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N-terminal polypeptide of retinoic acid induced protein 16 and preparation method and application of antibody thereof

A technology for inducing protein and polypeptide antibodies, applied in the field of biomedicine, can solve the problems that antibodies cannot satisfy chemical experiments and immunofluorescence experiments at the same time, RAI16 is expensive, and the cost of antibodies is high, and achieves good specificity, high titer, and low preparation cost. Effect

Inactive Publication Date: 2015-01-14
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention also solves the problem that the currently used RAI16 is expensive, the cost of the prepared antibody is too high, and the antibody cannot be used in western blotting, immunohistochemical experiments and immunofluorescence experiments at the same time

Method used

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  • N-terminal polypeptide of retinoic acid induced protein 16 and preparation method and application of antibody thereof
  • N-terminal polypeptide of retinoic acid induced protein 16 and preparation method and application of antibody thereof
  • N-terminal polypeptide of retinoic acid induced protein 16 and preparation method and application of antibody thereof

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Embodiment 1

[0030] Now, the present invention will be further described in conjunction with the embodiments, but the implementation of the present invention is not limited thereto. Example 1: Preparation of anti-RAI16 N-terminal polypeptide antibody

[0031] The anti-RAI16 N-terminal polypeptide antibody was prepared according to the following steps:

[0032] Analysis of the RAI16 N-terminal epitope: DNAStar software was used to analyze the amino acid sequence of the N-terminal of the RAI16 protein, and the analysis results were as follows figure 1 As shown, the 40-55 amino acids (polypeptide) at the N-terminal of the RAI16 protein have excellent hydrophilic structure, flexible region, antigenic index and surface probability structure. Determine the 16 amino acids at positions 40-55 of the RAI16 protein as the amino acid sequence of the synthetic polypeptide:

[0033] Cys-Thr-Asp-Glu-Ser-Thr-Pro-Ala-Lys-Lys-Thr-Asp-Ile-Pro-Trp-Arg

[0034] Synthesis of RAI16 polypeptide: synthesized by...

Embodiment 2

[0043] Example 2: Determination of the Titer of Anti-RAI16 N-terminal Polypeptide Antibody

[0044] In this embodiment, ELISA is used to detect the titer of the anti-RAI16 N-terminal polypeptide 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 200ul is added to each well Bovine serum with a concentration of 100ul was blocked at 37°C for 2h, and after washing, add double-diluted anti-RAI16 polypeptide antibody (normal rabbit serum was added to the control group), incubate at 37°C for 60min, and add to each well after washing three times. 1:5000 diluted HRP-labeled goat anti-rabbit IgG100ul, incubated at 37°C for 30min, washed 3 times, then developed TMB color, measured the absorbance value of the sample at A450 with a microplate reader, and calculated the antibody titer. The potency is 1:64000 (eg image 3 shown).

Embodiment 3

[0045] Example 3: Anti-RAI16 N-terminal polypeptide antibody is used for detection of RAI16 protein expression

[0046] 1. Western blotting to detect the expression of RAI16 in human liver cancer cell line HepG2 cells and hepatocellular carcinoma tissue samples

[0047] HepG2 cell and hepatocellular carcinoma tissue protein sample preparation: 1×10 7 HepG2 cells or 100mg hepatocellular carcinoma tissue (HCC, after homogenization), add lysate (50mM Tris-HCl, 150mM NaCl, 0.5mM EDTA, protease inhibitor cocktail) and lyse at 4°C for 30min, under the conditions of 4°C and 10000g After centrifugation for 20 min, the protein in the supernatant was quantified, aliquoted and stored at -80°C. In this embodiment, the immunoblotting analysis of the antibody is carried out, and the specificity of the antibody is identified by a synthetic polypeptide competition method. Suspend the prepared human HepG2 cells or hepatocellular carcinoma tissue in 5xSDS loading buffer, heat at 95°C for 10 m...

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Abstract

The invention relates to the technical field of biomedicine and provides an N-terminal polypeptide of retinoic acid induced protein 16 (RAI16). The N-terminal polypeptide has an amino acid sequence represented by SEQ ID NO: 1 and excellent hydrophylic structures, flexible regions, antigen indexes and surface probability structures. The invention further provides an antibody of the N-terminal polypeptide of the RAI16 and a preparation method of the antibody. The antibody of N-terminal polypeptide of the RAI16, prepared by the invention, is high in valence and good in specificity and can generate a specific conjugation reaction with natural human RAI16 molecules. Furthermore, the invention provides applications of RAI16 N-terminal polypeptide and the antibody thereof in the preparation of vaccines or diagnostic kits for preventing and treating acute lymphoblastic leukemia, lung cancer, liver cancer and the like.

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to a preparation method and application of a retinoic acid-inducible protein 16 N-terminal polypeptide and an antibody thereof. Background technique [0002] The NCBI accession number of retinoic acid induced protein 16 (RAI16) is NP_073586.5, the Swissprot accession number is Q86V87, 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. Treatment (Okuno M, Kojima S, Matsushima-Nishiwaki R, Tsurumi H, Muto Y, Friedman SL, Moriwaki H. Retinoids in cancer chemopreventi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K16/18C07K16/06A61K39/00A61K39/395A61P35/02A61P35/00G01N33/68G01N33/574
Inventor 王文任浩许刚戚中田
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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