CN107058235B - B cell screening method and application thereof in monoclonal antibody preparation - Google Patents
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Abstract
The application relates to a B cell screening method and application thereof in preparation of monoclonal antibodies. The B cell screening method comprises the following steps: s11, adding a signal peptide sequence for secretory expression at the N end of the Protein A Protein gene, and adding a transmembrane Protein gene sequence at the C end to obtain a fusion foreign gene; s12, transfecting the fused foreign gene into the immunized B cell to serve as a cell to be screened; and S13, adding fluorescence labeled antigen into the cells to be screened, and sorting and collecting the cells with fluorescence by using a flow cytometer to obtain positive B cells. The invention collects antibody molecules on the cell surface, screens cells to be fused in advance by utilizing the rapid and precise sorting function of a flow cytometer, greatly improves the success rate and the accuracy of the traditional method, and solves the problem of detecting monoclonal antibody cell strains in the later period.
Description
Technical Field
The invention belongs to the field of cell biology, and particularly relates to a B cell screening method and application thereof in preparation of monoclonal antibodies.
Background
Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone and directed only against a specific epitope, and are generally prepared by the hybridoma (hybridoma) technique, which is a technique of fusing a sensitized B cell having the ability to secrete a specific antibody and a myeloma cell having an unlimited proliferation ability into a B cell hybridoma based on a cell fusion technique. By culturing a single hybridoma having such a characteristic into a cell population, a monoclonal antibody, which is a specific antibody against one epitope, can be produced. The preparation of hybridoma is a common technical means in the field of biology, and the detection of fused cell strains in the conventional method wastes time and labor for identification, and the success rate is not high.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the detection of the fusion cell strain in the preparation process of the hybridoma cells is time-consuming and labor-consuming, and the success rate is not high.
In order to solve the technical problems, the technical scheme of the invention is as follows: provided is a B cell screening method, comprising the steps of:
s11, adding a signal peptide sequence for secretory expression at the N end of the Protein A Protein gene, and adding a transmembrane Protein gene sequence at the C end to obtain a fusion foreign gene;
s12, transfecting the fused foreign gene into the immunized B cell to serve as a cell to be screened;
and S13, adding fluorescence labeled antigen into the cells to be screened, and sorting and collecting the cells with fluorescence by using a flow cytometer to obtain positive B cells.
In the B cell screening method provided by the invention, in the step S11, an upstream primer and a downstream primer are designed, a signal peptide sequence for secretory expression is added to the N end, a transmembrane protein gene sequence is added to the C end, the three sequences are connected, the DNA of staphylococcus aureus is used as a template, and the upstream primer and the downstream primer are adopted for PCR amplification to obtain the fusion exogenous gene.
In the B cell screening method provided by the invention, the upstream primer and the downstream primer are designed as follows:
PROTEIN-BAMH I-F:
5’-CCAGGATCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGCGGCCAATGCTGCGCAACACGATGAA-3’；
PROTEIN-XHOI-R:
5’-CCGCTCGAGTCAGACACAGAAGAAGATGCCTAGCCCAATGAAAAGCAGGAGGCCGGCGACGCCCCCCAGCACAATCAGGGCGCCCCCCAGCACAATCAG-3’。
in the B cell screening method provided by the present invention, in step S12, the fusion foreign gene is linked to a lentiviral vector to obtain a lentiviral expression plasmid, and the immunized B cell is transfected.
In the method for screening B cells provided by the invention, the lentivirus expression plasmid and the lentivirus helper plasmid are cotransfected with B cells which are already immunized.
In the B cell screening method provided by the invention, spleen of an immunized animal is taken, and spleen cells obtained by separation are the B cells which are immunized.
In the B cell screening method provided by the present invention, in step S13, Protein a secreted and expressed is hung on a cell membrane through transmembrane Protein, antibody molecules are adsorbed by Protein a and then aggregated on the surface of the cell membrane, a fluorescence-labeled antigen is bound to the antibody to form a complex of Protein a + target antibody molecule + antigen, and this complex is linked to B cells, and positive B cells are collected by sorting using a flow cytometer because the antigen molecules carry fluorescence signals.
The invention also provides an application of the B cell screening method in monoclonal antibody preparation, and the positive B cells obtained by screening in the method are fused with myeloma cells to prepare the monoclonal antibody.
The invention also provides a preparation method of the monoclonal antibody, which comprises the following steps:
s21, adding a signal peptide sequence for secretory expression at the N end of the Protein A Protein gene, and adding a transmembrane Protein gene sequence at the C end to obtain a fusion foreign gene;
s22, transfecting the fused foreign gene into the immunized B cell to serve as a cell to be screened;
s23, adding fluorescence labeled antigen into the cells to be screened, and sorting and collecting the cells with fluorescence by using a flow cytometer to obtain positive B cells;
s24, fusing the positive B cells with myeloma cells, and using the obtained hybridoma cells for preparing monoclonal antibodies.
