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Carboxyl-terminal specific anti-human amyloid protein monoclonal antibody gene and its encoded polypeptide and application

A monoclonal antibody and amyloid technology, which is applied in the field of genetic engineering, can solve the problems of unsatisfactory differential diagnosis and treatment target effect, no Aβ42 monoclonal antibody, and inability to specifically recognize Aβ2 oligomers, etc., and achieve immune effect. Good, high titer of immune serum, and the effect of reducing side effects

Active Publication Date: 2014-05-07
BEIJING JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, some people have carried out the development of Aβ42-specific antibodies, but there is no Aβ42 monoclonal antibody with a conformational epitope
It cannot specifically recognize Aβ2 oligomers, so the target effect of differential diagnosis and treatment is not ideal, and a monoclonal antibody that specifically recognizes and binds to Aβ2 oligomers at the carboxyl terminal is needed

Method used

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  • Carboxyl-terminal specific anti-human amyloid protein monoclonal antibody gene and its encoded polypeptide and application
  • Carboxyl-terminal specific anti-human amyloid protein monoclonal antibody gene and its encoded polypeptide and application
  • Carboxyl-terminal specific anti-human amyloid protein monoclonal antibody gene and its encoded polypeptide and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of carboxy-terminal specific anti-human amyloid monoclonal antibody

[0034] 1. Test materials

[0035] 1. Aβ(35-42)(MVGGVVIA) (synthesized by Shanghai Sangon Bioengineering Co., Ltd.)

[0036] 2. Aβ42 peptide (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) and Aβ40 peptide (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) (synthesized by Shanghai Sangon Bioengineering Co., Ltd.)

[0037] 3. Hexafluoroisopropanol (1,1,1,3,3,3-hexafluoro-2-propannol, HFIP) (purchased from Sigma)

[0038] 4. Anhydrous dimethyl sulfoxide (DMSO) (purchased from Sigma)

[0039] 5. F12 medium (purchased from Sigma Company)

[0040] 6. Complete Freund's adjuvant (purchased from Sigma)

[0041] 7. Incomplete Freund's adjuvant (purchased from Sigma)

[0042] 8. RPMI1640 culture medium (purchased from Sigma Company)

[0043] 9. PEG4000 (polyethylene glycol) (purchased from Sigma)

[0044] 10. Fetal bovine serum (provided by Hangzhou Sijiqing Bioengineering Materials Co., Ltd.) ...

Embodiment 2

[0076] Embodiment 2 Indirect ELISA experiment

[0077] 1. Test materials

[0078] 1. ELISA plate (manufactured by SUNRISE)

[0079] 2. HRP-labeled goat anti-mouse IgG (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.)

[0080] 2. Test method

[0081] 1. Coating: take 10 μg / ml Aβ40 and Aβ42 oligomeric mixture as the coating antigen, dissolve in the coating buffer (pH9.6, 0.05mol / L carbonate buffer), add to the microtiter plate, each Well 100 μl, overnight at 4°C.

[0082] 2. PBS-T (Weighing NaCl8g, KCl0.2g, NaCl 2 HPO 4 1.44g, KH 2 PO 4 0.44g, Tween-200.05ml, add ddH 2 0 to 1 L, adjust the pH to 7.2-7.4) Wash the plate: 3 times, 5 min each time.

[0083] 3. Blocking: add PBS-T containing 0.2% BSA, 100 μl per well. Treat at 37°C for 2h.

[0084] 4. Add the carboxy-terminal specific anti-human amyloid monoclonal antibody diluted in PBS-T times to a 96-well plate, 100 μl per well. Treat at 37°C for 2h. At the same time, a negative control and a positiv...

Embodiment 3

[0098] Example 3 Western blot (Western blot) experiment

[0099] 1. Test method

[0100] Preparation of detection antigen Aβ oligomerization mixture: as shown in Example 1.

[0101] Take 5-10 μg sample, 5×sample buffer, mix well and load the sample, first make the protein pass through the stacking gel with a voltage of 100V. When the sample enters the separating gel, adjust the voltage to keep it constant at 120V. When the bromophenol blue swims to the bottom of the gel, end the electrophoresis, remove the gel, and stain it with Coomassie Brilliant Blue R-250 routinely; put the gel and nitrocellulose membrane into containers containing blotting buffer Equilibrate in the chamber for 10 minutes, put filter paper, gel, NC membrane, and filter paper in order to form a "sandwich" shape, pour the transfer buffer, with the gel side facing the negative electrode and the NC membrane facing the positive electrode, carefully avoiding and driving away air bubbles. Turn on the power, ma...

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Abstract

The invention discloses a carboxyl-terminal specific anti-human amyloid protein monoclonal antibody gene and its encoded polypeptide and an application, the gene comprises a heavy chain variable region gene and a light chain variable region gene, wherein a sequence of the heavy chain variable region is showed as SEQ ID NO:1, and a sequence of the light chain variable region is showed as SEQ ID NO:3. According to the gene provided by the invention, because a Abeta 42 oligomer can be specifically recognized, and Abeta 40 and Abeta 42 oligomers which have the difference of two amino acid can be distinguished, in an immunotherapy experiment, the combination of monoclonal antibody gene and Abeta 40 can be avoided, thereby the side reaction caused by the non specific recognition can be reduced; the polypeptide obtained according to the gene coding is amyloid protein carboxyl-terminal polypeptide, the immunization effect is good, the mice immunization serum has high valence, and its antigen recognition capability has conformational characteristics, the invention can be used for AD early stage diagnosis.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, more specifically, the present invention relates to a carboxy-terminal specific anti-human amyloid monoclonal antibody gene and its coded polypeptide and application. Background technique [0002] The deposition of β-amyloid plaques in the brains of patients with Alzheimer's disease (AD) is a typical lesion of the disease. Aβ40 and Aβ42 are the main components of β-amyloid plaques in the brain of AD patients. The difference between the two is that Aβ42 has two more carboxyl terminals than Aβ40, and Aβ42 is more likely to aggregate into multiple levels of polymers, which is the main pathogenesis of AD. factor. Therefore, research on the diagnosis and treatment of Aβ42 is more meaningful than that of Aβ40. [0003] At present, the development of Aβ42-specific antibodies has been carried out, but there is no Aβ42 monoclonal antibody with a conformational epitope. It cannot spe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/13C07K16/18C12N5/20G01N33/68G01N33/577A61K39/395A61K48/00A61P25/28
Inventor 张莹何金生张娜洪涛
Owner BEIJING JIAOTONG UNIV
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