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Hybridoma cell strain and anti-Wnt5a monoclonal antibody produced thereby as well as application thereof

A hybridoma cell line and monoclonal antibody technology, applied in the direction of microbiology, biological testing, biochemical equipment and methods, etc., can solve the problems of inconvenient standardization, large individual differences in the judgment of examiners, subjective errors, etc., and achieve strong specificity and The effect of sensitivity

Inactive Publication Date: 2014-02-05
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results of peripheral blood and bone marrow morphological examination are often used as the standard in the judgment of leukemia curative effect and prognosis, but there are subjective errors in the judgment of the results of this method, and the individual differences in the judgment of the tester are large, which is inconvenient to standardize; and the change of cell morphology always lags behind the gene. Changes in blood and protein levels are not conducive to early diagnosis; bone marrow specimen collection is traumatic. When monitoring the curative effect and prognosis of leukemia, it is necessary to repeatedly extract the patient's bone marrow for before and after control checks, which causes great pain to the patient and requires high technology. It is inconvenient to collect materials, and there are differences in the parts of each extraction, which cannot truly reflect the changes in the patient's condition

Method used

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  • Hybridoma cell strain and anti-Wnt5a monoclonal antibody produced thereby as well as application thereof
  • Hybridoma cell strain and anti-Wnt5a monoclonal antibody produced thereby as well as application thereof
  • Hybridoma cell strain and anti-Wnt5a monoclonal antibody produced thereby as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Preparation of Wnt5a antigen

[0025] The Wnt5a antigen is based on the total RNA of nucleated cells from human umbilical cord blood as a template, and the primer sequence is designed according to the Wnt5a sequence (SEQID NO: 1), and the two ends of the primer are connected to EcoR I and Sal I restriction sites

[0026] Upstream primer: (SEQ ID NO:2)

[0027] 5'-CCGGAATTCATGAAGAAGTCCATTGGAATATTAAG-3'

[0028] Downstream primer: (SEQ ID NO:3)

[0029] 5'-ACGCGTCGACCTACTTGCACACACAAACTGGTCC-3'

[0030] RT-PCR amplifies the gene that removes the Wnt5a signal peptide sequence (PCR parameters: 42°C for 2 hours, 95°C for 5min; 94°C for 5min; 58.8°C for 30s; 72°C for 60s, a total of 35 cycles; 72°C for 5 minutes) After double enzyme digestion with Sal I and EcoR I (purchased by Takara), it was ligated with the prokaryotic expression vector pET-42a(+), transformed into competent cells BL21(DE3), and the single clone was picked to extract the plasmid for sequencing...

Embodiment 2

[0032]Embodiment 2: the preparation of anti-Wnt5a hybridoma

[0033] 1. Peptide synthesis and coupling

[0034] A total of 6 peptides were synthesized; the wnt5a protein sequence was analyzed by software, and 6 specific epitopes were selected, each segment was about 10-20 amino acids (Zhongke Yaguang Synthesis).

[0035] Synthesize 6 polypeptides:

[0036] A1 (SEQ ID NO: 4): LGMNNPVQMSEVYIIGAQPLC

[0037] A2 (SEQ ID NO:5): HLYQDHMQYIGEGAKTGIKEC

[0038] A3 (SEQ ID NO: 6): CSRAARPKDLPRDWLWGG

[0039] A4 (SEQ ID NO:7): NSRFNSPTTQDLVYIDPSPDYC

[0040] A5 (SEQ ID NO:8): CSQLAGLSQGQKKL

[0041] A6 (SEQ ID NO:9): GRGYDQFKTVQTERC

[0042] Each polypeptide is coupled to hemocyanin KLH separately, and the coupling method is SULFO-GMBS.

[0043] 2. Animal immunization

[0044] After coupling with KLH respectively, mix and immunize A1; A2; A5; immunize 3 mice by mixing; immunize 3 mice separately with A3; A4; A6; the antigen concentration is 3mg / ml, and the immune buffer is 8M ur...

Embodiment 3

[0056] Example 3: Preparation of anti-Wnt5a monoclonal antibody by hybridoma cell line with deposit number CCTCC C2013112

[0057] The inventor used the hybridoma cell line Wnt5a to prepare mouse anti-Wnt5a monoclonal antibody, and the specific operation steps are as follows:

[0058] 1. Cell recovery

[0059] Quickly take out the cryopreservation tube from the -80°C refrigerator or liquid nitrogen tank; quickly put it into a 37°C water bath and stir quickly to make the cryopreservation solution melt into a liquid within 2 minutes. Add 3mL of serum medium to a 15mL centrifuge tube, draw the cryopreserved solution into the centrifuge tube, and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, suspend the cells with complete medium, and culture in a 6-well plate (3 mL) or bottle (5 mL).

[0060] 2. Ascites Preparation

[0061] Cells in logarithmic growth phase were washed with serum-free medium and suspended; counted approximately 5×10 5 , 1 mL. Suspended cells...

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Abstract

The invention relates to a hybridoma cell strain and an anti-Wnt5a monoclonal antibody produced by the hybridoma cell strain as well as an application of the hybridoma cell strain. Particularly, based on molecular cloning and monoclonal antibody technology, hybridoma cell strain Wnt5a which can secrete the monoclonal antibody capable of specially recognizing wnt5a is screened. The monoclonal antibody secreted by the hybridoma cell strain Wnt5a can be used for clinically testing leukemia lesion in human bones and blood.

Description

technical field [0001] The invention relates to the fields of immunochemistry, tumor biology and cell biology. Specifically, the present invention relates to a hybridoma that prepares a secreted protein wnt5a, which is related to human cell proliferation, differentiation, adhesion and migration, and a monoclonal antibody combined with it; the anti-wnt5a monoclonal antibody produced by the hybridoma and its The corresponding products can be applied to diagnostic reagents for human leukemia or related fields. Background technique [0002] Leukemia is the most common malignant tumor in children and young adults, accounting for about 3% of the total incidence of tumors. The number of leukemia patients in my country is about 3 to 4 per 100,000 population. Leukemia has the highest incidence of malignant tumors in children, and it is increasing at a rate of at least 30,000 to 40,000 per year. [0003] At present, the main methods of treating leukemia in the world are chemotherapy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/68G01N33/577G01N33/574
Inventor 司维柯张晓丽赵宸牛长春杨忠吴韦铷潘静
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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