Preparation method of CYP1A2 polypeptide and antihuman CYP1A2 polypeptide antibody

A CYP1A2 and peptide technology, applied in the field of peptides, can solve the problems of high price of human CYP1A2 and high cost of antibodies

Inactive Publication Date: 2008-11-19
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The purpose of the present invention is to solve the problem that the currently used human CYP1A2 is expensive, the cost of the prepared antibody is too high, and the antibody cannot be fully used in western blotting, immunohistochemical experiments and enzyme-linked immunosorbent assays, and provides a CYP1A2 polypeptide

Method used

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  • Preparation method of CYP1A2 polypeptide and antihuman CYP1A2 polypeptide antibody
  • Preparation method of CYP1A2 polypeptide and antihuman CYP1A2 polypeptide antibody
  • Preparation method of CYP1A2 polypeptide and antihuman CYP1A2 polypeptide antibody

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specific Embodiment approach 1

[0006] Embodiment 1: The amino acid sequence of the CYP1A2 polypeptide in this embodiment is: GlyArgGluArgArgProArgLeuSerAspArgProGlnLeuPro.

specific Embodiment approach 2

[0007] Specific embodiment two: In this embodiment, the anti-human CYP1A2 polypeptide antibody is prepared according to the following steps: (1) Analysis of CYP1A2 epitope: use DNAStar software analysis to determine the 15 amino acids at positions 351-365 of the CYP1A2 protein as the amino acid sequence of the synthetic polypeptide; ( 2) Synthesis of CYP1A2 polypeptide: Synthesized and purified by an automatic polypeptide synthesizer; (3) Cross-linking of CYP1A2 polypeptide and carrier protein: CYP1A2 polypeptide and carrier protein KLH are linked by glutaraldehyde, and then boric acid buffer with a pH value of 8.5 Dialyze for 8-12 hours, cross-link to form CYP1A2-KLH; (4) Preparation of rabbit anti-CYP1A2 polypeptide antibody: inject the emulsified CYP1A2-KLH subcutaneously on the back of the rabbit, and repeatedly boost the immunization until the antibody titer is equal to or higher than 1: 10000; (5) collecting and separating serum containing rabbit anti-human CYP1A2 polypep...

specific Embodiment approach 3

[0009] Specific embodiment three: the difference between this embodiment and specific embodiment two is: in step (two), a conventional C18 RP-HPLC column is used for purification. Other steps are the same as the second embodiment.

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Abstract

The present invention relates to a preparation method of CYP1A2 polypeptide and antihuman CYP1A2 polypeptide antibody. The amino acid sequence of CYP1A2 polypeptide is GRERRPRLSDRPQLP. The preparation method of antihuman CYP1A2 polypeptid antibody includes the following steps: (1) analysis of CYP1A2 antigen epitope; (2) synthesis of CYP1A2 polypeptide; (3) CYP1A2 polypeptide and carrier protein crosslinking; (4) preparing rabbit anti-CYP1A2 polypeptide antibody; and (5) collecting and separating to obtain serum containing antibody, purifying antibody so as to obtain anti-human CYP1A2 polypeptide antibody.

Description

technical field [0001] The present invention relates to a polypeptide. Background technique [0002] Human CYP1A2 (CYP4501A2) is a member of the CYP1A subfamily, mainly expressed in the liver. Many drugs are metabolized by CYP1A2. Aromatic amines, heterocyclic amines and some halogenated hydrocarbons are important substrates of CYP1A2. CYP1A2 plays an important role in the process of drug metabolism and activation of pre-carcinogens, and its activity is closely related to the efficacy or toxicity of many drugs and the susceptibility of some tumors. CYP1A2 can catalyze the activation of many pro-carcinogens and pro-mutogens such as aflatoxin B, acryloylnitrosamines and aromatic hydrocarbons. Currently used human CYP1A2 (ab3569: http: / / www.abcam.com / datasheet=3569 provides rabbit anti-human CYP1A2 gene recombinant expression all amino acid antibodies for western blot experiments. ab4233: http: / / www.abcam.com / datasheet=4233 provides rabbit anti-human CYP1A2 gene recombinan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K14/47C07K16/28
Inventor 唐晓波朱大岭刘海英杨微武正华王志刚于磊纪宏宇戴丽娟
Owner HARBIN MEDICAL UNIVERSITY
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