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Antigen peptide identified by p53 antoantibody, kit and application thereof in preparing tumor detection kit

An autoantibody, antigen polypeptide technology, applied in the fields of peptides, measuring devices, instruments, etc., can solve the problems of high purification difficulty and high cost of kits, and achieve the effect of simple preparation method, not easy to degrade, and lower use cost.

Inactive Publication Date: 2010-03-31
BEIJING TUBERCULOSIS & THORACIC TUMOR RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the defects of p53 pure protein expression and purification in the existing p53 kits, which are difficult and the cost of the kit is too high, and propose a highly sensitive and specific p53 autoantibody-recognized antigenic polypeptide

Method used

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  • Antigen peptide identified by p53 antoantibody, kit and application thereof in preparing tumor detection kit
  • Antigen peptide identified by p53 antoantibody, kit and application thereof in preparing tumor detection kit
  • Antigen peptide identified by p53 antoantibody, kit and application thereof in preparing tumor detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1P53

[0071] Example 1 Screening of linear antigen sequences recognized by P53 autoantibodies

[0072] 1. The specific implementation steps of the screening process of the core sequence:

[0073] According to the amino acid sequence of p53 protein, the 12-peptide peptide chip was synthesized on the surface of activated cellulose membrane by point synthesis technology at intervals of 3 amino acids. Because the wild-type p53 protein contains 393 amino acid residues, its full length includes 128 12-peptide polypeptide chains overlapping 9 amino acid sequences, that is, the polypeptide array includes 128 spots. Therefore, the peptide array we designed is 32×4, that is, there are 32 points in each row and 4 points in each column, such as figure 1 shown.

[0074] According to the operating instructions of the ASP SL peptide synthesizer, put 20 FMOC-group-protected natural amino acid solutions into the corresponding positions on the machine. After the predetermined program is input int...

Embodiment 2p53

[0099] Determination of embodiment 2p53 polypeptide core sequence

[0100]In order to determine the core sequences of the polypeptides of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, the inventors used two methods: 1. Based on the full-length sequence, one amino acid was sequentially reduced from the N-terminus and C-terminus of the polypeptide Synthetic series of peptides. The core sequence used to determine the sequences shown in SEQ ID No.1 and SEQ ID No.3. 2. For the sequence of SEQ ID No.2, the inventors used the 4-12 synthetic peptide library method based on the 21-peptide polypeptide sequence containing the core sequence to determine its core sequence. Incubate the synthesized series of polypeptides with positive serum according to the above-mentioned experimental method, and develop color to determine the core sequence of the polypeptide. Two experimental methods successfully determined the core sequences of the three polypeptides. All experiments also showed that the ...

Embodiment 3

[0118] Embodiment 3 The preparation and use method of p53 kit of the present invention

[0119] Those skilled in the art can entrust a biological company to complete the synthesis of the polypeptide described in the present invention. This example was synthesized by Shanghai Huada Tianyuan Biotechnology Co., Ltd.

[0120] 1. Preparation of kits

[0121] 1. Synthesize the antigenic polypeptide according to the above core sequence.

[0122] 2. Dilute the antigenic polypeptide to 10 μg / ml with 0.05M, pH 9.0 carbonate coating buffer, add 0.1ml to the reaction well of each microtiter plate, and leave overnight at 4°C. The next day, the solution in the well was discarded, and washed 3 times with washing buffer, 5 minutes each time.

[0123] 3. Dry and seal for storage.

[0124] Preparation of reagents:

[0125] 1. Wash buffer (PH7.4PBS 20×): 3M

[0126] K H 2 PO 4 0.2 grams

[0127] Na 2 HPO 4 12H 2 O 2.9 g

[0128] NaCl 8.0g

[0129] KCl 0.2 g

...

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Abstract

The invention discloses an antigen peptide identified by p53 antoantibody, and an active fragment of the peptide is as follows: (1) a sequence shown in SEQ ID No.4 is F-S-D-L-W-K; (2) a sequence shownin SEQ ID No.5 is D-I-E-Q-W-F-T; (3) a sequence show in SEQ ID No.6 is Y-E-P-P-E. The invention finds out a linear antigenic determinant and a core sequence thereof identified by the p53 antoantibody. The kit of the invention synthesizes the antigen peptide identified by the p53 antoantibody into a 96 pore plate, can reduce synthesized cost of each sample; if the kit is produced in mass, the detection cost of each patient can be half reduced. Meanwhile, the peptide is shorter and can not be easily degraded, and the valid period of the kit can be prolonged to two years, thus having considerable value in use.

Description

technical field [0001] The invention relates to a polypeptide and a kit for its preparation, in particular to an antigen polypeptide kit recognized by p53 autoantibody and its application in the preparation of a kit for detecting tumors. Background technique [0002] Lung cancer is the leading cause of death among various malignant tumors. Most of the patients are found in the advanced stage (III or IV stage), and the overall five-year survival rate is less than 5%. Therefore, it is of great significance to develop new rapid and simple early diagnosis methods for non-small cell lung cancer. Tumors are uncontrolled cell growth caused by abnormal cell differentiation and proliferation. During the malignant transformation of tumors, new proteins must appear, and some normal proteins will also be overexpressed, which will trigger the body's immune response and lead to the production of serum autoantibodies. . Studies have shown that autoantibodies in tumor patients are expect...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K7/08G01N33/574
Inventor 岳文涛许绍发王玥唐恺张丽娜
Owner BEIJING TUBERCULOSIS & THORACIC TUMOR RES INST
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