Anti-pax-5 protein monoclonal antibody and its cell line, preparation method and application

A monoclonal antibody, pax-5 technology, applied in the field of biomedical engineering, can solve the problems of impact, antigenic peptide sequence is not exactly disclosed, etc.

Active Publication Date: 2021-06-04
FUZHOU MAIXIN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the antibody has been commercially sold, and the sequence of the selected antigenic peptide in the preparation has not been disclosed exactly. It can be seen from its product information that its antigenic peptide is located within the range of amino acids 250-350 (commodity information: http: / / www. abcam.cn / pax5-antibody-ab183575.html), the antibodies obtained by immunization at different protein positions, due to the different protein structures and localization on cells, the interaction with other molecules is different, and even the way of antigen repair in immunohistochemical detection Different will bring obvious differences, directly affect the interpretation and judgment of disease diagnosis and prognosis evaluation

Method used

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  • Anti-pax-5 protein monoclonal antibody and its cell line, preparation method and application
  • Anti-pax-5 protein monoclonal antibody and its cell line, preparation method and application
  • Anti-pax-5 protein monoclonal antibody and its cell line, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Polypeptide Synthesis and Chemical Coupling with Carrier Protein

[0029] Select the Pax-5 protein sequence numbered as P15391 as a standard sequence from the Uniprot database (http: / / www.uniprot.org), and its sequence is shown in the sequence table SEQID1, by BLAST (https: / / blast.ncbi.nlm .nih.gov / Blast.cgi) tool to compare its sequence differences with other proteins of the same family, and use the Protean module of DNASTAR8.0 software (www.dnastar.com) to conduct secondary structure, antigenicity and surface accessibility analysis .

[0030] The 378th to 391st amino acids of the C-terminal of the Pax-5 protein were selected as the antigenic peptide, and a cysteine ​​was added to the C-terminal of the sequence for coupling with the carrier protein. The antigenic peptide was named Pax5-PEP , and its sequence is shown as SEQID2 in the sequence listing.

[0031] (1) Dissolve 20mg of SMCC ((N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester, 4-(...

Embodiment 2

[0041] Example 2: Establishment of 20G4 hybridoma cell line

[0042] 1. Immunity

[0043] The immunogen obtained in Example 1 was emulsified with complete Freund's adjuvant (Sigma Company), and immunized with 4-6 week-old female Balb / c mice or ICR mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. ), the abdomen was subcutaneously injected into each mouse at 6 points, and the dose was 60 μg / only. Immunization was boosted once every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Company) at a dose of 30 μg per mouse. 7 days after the third booster immunization, indirect ELISA (wavelength 450nm) was used to detect the polyantibody titer of the anti-immunogen in the mouse serum. 50μg / only.

[0044] 2. Cell Fusion

[0045] Aseptically prepare the mouse splenocyte suspension that meets the immune standard, mix it with mouse myeloma cell sp2 / 0 (ATCC) at a ratio of 5:1, and centrifuge at 1500 rpm for 5 min. After...

Embodiment 3

[0050] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method

[0051] 1. Ascites preparation

[0052] The cells in the logarithmic growth phase were washed with serum-free medium and suspended, counted about 5×10 5 , 1ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The removed ascites was centrifuged at 4°C at 4000 rpm for 10 min. Carefully suck out the ascites in the middle and collect in a centrifuge tube, and store at 4°C or -20°C.

[0053] 2. Purification of monoclonal antibodies

[0054] Antibody was purified from ascitic fluid by HiTraprProtein A FF (GE Company) affinity chromatography according to the instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by Bradford method. Purified antibodies were stored at -20°C.

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Abstract

The invention relates to a monoclonal antibody 20G4 capable of recognizing human B cell-specific activation protein Pax-5, a secreting cell line, a preparation method of an immunogen and an application in immunoassay. Pax‑5, also known as B cell-specific activation protein, is expressed in the early stage of B cell differentiation, and Pax‑5 is expressed in 97% of Hodgkin's lymphoma Reed‑Sternberg cells, and it has been It has been used as a marker of hematopoietic tumor cells of B cell origin. The present invention provides an immunogen of Pax-5 protein for immunizing animals, the monoclonal antibody cell line 20G4 obtained by screening the immunogen after immunizing mice, and discloses the DNA of the variable region of the heavy chain and the light chain and amino acid sequence.

Description

technical field [0001] The present invention relates to the field of biomedical engineering, in particular to an anti-Pax-5 protein monoclonal antibody and its cell line, preparation method and application. Background technique [0002] Pax protein is a transcription factor protein that plays a transcriptional regulatory role in different pathways of development. This family protein functions through a domain of about 128 amino acids that can be combined with a degenerate conserved DNA sequence. This domain is called It is a paired domain or a paired box, and the two globular subdomains of the N-terminal domain and the C-terminal domain are connected by a connecting peptide. The protein interacts with TLE4 and death associated protein 6 (Death associated protein 6). [0003] Pax-5, also known as B cell-specific activator protein (BSAP), is expressed in the early stage of B cell differentiation, and is also expressed in the CNS and testis. Therefore, it may not only be relat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N5/20G01N33/68C12R1/91
CPCC07K16/18C07K2317/33C07K2317/56C07K2317/92G01N33/68
Inventor 史永勤杨清海陈惠玲陈昌星王小亚
Owner FUZHOU MAIXIN BIOTECH CO LTD
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