In the method for producing a monoclonal antibody according to the present invention, in step S24, the positive B cells are fused with myeloma cells, and then cultured by a dilution method to obtain a monoclonal cell line, and an antibody secreted by the monoclonal cell line is collected.
The implementation of the invention has the following beneficial effects: the invention designs and secretes and expresses Protein A, adds transmembrane Protein at the N end of Protein A, after transfecting immunized B cell, Protein A is naturally connected with cell membrane through transmembrane Protein, and antibody molecule generated by immunization is gathered on the cell surface, so that the antibody is easier to be identified and detected.
Drawings
FIG. 1 shows positive B cells obtained by flow cytometry screening in example 2.
Detailed Description
The present invention will be described in detail with reference to examples.
The invention has the innovation points that antibody molecules are gathered on the surface of the cell, and the cells to be fused are screened in advance by utilizing the rapid and precise sorting function of a flow cytometer, so that the success rate and the accuracy of the traditional method are greatly improved, and the problem of detecting the monoclonal antibody cell strain in the later period is solved.
In a preferred embodiment of the method for screening B cells of the present invention, the method comprises the following steps:
s11, adding a signal peptide sequence for secretory expression at the N end of the Protein A Protein gene, and adding a transmembrane Protein gene sequence at the C end to obtain a fusion foreign gene;
s12, transfecting the fused foreign gene into the immunized B cell to serve as a cell to be screened;
and S13, adding fluorescence labeled antigen into the cells to be screened, and sorting and collecting the cells with fluorescence by using a flow cytometer to obtain positive B cells.
The Protein A is expressed on the surface of a B cell membrane in a transmembrane expression mode by utilizing the characteristic that the Protein A can be jointed with an antibody, a monoclonal antibody molecule is secreted from a B cell during formation and is just intercepted by the Protein A, and a fluorescence-labeled antigen is combined with the antibody molecule to form a complex of the Protein A, a target monoclonal antibody molecule and the antigen, wherein the complex is connected with the B cell. The B cell screening method of the invention advances the preparation and screening process of monoclonal antibody, utilizes the sorting of flow cells to directly screen out positive cells, and then carries out fusion, thereby simplifying experimental operation and improving experimental efficiency.
In another preferred embodiment of the B cell screening method of the present invention, based on the above embodiment, in step S11, an upstream primer and a downstream primer are designed, a signal peptide sequence for secretory expression is added to the N-terminal, a transmembrane protein gene sequence is added to the C-terminal, these three sequences are connected, and DNA of staphylococcus aureus is used as a template, and the upstream primer and the downstream primer are used for PCR amplification to obtain the fusion foreign gene. It should be noted that other means capable of linking these three sequences are fully applicable to the present invention, for example, the fusion foreign gene is synthesized by whole gene, so that the Protein A Protein gene is added with a signal peptide sequence for secretory expression at its N-terminal and a transmembrane Protein gene sequence at its C-terminal. Preferably, the upstream primer and the downstream primer are designed as follows:
PROTEIN-BAMH I-F:
5’-CCAGGATCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGCGGCCAATGCTGCGCAACACGATGAA-3’；
PROTEIN-XHOI-R:
5’-CCGCTCGAGTCAGACACAGAAGAAGATGCCTAGCCCAATGAAAAGCAGGAGGCCGGCGACGCCCCCCAGCACAATCAGGGCGCCCCCCAGCACAATCAG-3’。
in another preferred embodiment of the method for screening B cells of the present invention, based on the above embodiment, in step S12, the fusion foreign gene is ligated to a lentiviral vector to obtain a lentiviral expression plasmid, and the immunized B cells are transfected. Preferably, the lentiviral expression plasmid is co-transfected with a lentiviral helper plasmid into an already immunized B cell. Separating immunized B cells directly from animal tissues, such as spleen of immunized animals, and separating the obtained spleen cells; of course, B cells isolated from other lymphoid tissues are also fully suitable for use in the present invention, e.g., B cells isolated from lymph nodules beneath the mucosa of the digestive tract.
The invention also provides an application of the B cell screening method in monoclonal antibody preparation, and the positive B cells screened by the method are fused with myeloma cells to prepare the monoclonal antibody. The specific application process is a preparation method of the monoclonal antibody, and the preparation method comprises the following steps:
s21, adding a signal peptide sequence for secretory expression at the N end of the Protein A Protein gene, and adding a transmembrane Protein gene sequence at the C end to obtain a fusion foreign gene;
s22, transfecting the fused foreign gene into the immunized B cell to serve as a cell to be screened;
s23, adding fluorescence labeled antigen into the cells to be screened, and sorting and collecting the cells with fluorescence by using a flow cytometer to obtain positive B cells;
s24, fusing the positive B cells with myeloma cells, and using the obtained hybridoma cells for preparing monoclonal antibodies.
