Mouse anti-human P53 monoclonal antibody and hybridoma cell strain capable of secreting monoclonal antibody

A technology of hybridoma cell line and monoclonal antibody, applied in the field of bioengineering

Inactive Publication Date: 2017-05-31
TIANJIN SUNGENE BIOTECH
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cells with a defective p53 gene do not have this control and continue to divide even under unfavorable conditions

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Mouse anti-human P53 monoclonal antibody and hybridoma cell strain capable of secreting monoclonal antibody
  • Mouse anti-human P53 monoclonal antibody and hybridoma cell strain capable of secreting monoclonal antibody
  • Mouse anti-human P53 monoclonal antibody and hybridoma cell strain capable of secreting monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Obtaining of hybridoma cell line 6C4 and the monoclonal antibody it produces

[0022] 1. Antigen preparation

[0023] (1) Obtain the target gene

[0024] In this example, according to the coding frame of the P53 gene sequence (BC003596.1), design a pair of specific primers:

[0025] Primer 1: 5'-CGCGGATCCATGGAGGAGCCGCAGTCAG-3' (SEQ ID NO: 1)

[0026] Primer 2: 5'-CCGCTCGAGGGCCCTTCTGTCTTGAACATG-3'; (SEQ ID NO:2)

[0027] Trizol Reagent reagent was used to extract total RNA from human adipose tissue, the total RNA was reverse transcribed into cDNA, and the P53 gene was amplified by PCR using cDNA as a template.

[0028] (2) Construction of recombinant expression vector

[0029] The PCR product obtained in step (1) was double digested and recovered, and ligated into the expression vector PET28a under the action of T4 DNA ligase to construct the recombinant plasmid PET28a-P53.

[0030] (3) Obtain expression strains containing recombinant expression plasmids ...

Embodiment 2

[0051] Example 2: Identification and Application of Monoclonal Antibody

[0052] 1. Identification of mouse anti-P53 monoclonal antibody

[0053] Cleavage and extract cos-7 cell lysate, load the sample into 10% SDS-PAGE gel wells, 20 μg / well, transfer the protein in the gel to PVDF membrane after electrophoresis, after blocking, perform immunostaining with anti-P53 antibody , add the P53 monoclonal antibody prepared and purified in step (3) of Example 1, the working concentration is set to 1:40000, overnight at 4°C, add HRP-labeled goat anti-mouse antibody, the working concentration is 1:5000, react at 37°C for 1h , resulting in a band with a molecular weight of 55KDa (see image 3 ), which is consistent with the literature reports, proving that this antibody is a specific antibody against P53; the picture shows that the band is still clear after the antibody is diluted at 1:40000, which proves that the monoclonal antibody has a very high titer.

[0054] 2. Application detec...

Embodiment 3

[0070] Example 3: Sequencing of variable regions of monoclonal antibodies

[0071] Synthesize the following primers based on the constant region sequence of the antibody gene:

[0072] zh08 5'-GGGGATATCCACCATGRACTTCGGGYTGAGCTKGGTTTT-3' (SEQ ID NO: 3)

[0073] zhr11 5'-GACHGATGGGGSTGTYGTGCTAGCTGNRGAGACDGTGA-3' (SEQ ID NO: 4)

[0074] zl01 5'-GGGGATATCCACCATGGAGACAGACACACTCCTGCTAT-3' (SEQ ID NO:5)

[0075] zlr05 5'-GGATACAGTTGGTGCAGTCGACTTACGTTTKATTTCCARCTT-3' (SEQ ID NO: 6)

[0076] Trizol Reagent reagent extracts 5×10 6 The total RNA of hybridoma cell 6C4 was reverse transcribed into cDNA. Use zh08 and zhr11 as primers to amplify the variable region of the heavy chain of the monoclonal antibody P53 by PCR, and use zl01 and zlr05 as primers to amplify the variable region of the light chain of the monoclonal antibody P53 by PCR. The PCR reactions all adopt hot start, and the reaction conditions are: 94°C for 5 minutes; 94°C for 45 seconds, 60°C for 45 seconds, 72°C for 1 mi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a mouse anti-human P53 monoclonal antibody and a hybridoma cell strain capable of secreting monoclonal antibody. The cloning number of the hybridoma cell strain capable of secreting the P53 monoclonal antibody is 6C4, and the collection number is CGMCC No. 6916. The invention further provides a monoclonal antibody produced by the hybridoma cell strain 6C6. The antibody comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10. The antibody is mainly used for researching various tumors, and can be used as one of indexes of tumor prognosis.

Description

technical field [0001] The invention relates to a hybridoma cell line 6C4 secreting a P53 monoclonal antibody obtained by immunizing mice with a prokaryotically expressed P53 protein, and belongs to the technical field of bioengineering. Background technique [0002] P53 gene is a tumor suppressor gene (or tumor suppressor gene), which is one of the earliest tumor suppressor genes discovered. The P53 gene is located on the short arm of human chromosome 17 (17P13), about 20,000 bases long, and is a 53KD phosphate-rich nucleoprotein composed of 393 amino acids (hence the name P53). Divided into wild and mutant subtypes. [0003] P53 protein is mainly distributed in the nucleoplasm, can specifically bind to DNA, and its activity is regulated by post-translational modifications such as phosphorylation, acetylation, methylation, and ubiquitination. P53 protein plays an important role in cell cycle capture, DNA repair, cell senescence, differentiation, apoptosis and other proces...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N5/20C07K16/18C07K16/32
CPCC07K16/18C07K16/32C07K2317/21
Inventor 何小丹刘晨
Owner TIANJIN SUNGENE BIOTECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap