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Preparation and application of retinoic acid induced protein 16 specific polyclonal antibody

An antibody and antigen protein technology, applied in the field of biomedicine, can solve the problems of not being able to specifically recognize the RAI16 protein, not being able to knock out mouse validation, etc., and achieve the effects of good specificity, high titer, and low preparation cost.

Active Publication Date: 2021-08-20
THE NAVAL MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Scientific research on RAI16 protein has gradually revealed its important role in cell physiology and pathology. However, none of the mainstream commercialized antibodies against RAI16 protein can pass the verification of RAI16 gene knockout mice, and the identified protein bands are all It is non-specific binding, which proves that it cannot specifically recognize RAI16 protein

Method used

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  • Preparation and application of retinoic acid induced protein 16 specific polyclonal antibody
  • Preparation and application of retinoic acid induced protein 16 specific polyclonal antibody
  • Preparation and application of retinoic acid induced protein 16 specific polyclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Preparation of anti-RAI16 92-292AA antibody

[0060] Anti-RAI16 92-292AA antibody was prepared as follows:

[0061] (1) Analysis of RAI16 antigenic epitope: using DNAStar software to analyze the amino acid sequence of RAI16 protein, the analysis results are as follows figure 1 As shown, the 92-292 amino acids of RAI16 protein have excellent hydrophilic structure, flexible region, antigenic index and surface probability structure. Determine the 201 amino acids at positions 92-292 of the RAI16 protein as the expressed immunogen protein sequence:

[0062] Its amino acid sequence is as follows:

[0063] lctlgkaeyppgmrqqvfqffskvlsqvqhpllhylsvhrpvqkllrlggtvpgsltekeevqftsvlcskiqqdpellayilegkkiigkkktarestappkdiagyrdkdcphsdalnrdpgldkehcgvpalsihlpaetegpengpgesnlitsllglckskksrlalkaqenilltqvastvast

[0064] (2) RAI16 92-292AA protein expression: Construct 92-292AA expression plasmid, use Escherichia coli to express, use affinity chromatography column and HPLC column t...

Embodiment 2

[0067] Example 2: Determination of Anti-RAI16 92-292AA Protein Antibody Titer

[0068] In this embodiment, ELISA is used to detect the titer of the anti-RAI16 92-292AA protein antibody: the ELISA detection plate is coated with RAI16-BSA at a concentration of 1mg / L, 100ul per well, incubated at 4°C for 8-12h, and then each well is Add 200ul of bovine serum with a concentration of 100ul, block at 37°C for 2h, add double-diluted anti-RAI16 92-292AA protein antibody after washing (normal rabbit serum is added to the control group), incubate at 37°C for 60min, 3 times After washing, add 100 ul of HRP-labeled goat anti-rabbit IgG diluted 1:5000 to each well, incubate at 37°C for 30 min, wash 3 times and develop TMB color, measure the absorbance value of the sample at A450 with a microplate reader, and calculate the antibody efficacy Valence, the antibody titer of this embodiment is 1:64000 (such as image 3 shown).

Embodiment 3

[0069] Example 3: Anti-RAI16 92-292AA protein antibody is used for the detection of RAI16 protein expression

[0070] Western blotting was used to detect the expression of RAI16 protein.

[0071] 1×10 7 Cells or 100mg of mouse colon tissue were added to the lysate (50mM Tris-HCl, 150mM NaCl, 0.5mM EDTA, protease inhibitor cocktail) and lysed at 4°C for 30min, centrifuged at 4°C and 10000g for 20min, and the supernatant was quantified followed by protein samples. Suspend the prepared cell or colon tissue protein samples in 5xSDS loading buffer, heat at 95°C for 10 minutes, place on ice to cool for 10 minutes before loading the samples; after separation by SDS-PAGE gel, transfer to PVDF membrane by wet transfer method; After blocking with skimmed milk powder (at room temperature for 2 hours), add 1:1000 diluted purified RAI16 antibody overnight at 4°C; wash the membrane 3 times with PBST, then add 1:2000 HRP-labeled goat anti-rabbit IgG and incubate at room temperature for 1 h...

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Abstract

The invention provides preparation and application of a retinoic acid induced protein 16 (RAI 16) specific polyclonal antibody.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a preparation method and application of retinoic acid-induced protein 16 immunogen protein expression and antibody thereof. Background technique [0002] The NCBI accession number of retinoic acid induced protein 16 (RAI16) is: NP_919326.1, the Swissprot accession number is Q80YR2, and the IPI number is IPI00654780.2. RAI16 is a protein molecule induced by retinoic acid, and its structure and function are still unclear. RAI16 was originally isolated when retinoic acid induced cell differentiation, and retinoic acid has the functions of inhibiting cell proliferation, promoting cell apoptosis and promoting cell differentiation, and has been used in the treatment of various cancers such as acute lymphoblastic leukemia, lung cancer and liver cancer. treat. Therefore, the retinoic acid-induced protein RAI16 may also participate in a series of physiological processes, playing an ...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K16/18C07K16/06A61K39/00A61K39/395A61P35/02A61P35/00G01N33/574G01N33/68
CPCC07K14/4702C07K16/18C07K16/06C07K16/065A61K39/0011A61K39/39525A61K39/3955A61K39/39558A61P35/02A61P35/00G01N33/57426G01N33/57438G01N33/57423G01N33/57484G01N33/68A61K2039/804A61K2039/86A61K2039/844G01N2333/4703Y02A50/30
Inventor 王文丁翠玲苗根钱春霖
Owner THE NAVAL MEDICAL UNIV OF PLA
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