Preferably, the positive B cells are fused with myeloma cells, and then cultured by dilution to obtain a monoclonal cell line, and the antibody secreted therefrom is collected.
Example 1
Firstly, designing a primer to amplify a Protein A gene sequence, wherein two ends of the primer are respectively provided with a secretory signal peptide and a transmembrane Protein DNA fragment, and simultaneously introducing a restriction enzyme site, wherein the primer is designed as follows:
PROTEIN-BAMH I-F:
5’-CCAGGATCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGCGGCCAATGCTGCGCAACACGATGAA-3’；
PROTEIN-XHOI-R:
5’-CCGCTCGAGTCAGACACAGAAGAAGATGCCTAGCCCAATGAAAAGCAGGAGGCCGGCGACGCCCCCCAGCACAATCAGGGCGCCCCCCAGCACAATCAG-3’。
introducing a BamH I enzyme cutting site in an upstream primer, introducing Xho I in a downstream primer, and directly taking a staphylococcus aureus bacterial liquid as a template to amplify a Protein A gene.
Second, construction of lentivirus expression plasmid
Constructing pLVX-puro lentivirus system, and simultaneously double digesting pLVX-puro and ProteinA with BamH I and Xho I to construct pLVX-puro-Protein A lentivirus expression plasmid,
preparation of Protein A slow virus liquid
The plasmid pLVX-puro-Protein A and the packaging plasmids pSPAX2 and pCMV-VSV-G are transfected into 293T cells together, and after 24 hours, the supernatant is collected, namely the Protein A slow virus liquid.
Example 2
The preparation method of the complete monoclonal antibody is illustrated by taking the preparation of the human glycoprotein Tamm-Horsfall (THP) monoclonal antibody as an example.
One, synthesis of amino acid sequence
According to the THP protein sequence found in the (NCBI) database, through protein analysis, two segments of protein sequences with stronger THP antigenicity are selected to synthesize polypeptides, the sequences are respectively synthesized in two groups, one group is used for directly synthesizing the polypeptides (marked as THP), PBS is used for preparing 2 mu g/mu l for storage, the other group is used for coupling a fluorescent label FITC (marked as THP-FITC) at the C end of the synthesized polypeptides, PBS is used for preparing 2 mu g/mu l for light-shielding storage, the polypeptides THP are used as immunogen to directly immunize mice to generate antibodies, and the polypeptides THP-FITC is used as a marker for immunofluorescence detection.
Second, mouse immunization
The conventional immunization method of BALB/c mice is adopted, the complete adjuvant emulsified polypeptide is adopted for immunization of the first needle, 50 micrograms of polypeptide is adopted for immunization of each mouse, three mice are totally immunized, meanwhile, the non-immunized mice are taken as a reference, the incomplete adjuvant emulsified polypeptide is adopted for the second needle and the third needle, the immunization time interval of each needle is two weeks, and blood is taken by a tail breaking method after each immunization of the two needles for antibody detection.
Preparation of mouse splenocytes
The immunized mice are taken, blood is collected by an eyeball picking method, the mice are placed in a refrigerator at 4 ℃ and are kept still overnight, and the second separated serum is used as an antibody positive control, and meanwhile, the mice are killed by cervical dislocation. Soaking in 75% ethanol for 5 min for sterilization, cutting off abdomen on a super clean bench, taking out spleen, placing in 1460 culture medium containing serum, removing peripheral connective tissue, injecting culture medium into spleen with sterile syringe to expand spleen, placing spleen cells on 400 mesh sieve, squeezing spleen with inner core of syringe, and filtering spleen cells through the sieve.
Fourth, Protein A slow virus liquid infected cell
Adding Protein A slow virus liquid into prepared mouse spleen cells to infect the cells, continuously culturing for 24 hours, removing the culture medium, and replacing the fresh culture medium.
Fifth, immunofluorescence reaction of mouse splenocyte
Polypeptide THP-FITC is added into spleen cells of mice infected by Protein A lentivirus in a ratio of 1: 500, meanwhile, spleen cells of non-immune mice are treated as a negative control, the reaction is carried out for 2 hours in a carbon dioxide incubator, the cells are collected, the cells are centrifuged at 1000r/min for 5 minutes, washed with PBS for 3 times, and the cells are resuspended in 1ml of PBS to prepare for screening by a flow cytometer.
Sixth, screening by flow cytometer
Screening the prepared mouse splenocytes by a flow cytometer, collecting positive cells, and culturing the positive cells in a 1640 culture medium, wherein FIG. 1 shows the positive cells obtained by screening.
