CN112501124A - Preparation method and application of cell strain for stably expressing human transferrin receptor 1 - Google Patents

Preparation method and application of cell strain for stably expressing human transferrin receptor 1 Download PDF

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CN112501124A
CN112501124A CN202011367027.6A CN202011367027A CN112501124A CN 112501124 A CN112501124 A CN 112501124A CN 202011367027 A CN202011367027 A CN 202011367027A CN 112501124 A CN112501124 A CN 112501124A
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韩照中
潘红芽
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Linnuo Shanghai Pharmaceutical Technology Co ltd
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Abstract

The invention discloses a cell line for stably expressing human transferrin receptor 1 and application thereof in drug screening, and provides a construction method and an application example of the cell line, which comprises (1) inserting an hTFR1 gene into an eukaryotic expression vector; (2) introducing the vector obtained in the step 1 into host cells, and performing pressurized screening to obtain a cell line for stably expressing the hTfR 1; 3) and (3) using the cell line obtained in the step 2 for drug screening.

Description

Preparation method and application of cell strain for stably expressing human transferrin receptor 1
Technical Field
The invention relates to a preparation method of a cell strain for stably expressing human transferrin receptor 1(TfR1) and application thereof in drug screening
Background
Transferrin (Tf) is a family of monomeric glycoproteins that are widely found in vertebrate body fluids and their cells, and are synthesized predominantly in the liver, with a half-life of 8 days and a serum concentration of 200-300 mg/dL. Mature Tf consists of 679 amino acids and has a molecular mass of about 80 kDa. The crystal structure shows that amino acid residues 1-336 of Tf constitute its N-terminal domain and residues 337-679 constitute its C-terminal domain. The N-terminal and C-terminal domains are composed of two small subunits with the same size. The space between the small subunits is the Fe binding site, and each molecule of transferrin can carry 2 ferric ions (Fe)3+). Transferrin and Fe3+ the interaction is dependent on pH, at pH 7.4 transferrin and Fe3+ high binding, both separated at acidic pH.
The transferrin receptor (CD7l) is an essential protein involved in iron absorption and regulation of cell growth. Human iron is transported via Tf and tissue cells achieve iron uptake via Tf-TfR mediated internalization. TfR1 is a type II transmembrane glycoprotein cross-linked by two disulfide bonds to form a homodimer (180 kDa). The monomer (760 amino acids, molecular weight 90-95kDa) contains a large extracellular C-terminal region (671 amino acids), a single transmembrane region (28 amino acids) and a short N-terminal intracellular regulatory region (61 amino acids). The C-terminal region is an outer functional region that contains the binding site for Tf. The outer functional region contains 3N-and one O-glycosylation sites, the glycosylation of which is essential for the function of TfR 1.
In the process of high-throughput screening and evaluating whether a drug (including small molecule compounds, short peptides, genetically engineered proteins or antibodies and the like) which is combined with transferrin or targets TfR1 can be effectively combined with TfR1, a rapid in vitro cell model for transferrin-TfR1 or drug-TfR 1 combination is needed. The invention establishes an HEK293F-hTFR1 cell model, and is convenient for making a cell screening model for effectively combining a tfR1 with a transferrin-mediated or TfR 1-targeted drug antibody or other drug molecules.
Disclosure of Invention
The invention provides a preparation method and application of a cell strain for stably expressing human transferrin receptor 1.
The invention provides a preparation method of a cell strain for stably expressing human transferrin receptor 1, which comprises the following steps:
cloning the human transferrin receptor 1 gene to a eukaryotic expression vector.
The eukaryotic expression vector has the following characteristics: the human transferrin receptor encoding gene TfR1 and the green fluorescein gene (eGFP) are under the control of the same transcription regulatory element and can be co-expressed in mammalian cells.
The eukaryotic expression vector has all regulation elements required for transcription and translation for driving exogenous gene expression in eukaryotic cells, including a promoter, an enhancer, a transcription initiation region, a polyA processing and transcription termination signal, a ribosome binding region, a translation initiation signal, a translation termination signal and the like; TfR1 is under the same transcriptional and translational regulatory elements as eGFP, i.e., in the same transcript or mRNA.
The expression vector contains a self-splicing peptide T2A sequence between the humanized TfR1 and an eGFP coding gene, so that the expression vector drives the co-expression of TfR1 and eGFP by a CMV promoter and a gene transcription regulation element thereof in the same Open Reading Frame (ORF). The translated polypeptide sequence is cleaved at T2A from the cleavage site into TfR1 and eGFP. TfR1 constitutes the mature homodimeric TfR1 receptor by way of a disulfide bond.
