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B cell screening method and application of B cell screening method in preparing monoclonal antibody

A monoclonal antibody and screening method technology, applied in the field of cell biology, can solve the problems of low success rate, time-consuming and laborious detection of fusion cell lines, etc., and achieve the effect of improving experimental efficiency.

Active Publication Date: 2017-08-18
SHENZHEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved by the present invention is: the detection of fusion cell lines in the preparation process of hybridoma cells is time-consuming and laborious, and the success rate is not high

Method used

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  • B cell screening method and application of B cell screening method in preparing monoclonal antibody

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preparation example Construction

[0040] The present invention also provides an application of the above-mentioned B cell screening method in the preparation of monoclonal antibodies. The positive B cells screened in the above method are fused with myeloma cells for the preparation of monoclonal antibodies. The specific application process is a preparation method of a monoclonal antibody, including the following steps:

[0041] S21, adding a secreted and expressed signal peptide sequence to the N-terminus of the Protein A protein gene, and adding a transmembrane protein gene sequence to the C-terminus to obtain a fusion exogenous gene;

[0042] S22. Transfecting the fused exogenous gene into B cells that have been immunized as cells to be screened;

[0043] S23. Adding fluorescently labeled antigens to the cells to be screened, sorting and collecting the fluorescent cells using a flow cytometer to obtain positive B cells;

[0044] S24. Fusing the positive B cells with myeloma cells, and the resulting hybridoma ...

Embodiment 1

[0047] 1. Design primers to amplify the Protein A gene sequence. The two ends of the primers respectively have a secretion signal peptide and a transmembrane protein DNA fragment. At the same time, an enzyme cleavage site is introduced. The primers are designed as follows:

[0048] PROTEIN-BAMH I-F:

[0049] 5'-CCAGGATCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGCGGCCAATGCTGCGCAACACGATGAA-3';

[0050] PROTEIN-XHOI-R:

[0051] 5'-CCGCTCGAGTCAGACACAGAGAAGATGCCTAGCCCAATGAAAAGCAGGAGGCCGGCGACGCCCCCCAGCACAATCAGGGCGCCCCCCAGCACAATCAG-3'.

[0052] A BamH I restriction site was introduced into the upstream primer, Xho I was introduced downstream, and the Protein A gene was amplified directly using Staphylococcus aureus liquid as a template.

[0053] 2. Construction of lentiviral expression plasmid

[0054] Constructed with the pLVX-puro lentiviral system, and simultaneously cut pLVX-puro and ProteinA with BamH I and Xho I to construct a pLVX-puro-Protein A len...

Embodiment 2

[0058] The preparation method of the complete monoclonal antibody of the present invention is illustrated by taking the preparation of human glycoprotein Tamm-Horsfall (THP) monoclonal antibody as an example.

[0059] 1. Synthetic amino acid sequence

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Abstract

The invention relates to a B cell screening method and application of the B cell screening method in preparing a monoclonal antibody. The B cell screening method comprises the steps of S11, adding a signal peptide sequence of secretory expression at the N end of a Protein A protein gene, and adding a membrane-spanning gene sequence at the C end to obtain a fused exogenous gene; S12, using the fused exogenous gene to conduct transfection on a B cell of which immunity is completed, and adopting the B cell as a cell to be screened; S13, adding an antigen for labeling fluorescent into the cell to be screened, using a flow cytometry to sort and collect cell carrying fluorescent to obtain a positive B cell. According to the B cell screening method and application of the B cell screening method in preparing the monoclonal antibody, antibody molecules are gathered on the surface of the cell, through a fast and precise sorting function of the flow cytometry, a cell to be fused is screened in advance, thus the success rate and accuracy rate of a traditional mode are sharply increased, and the problem of detection of a monoclonal antibody cell strain in a later period is solved at the same time.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a B cell screening method and its application in the preparation of monoclonal antibodies. Background technique [0002] Monoclonal antibody is a highly uniform antibody produced by a single B cell clone that only targets a specific epitope. It is usually prepared by hybridoma technology. Hybridoma (hybridoma) antibody technology is based on cell fusion technology. Sensitized B cells with the ability to secrete specific antibodies and myeloma cells with the ability to multiply indefinitely are fused into B cell hybridomas. Using a single hybridoma cell with this characteristic to cultivate a cell population, a specific antibody against an antigenic epitope, that is, a monoclonal antibody, can be prepared. The preparation of hybridoma cells is a commonly used technique in the field of biology. The detection and identification of fused cell lines in conventional methods is ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62C12N15/867C07K16/00
CPCC07K14/31C07K16/00C07K2319/02C07K2319/03C12N15/86C12N2740/15043
Inventor 易俊波周兆平买制刚刘立忠邹永东
Owner SHENZHEN UNIV
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