Screening method of single antigen specific transgenic hybridoma cells

A hybridoma cell and transgenic technology, applied in chemical instruments and methods, biochemical equipment and methods, specific peptides, etc., can solve the problems of normal function and vitality damage, fat chain instability, loss, etc., to improve the rate of single cells and positive rate, high sensitivity, promising effect

Pending Publication Date: 2022-03-04
CHINA AGRI UNIV
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Problems solved by technology

However, the aliphatic chains coupled on the cell surface are very unstable and will be lost with the division of hybridoma cells, and the chemical covalent modification will also cause irreversible damage to the normal function and vitality of hybridoma cells

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  • Screening method of single antigen specific transgenic hybridoma cells
  • Screening method of single antigen specific transgenic hybridoma cells
  • Screening method of single antigen specific transgenic hybridoma cells

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preparation example Construction

[0044]The preparation method of transgenic Sp2 / 0 myeloma cells is as follows: the signal peptide of EGF-R is inserted into the N-terminus of the bioreceptor peptide AVI-tag sequence, and the transmembrane region of EGF-R is inserted into the C-terminus. Transfected into the genome of Sp2 / 0 myeloma cells, screened with cell culture medium containing 10% (v / v) FCS, 2mM glutamine, and 2μg / mL puromycin for 21 days, and used conventional complete medium to counteract purine-resistant Myeloma transgenic Sp2 / 0 myeloma cells were expanded and cultured.

[0045] The screening method of a single transgenic Sp2 / 0 myeloma cell is as follows: first, the transgenic Sp2 / 0 myeloma cell is biotinylated in vitro, and the transgenic Sp2 / 0 myeloma cell is treated with 2% BSA and 2mM PE-streptavidin conjugate (PE is fluorescein , that is, phycoerythrin (P-phycoerythrin, PE)) resuspended biotinylated transgenic Sp2 / 0 myeloma cells in PBS, incubated at 4°C for 20 minutes, washed 3 times with PBS, an...

Embodiment 1

[0053] Embodiment 1 Preparation of transgenic Sp2 / 0 myeloma cells

[0054] 1. Insert the signal peptide of EGF-R (amino acids 1-24) at the N-terminus of the biotin receptor peptide (AVI-tag) sequence, and insert the transmembrane of EGF-R (amino acids 25-668) at the C-terminus area( figure 1 ). The amino acid sequence of the EGF-R-AVI-tag fusion protein is shown in SEQ ID NO:1.

[0055] 2. One day before transfection, 0.5×10 5 -1.0×10 5 Sp2 / 0 myeloma cells were plated in a 6-well plate to achieve an optimal cell density of 70-80%.

[0056] 3. On the day of transfection, discard the medium supernatant, add 2 mL of complete medium, mix 0.2 μg transposase plasmid (mPB, helper plasmid) and 2 μg AVI-tag-EGF-R plasmid (pPB EF1a, donor plasmid) , dilute to 100 μL of Opti-MEM buffer, add Reagent, incubated at room temperature.

[0057] 4. Add the incubated solution to the Sp2 / 0 myeloma cells drop by drop. After 18-24 hours, add the selection medium containing 2 μg / mL puromycin...

Embodiment 2

[0058] Example 2 Screening of a single transgenic Sp2 / 0 myeloma cell

[0059] 1. Put 4×10 6 transgenic Sp2 / 0 myeloma cells with optimized 5mM MgCl 2 , 10 mM ATP, 5 μM biotin and 0.85 μg BirA in PBS, at 37 °C, 5% CO 2 Incubate under certain conditions for 30 min to achieve in vitro biotinylation of transgenic Sp2 / 0 myeloma cells.

[0060] 2. Use 5mM MgCl 2 Wash the biotinylated transgenic Sp2 / 0 myeloma cells with PBS, resuspend the cells in PBS containing 2% BSA, add 2mM PE-streptavidin conjugate, incubate at 4°C for 20 minutes, and enrich by FACS and preparation of a single PE-streptavidin-labeled transgenic Sp2 / 0 myeloma cell ( figure 2 , A and B).

[0061] The top ten transgenic Sp2 / 0 myeloma cells with good growth status were selected, and the foreign protein core element AVI-tag on the surface of the transgenic Sp2 / 0 myeloma cells was analyzed by real-time fluorescent quantitative PCR technology and Western blotting. mRNA and protein expression, so as to select the ...

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Abstract

The invention discloses a single antigen specific transgenic hybridoma cell screening method, which comprises the following steps: preparing a transgenic Sp2 / 0 myeloma cell expressing EGF-R-AVI-tag fusion protein, and combining with a splenocyte fusion technology of a traditional hybridoma technology to obtain a transgenic hybridoma cell capable of displaying a secreting type specific antibody on a cell membrane. And screening of a single specific transgenic hybridoma cell is realized by using a single cell sorting system. The invention provides an accurate and high-throughput transgenic hybridoma monoclonal antibody production platform based on a transgenic technology. Compared with the traditional hybridoma technology, the preparation time and cost of a single antigen-specific hybridoma cell are remarkably shortened, the single cell rate and positive rate are improved, and the titer and affinity of the prepared monoclonal antibody are also remarkably improved. The transgenic hybridoma antibody preparation technology provided by the invention can be used for preparing rare monoclonal antibodies with high specificity, high sensitivity and high stability, and has a wide application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for screening single antigen-specific transgenic hybridoma cells. Background technique [0002] Monoclonal antibody (Monoclonal antibody, mAb) is widely used in biology, medicine, environmental testing and food science and other fields. In recent years, phage display technology and single B cell technology have provided a broad space for the development of monoclonal antibodies, but these methods are still in their infancy and have certain limitations for the widespread production of antibodies. Therefore, traditional hybridoma technology is still the most important method for preparing monoclonal antibodies. In traditional hybridoma technology, screening antigen-specific hybridoma cells by limiting dilution method is time-consuming, labor-intensive and inefficient. At the same time, in the early stage of hybridoma cells, the antibody secretion is extremely low, making it d...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/85C12N5/20C07K16/00
CPCC07K14/485C12N15/85C07K16/00C07K2319/00
Inventor 王战辉沈建忠温凯余文博于雪芝江海洋李园李培培
Owner CHINA AGRI UNIV
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