To facilitate observation of transfection efficiency and gene expression in cells, eukaryotic expression vectors may be supplemented with genes for enzymes or light-emitting compounds that produce a color change, such as GFP. A schematic diagram of the open reading frame of the vector is shown in the attached figure 1:
and (3) introducing the expression vector obtained in the step into host cells, and screening to obtain a human transferrin receptor 1 cell strain which is stably expressed and has functional activity.
The mode of introducing the expression vector into the cells comprises introducing the expression vector into mammalian cells by a chemical mode (cell transfection mediated by a microsphere structure taking lipid components such as liposome, lipofectamine and the like as a main body), a physical mode or a biological mode;
physical means of introducing the expression vector into a cell include, but are not limited to, introducing the DNA into a mammalian cell by electroporation;
the biological method for introducing the expression vector into the cell includes, but is not limited to, encapsulating the constructed vector into a virus particle, and introducing a foreign gene segment by means of infecting a mammalian cell with the virus particle;
screening of Positive cell lines
The screening of the above cells can be achieved by:
based on single cell separation technology such as flow cell sorting, the expression of fluorescein genes (such as green, yellow and red fluorescent proteins GFP, YFP, RFP and the like) related to the constructed vector can be used for selecting high-expression cell strains according to the fluorescence intensity;
the cells can be selected by expressing antibiotic resistance genes related to gene segments in an expression vector and removing cells without gene integration or with relatively low expression level by using antibiotics; high-expression cell strains can be selected by gradually increasing the concentration of antibiotics; obtaining the monoclonal cells by a limiting dilution method.
Identification of human transferrin receptor 1 stably expressing cell lines:
the expression quantity of the transferrin receptor in the human transferrin receptor 1 high-expression cell strain can be determined by FACS detection of cells by utilizing a transferrin receptor specific antibody, or by detecting the content of hTfR1 in cell lysates by Western printing and dyeing (western blot) or enzyme-linked immunosorbent assay (ELISA) by using a corresponding antibody;
the stable expression cell strain of human transferrin receptor 1 prepared by the method can be applied to the following aspects:
screening positive clones in phage display by FACS (FACS) by applying a HEK293F-hTfR1 cell model;
the screening method is used for screening the drug molecules mediated by transferrin or targeting TfR1, and comprises small molecule compounds, short peptides, genetically engineered proteins or antibodies and the like.
A cell strain HEK293F-hTfR1 stably expressing human transferrin receptor was deposited at the China center for type culture Collection (CGMCC) at 04.08.2020. The deposit unit code: CCTCC China center for type culture Collection; and (4) storage address: wuhan university in China; preservation state: survival; the preservation date is as follows: 8, month 4 in 2020; the preservation number is: c2020143; and (3) classification and naming: human TfR1 overexpresses the human embryonic kidney cell line HEK293F-hTfR 1.
Description of the drawings:
FIG. 1: human transferrin receptor 1 gene eukaryotic expression vector open reading frame diagram.
FIG. 2: hTfR1 homodimer expression plasmid: the self-splicing peptide T2A sequence is included between the hTFR1 and the eGFP encoding gene, and the hTFR1 and the eGFP are co-expressed in the same Open Reading Frame (ORF) driven by the CMV promoter and its gene transcription regulatory elements. The translated polypeptide sequence was cleaved at T2A from the cleavage site into hTF 1 and eGFP. TfR1 constitutes the mature homodimeric hTfR1 receptor by way of a disulfide bond.
FIG. 3: FACS measures hTfR1 expression after HEK293F cell transfection: the positive rate of the hTFR1 cells at 48 hours after transfection is 9.09%; the 72-hour positive rate was 15.88%.
FIG. 4: FACS detection of HEK293F-hTfR1 cells GFP positive cells: the GPF positive cells of the HEK293F-hTfR1 account for 19.27 percent.
FIG. 5: FACS detects Tf binding to HEK293F-hTfR1 cells: tf can bind to HEK293F-hTfR1 cells, and the positive cell rate is 21.99%.
FIG. 6: FACS screening of anti-transferrin phage positive clones: the double positive rate of GFP/M13 of phage clones A2, E11, G9, D5, A5 and F10 under the condition of Tf is more than 5%, which indicates that the clones can be mediated by Tf to bind to TfR 1.
FIG. 7: screening for Tf binding proteins using stably expressing HEK293-hTfR1 cells: the double positive rates of Tf-binding proteins SLN0042, SLN0044 and SLN0049GFP/OVA are respectively as follows: 7.12%, 11.34% and 20.95%, can bind to Tf and bind to the cell surface via Tf-TfR 1.