Seventhly, cell fusion
The positive cells were fused with bone marrow hybridoma SP2/0, and 2 weeks later, a monoclonal cell line was obtained by dilution.
Eighth, detection of monoclonal antibody Strain
Culturing the monoclonal cell strain, detecting the antibody secreted by the monoclonal cell strain, and displaying the THP monoclonal antibody expression according to the detection result.
The foregoing examples further illustrate the present invention but are not to be construed as limiting thereof. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Claims (7)
1. A method for B cell screening, comprising the steps of:
s11, designing an upstream primer and a downstream primer, adding a signal peptide sequence for secretory expression at the N end of a Protein A gene, adding a transmembrane Protein gene sequence at the C end, connecting the three sequences, and performing PCR amplification by using the upstream primer and the downstream primer by using DNA of staphylococcus aureus as a template to obtain a fused exogenous gene;
s12, connecting the fusion foreign gene with a lentiviral vector to obtain a lentiviral expression plasmid, and cotransfecting the B cell which is immunized with the lentiviral expression plasmid and a lentiviral helper plasmid to be used as a cell to be screened;
and S13, adding fluorescence labeled antigen into the cells to be screened, and sorting and collecting the cells with fluorescence by using a flow cytometer to obtain positive B cells.
2. The method for screening B cells according to claim 1, wherein the forward primer and the reverse primer are designed as follows:
PROTEIN-BAMH I-F:
5’-CCAGGATCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGCGGCCAATGCTGCGCAACACGATGAA-3’；
PROTEIN-XHOI-R:
5’-CCGCTCGAGTCAGACACAGAAGAAGATGCCTAGCCCAATGAAAAGCAGGAGGCCGGCGACGCCCCCCAGCACAATCAGGGCGCCCCCCAGCACAATCAG-3’。
3. the method of claim 1, wherein the spleen of the immunized animal is collected and the spleen cells isolated are the immunized B cells.
4. The method for screening B cells according to claim 1, wherein in step S13, the Protein A secreted and expressed is hung on the cell membrane via transmembrane Protein, the antibody molecules are adsorbed by the Protein A and then aggregated on the surface of the cell membrane, the fluorescence-labeled antigen is bound to the antibody to form a complex of Protein A + target antibody molecule + antigen, and this complex is linked to B cells, and positive B cells are collected by flow cytometry because the antigen molecules carry fluorescence signals.
5. Use of a method of screening B cells for the preparation of monoclonal antibodies, wherein positive B cells obtained by screening according to any one of claims 1 to 4 are fused with myeloma cells for the preparation of monoclonal antibodies.
6. A method for preparing a monoclonal antibody, comprising the steps of:
s21, designing an upstream primer and a downstream primer, adding a signal peptide sequence for secretory expression at the N end of a Protein A gene, adding a transmembrane Protein gene sequence at the C end, connecting the three sequences, and performing PCR amplification by using the upstream primer and the downstream primer by using DNA of staphylococcus aureus as a template to obtain a fused exogenous gene;
s22, connecting the fusion foreign gene with a lentiviral vector to obtain a lentiviral expression plasmid, and cotransfecting the B cell which is immunized with the lentiviral expression plasmid and a lentiviral helper plasmid to be used as a cell to be screened;
s23, adding fluorescence labeled antigen into the cells to be screened, and sorting and collecting the cells with fluorescence by using a flow cytometer to obtain positive B cells;
s24, fusing the positive B cells with myeloma cells, and using the obtained hybridoma cells for preparing monoclonal antibodies.
7. The method of producing a monoclonal antibody according to claim 6, wherein in step S24, the positive B cells are fused with myeloma cells, and then cultured by dilution to obtain a monoclonal cell line, and the antibody secreted therefrom is collected.
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| CN111303294A (en) * | 2018-12-11 | 2020-06-19 | 澳世科技(深圳)有限公司 | Method for rapidly screening antibody expression cells in cell surface display mode |
| CN110016462B (en) * | 2019-02-20 | 2020-04-24 | 优睿赛思(武汉)生物科技有限公司 | Method for efficiently separating single antigen-specific B lymphocytes from spleen cells |
| CN114651065B (en) * | 2019-09-03 | 2024-08-20 | 五松尖端医疗产业振兴财团 | Signal peptide ultra-high speed screening method by introducing single bar code system for improving protein productivity |
| CN112094810B (en) * | 2020-08-26 | 2022-07-05 | 中国农业大学 | Single B cell screening method and application thereof in preparation of small molecule monoclonal antibody |
| CN115166241B (en) * | 2022-08-22 | 2023-03-24 | 广东忠信生物科技有限公司 | Efficient screening technology for simultaneously screening memory B cells and plasma cells and application |
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