment, the crosslinking agent is maleimide.

[0045] S20, providing the AG15 polypept...

Embodiment 1

[0063] Preparation of complete antigen of AG15 polypeptide.

[0064] 1. Open a tube of maleimide-activated KLH and add 1mL of 20mM sodium phosphate buffer (230mM NaCl, 2mM EDTA, 80mM sucrose, pH6.6) to reconstitute the maleimide-activated KLH to prepare 5mg / mL solution, use it as soon as possible;

[0065] 2. Reconstitute the coupling buffer with 10mL double distilled water to prepare sodium phosphate buffer (100mM EDTA, 80mM sucrose, pH6.6);

[0066] 3. Dissolve 4 mg of AG15 polypeptide containing cysteine ​​in 0.5 mL of coupling buffer or double distilled water, and keep 50 μL of the peptide solution for determining the coupling efficiency (total cys), and store it at 2-8 °C;

[0067] 4. Immediately mix the AG15 polypeptide solution with the maleimide-activated KLH in the reaction tube and stir;

[0068] 5. Stir the reaction for 2 hours at room temperature or overnight at 2-8°C;

[0069] 6. Reserve 100 μL of the coupling reaction solution to determine the coupling effici...

Embodiment 2

[0071] Separation and purification of complete antigen of AG15 polypeptide.

[0072] 1. Dissolve a packet of PBS in 1L double distilled water, add 0.01% sodium azide, and store for a long time;

[0073] 2. Fix the Sephadex G-25M gel column on a suitable beaker;

[0074] 3. Remove the cover of the gel column, cut off the bottom of the gel column, let the excess liquid flow out, and prevent the liquid in the column from draining;

[0075] 4. Equilibrate the gel column with 30mL PBS. If the gel column is not used immediately, please cover the upper and lower covers and store it at 2-8°C;

[0076] 5. Add the reacted liquid to the gel column, and collect the liquid flowing out from the lower end;

[0077] 6. Wash with a total volume of 10mL PBS, collect the eluate, collect 0.5mL-1.0mL each time, and measure the absorbance value at 280nm;

[0078] 7. Collect the protein-containing eluate and freeze it at -20°C.

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Abstract

The invention relates to a complete antigen of AG15 polypeptide. The complete antigen of AG15 polypeptide is formed by the coupling of AG15 polypeptide and keyhole limpet hemocyanin activated by a crosslinking agent, wherein the sequence of AG15 polypeptide is the sequence as shown in SEQ ID No.1. The complete antigen of such AG15 polypeptide is relatively high in immunogenicity and can be put into use as the complete antigen. The invention also discloses a preparation method of the complete antigen of AG15 polypeptide, and an antibody of the complete antigen of AG15 polypeptide. The antibody is specially bound with the complete antigen of AG15 polypeptide. The antibody of the complete antigen of AG15 polypeptide can be used for detecting mice osteocalcin protein in serum or tissue.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a complete antigen of AG15 polypeptide, its preparation method and antibody. Background technique [0002] Osteocalcin protein is secreted by osteoblasts, and its content changes with age. The state of osteoblasts can be understood through the content of serum osteocalcin, which is of great significance for the judgment of osteoporosis. [0003] The AG15 polypeptide is a conservative sequence in the mouse osteocalcin protein molecule, which is an antigenic polypeptide after sequence analysis. However, the AG15 polypeptide itself has low immunogenicity and cannot be used as a complete antigen. Contents of the invention [0004] Based on this, it is necessary to provide a complete antigen of AG15 polypeptide. [0005] In addition, it is also necessary to provide a method for preparing the complete antigen of the AG15 polypeptide and an antibody specifically binding to the complete ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/795C07K14/47C07K16/18
CPCC07K14/795C07K14/47C07K16/18
Inventor 任培根郭成志李健郭小勇
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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