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Epitope peptide monoclonal antibody for tumor-related gene TP53 and application thereof

A monoclonal antibody and epitope polypeptide technology, applied in the direction of p53 protein, peptide, peptide source, etc., can solve the problems of dysfunction, high deletion rate of TP53 gene, etc., and achieve high sensitivity and high specificity

Active Publication Date: 2016-04-06
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In breast cancer, lung cancer, colon cancer, liver cancer and other tumors, the deletion rate of TP53 gene is high and the function is obviously dysfunctional

Method used

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  • Epitope peptide monoclonal antibody for tumor-related gene TP53 and application thereof
  • Epitope peptide monoclonal antibody for tumor-related gene TP53 and application thereof
  • Epitope peptide monoclonal antibody for tumor-related gene TP53 and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1, establishment of hybridoma cell line

[0019] 1. Experimental materials

[0020] 1. Immunogen: In this case, the highly conserved protein sequence of the TP53 gene is used as the target protein to design and synthesize the epitope polypeptide. The TP53 epitope polypeptide: PGTRVRQSQHMTELIRVEC is prepared by chemical synthesis, and the purity requirement is greater than 95%. Epitope polypeptides were coupled with KLH to prepare immunogens.

[0021] 2. Culture medium: DMEM medium was purchased from Hyclone Company; HAT, HT selection medium and pristane were purchased from Sigma Company.

[0022] 3. Experimental animals: Balb / c mice, 8-12 weeks old, female, SPF grade animal culture.

[0023] 4. Other materials: Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma Company; PEG4000 was purchased from Fluka Company; HRP-goat anti-mouse IgG antibody was purchased from JacksonImmune Company; other reagents were domestic analytica...

Embodiment 2

[0052] Embodiment 2 Preparation of monoclonal antibody against TP53 epitope polypeptide

[0053] Primary antibody preparation

[0054] Adult BALB / c mice were selected, and pristane was inoculated intraperitoneally, 0.5ml per mouse. After 7-10 days, the 16th generation hybridoma cells were inoculated intraperitoneally, 1×10 per mouse 6 -2×10 6 indivual. After an interval of 5 days, when the abdomen is obviously enlarged and the skin feels tense when touched with hands, the ascites can be collected with a No. 9 needle.

[0055] Centrifuge the ascitic fluid (13000r / min for 30 minutes), remove cell components and other precipitates, and collect the supernatant. Purify with ProteinG~SepharoseCL-4B, the upper column liquid is 20mM PBS buffer, and the eluent of column chromatography is: pH2.7, 20mM glycine buffer, to obtain the monoclonal antibody of TP53 epitope polypeptide (will be determined by the epitope The monoclonal antibody produced by the peptide sequence is called clone...

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Abstract

The invention provides a monoclonal antibody for a gene TP53. A highly-conserved protein sequence of the gene TP53 is target protein. Epitope peptide is synthesized through design and coupled with carrier protein to be used as immunogen. The monoclonal antibody is prepared from the immunogen. According to the monoclonal antibody and the epitope peptide synthetized through design, the monoclonal antibody can be used for detecting quantitative expression of human blood or tissue TP53 antibodies, has the advantages of being high in specificity and sensitivity and can be widely applied in clinical detection and experimental research.

Description

Technical field: [0001] The present invention relates to a hybridoma cell line and the monoclonal antibody and application thereof, especially [0002] It involves the highly conserved protein sequence of the TP53 gene as the target protein, designing and synthesizing epitope polypeptides, coupling with carrier proteins as immunogens, preparing monoclonal antibodies obtained through the immunogens and hybridoma cell lines secreting the antibodies and application of the antibody. technical background: [0003] TP53 is a cell cycle inhibitory protein encoded by the TP53 gene, consisting of 393 encoded amino acids. The TP53 protein contains multiple functional domains, which are important structures for the TP53 gene to perform functions. The TP53 gene is located on human chromosome 17p13.1, with a total length of 16kb and a molecular weight of 53KD. It has 10 introns and 12 exons, of which the first exon does not encode. The TP53 gene is a negative regulator of normal cell ...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K14/47C12N5/20G01N33/68G01N33/577C12R1/91
CPCC07K14/4746C07K16/18C07K2317/56G01N33/577G01N33/68G01N2333/4748
Inventor 张连波孙世龙刘颖
Owner JILIN UNIV
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