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Anti-p16 monoclonal antibody and preparation method and application thereof

A technology of monoclonal antibody and p16, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as inactivation, abnormal cell proliferation, and malignant cell proliferation, and achieve accurate diagnosis, specificity and The effect of high sensitivity

Active Publication Date: 2015-10-28
FUZHOU MAIXIN BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Once p16 gene deletion, mutation, etc. lead to loss of function, CDK4 cannot be inhibited, eventually leading to malignant proliferation of cells and accelerating tumorigenesis
Because p16 inhibits the activity of cyclin D / CDK4 by competing with cyclin D to bind to CDK4 and CDK6, when the inhibitory effect of p16 is removed, cells will proliferate abnormally, excessive activation of cyclins and / or inactivation of inhibitory proteins will lead to CDKs Excessive action, causing abnormal proliferation of cells

Method used

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  • Anti-p16 monoclonal antibody and preparation method and application thereof
  • Anti-p16 monoclonal antibody and preparation method and application thereof
  • Anti-p16 monoclonal antibody and preparation method and application thereof

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Experimental program
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Embodiment

[0040] Step 1: Preparation of recombinant P16 fusion protein

[0041] 1. Gene cloning

[0042] According to the protein sequence of the accession number P427712 in the Uniprot database and the corresponding nucleic acid sequence of NP_000068 in Genbank, the specific upstream primer prIHC-13F: 5'-CGGGATCCGGTGGTGGTATGGAGCCGGCGGCGGGGAG-3' and the downstream primer prIHC-13R: 5'-CCGCTCGAGATCGGGGATGTCTGAGGGACCTT were designed -3', amplified from the reverse transcription product in lymphocytes including a gene fragment encoding a full-length 156 amino acids.

[0043] During the PCR process, BamHI and XhoI restriction sites were added to the 5' and 3' ends of the gene respectively; the PCR products were separated by agarose gel electrophoresis and recovered, and the recovered fusion protein gene and the plasmid vector pET for expression were respectively -30-GST was digested with BamHI and XhoI, recovered by electrophoresis again, and ligated with T4 DNA ligase; the ligated product...

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Abstract

The invention discloses an anti-p16 monoclonal antibody. A heavy chain variable region amino acid sequence of the anti-p16 monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No.2. A light chain variable region amino acid sequence of the anti-p16 monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No.3. The heavy chain variable region amino acid sequence of the anti-p16 monoclonal antibody is the amino acid sequence shown in SEQ ID No.4. The light chain variable region amino acid sequence of the anti-p16 monoclonal antibody is the amino acid sequence shown in SEQ ID No.5. The monoclonal antibody generated through hybridoma (30859-S17) secretion can recognize recombinant protein p16 molecules and lymphocytes expressing p16, and various kinds of tumor tissue of skin squamous-cell carcinoma, pancreatic cancer, melanoma, lymphoma, esophageal carcinoma, cervical cancer and the like of high-expression p16 protein can be detected.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a monoclonal antibody capable of recognizing human p16 protein molecules, a preparation method and application thereof. Background technique [0002] The p16 molecule is located in the nucleus and cytoplasm, and can specifically bind to the key enzyme of the cell division cycle (Cell Division Cycle) CDK4 or the complex of CDK4 and Cyclin, participate in the regulation of G1-S transition, and inhibit the activity of CDK4. It prevents cells from entering the S phase and plays a negative regulatory role in the cell proliferation cycle controlled by the RB gene. Once p16 gene deletion, mutation, etc. lead to loss of function, CDK4 cannot be inhibited, eventually leading to malignant proliferation of cells and accelerating tumorigenesis. Because p16 inhibits the activity of cyclin D / CDK4 by competing with cyclin D to bind to CDK4 and CDK6, when the inhibitory effect of...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13G01N33/68G01N33/577G01N33/574
Inventor 杨清海陈惠玲王小亚
Owner FUZHOU MAIXIN BIOTECH CO LTD
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