Detailed Description
The present invention is further illustrated by the following specific examples.
The methods used in the following examples are conventional methods unless otherwise specified.
Example 1 construction of recombinant eukaryotic expression vector pcDNA3.1-TfR1
A HUMAN transfer receptor 1(UniProtKB-P02786(TFR1_ HUMAN) with the sequence of Seq 1 is inserted into the multi-cloning site of a eukaryotic expression vector pcDNA3.1, and an eGFP sequence comes from the gene coding sequence (CDS) of P42212.1 with the sequence of Seq 2.
The two are connected through a self-cutting peptide T2A sequence, the sequence of T2A is Seq 3, so that the co-expression of TfR1 and eGFP is driven by a CMV promoter and a gene transcription regulatory element thereof in the same Open Reading Frame (ORF). The translated polypeptide sequence is cleaved at T2A from the cleavage site into TfR1 and eGFP. The form of TfR1 bound by disulfide bonds constitutes the mature homodimeric TfR1 receptor. The carrier is shown in figure 2 in the specification.
The host cell HEK293F is a suspension culture human embryonic kidney cell. The expression sequence was codon optimized for the cell. The synthetic gene fragment and the pcDNA3.1 vector are subjected to double enzyme digestion by BamH I and Xba I, T4 ligase connection, DH 5 alpha competent cell transformation and amplification, and plasmids are extracted for later use.
Example 2 establishment of a HEK293F-hTfR1 Stable cell line
HEK293F cells were cultured in Opimei (OPM-CD05, 81075-001) medium. Subculturing according to conventional method. The cells were cryopreserved in culture medium containing 10% DMSO, 1X 107Cells/ml per vial.
pcDNA3.1-TfR1-T2A-eGFP transfected HEK293F cells: day before transfection D0, inoculation 1.5X 106From/ml cells to 125ml cell shake flasks. Cells transfected with D1 after 24 hours: according to LipofectamineTM2000Transfection Reagent (Invitrogen, Cat #11668027), DNA LipofectamineTMMix at room temperature for 5min at 2000 ═ 1:5, and add to the cells.
Establishment of cell line stably expressing HEK293-hTfR 1: d3, cells were passaged and pressure screened by adding 400ug/ml G418. D7, cell change fluid, G418400 ug/ml. D10, control cells all dead, HEK293F-hTfR1 cells were instead cultured in medium containing G418200 ug/ml. And (3) identifying positive expression of TfR1 by FACS, and carrying out conventional passage, amplification culture and liquid nitrogen preservation for later use.
EXAMPLE 3 identification of cell line stably expressing HEK293F-hTfR1
Detection of expression of TfR1 by HEK293F-hTfR1 cells by flow cytometry:
pcDNA3.1-TfR1-T2A-eGFP was transfected into HEK293F cells, and the cells were harvested at 48 hours and 72 hours after transfection, respectively. Blocking the cells with 1% BSA in PBS for 30min at room temperature; centrifuging at 300g, and discarding the supernatant; anti-TfR1(Abcam, ab214039)1:100 dilution, shake-shake incubation at 4 ℃ for 1 hour; precooling PBS to wash the cells for 3 times; second antibody Alexa
Figure BDA0002803446180000032
647Donkey anti-rabbitIgG (biolegend,406414) diluted 1:400, incubated at 4 ℃ for 30min in the absence of light; precooling PBS to wash the cells for 3 times; FACS buffer 200ul resuspended cells and tested on the machine. CytExpert software analyzes data. The experimental results are shown in the attached figure 3: the positive rate of the hTFR1 cells at 48 hours after transfection is 9.09%; the 72-hour positive rate was 15.88%.
Detecting GFP positive cells in the cells stably expressing HEK293F-hTFR1 by a flow cytometer: collecting HEK293F and HEK293F-hTFR1 cells respectively, centrifuging at 300g, and discarding the supernatant; FACS buffer 200ul resuspended cells and tested on the machine. CytExpert software analyzes data. The experimental results are shown in the attached figure 4: the GPF positive cells of the HEK293F-hTfR1 account for 19.27 percent.
EXAMPLE 4 detection of biological function of cells stably expressing HEK293-hTfR1
Flow cytometry detection of transferrin binding to HEK293F-hTfR1 cells: collecting HEK293F and HEK293F-hTFR1 cells respectively, centrifuging at 300g, and discarding the supernatant; blocking the cells with 1% BSA in PBS for 30min at room temperature; centrifuging at 300g, and discarding the supernatant; 1, diluting anti-Tf (abcam, ab82411) at a ratio of 100, and incubating for 1 hour at a temperature of 4 ℃ in a shaking table; centrifuging at 300g, and discarding the supernatant; precooling PBS to wash the cells for 3 times; second antibody Alexa
Figure BDA0002803446180000031
647Donkey anti-rabbitIgG (biolegend,406414) diluted 1:400, incubated at 4 ℃ for 30min in the absence of light; precooling PBS to wash the cells for 3 times; FACS buffer 200ul resuspended cells and tested on the machine. CytExpert software analyzes data. The experimental results are shown in the attached figure 5: tf can bind to HEK293F-hTfR1 cells, and the positive cell rate is 21.99%.
Example 5 application of cells stably expressing HEK293-hTfR1 in drug screening
Example 1: screening positive clones of anti-transferrin antibody in phage display library with cells stably expressing HEK293-hTfR 1: collecting HEK293F and HEK293F-hTFR1 cells respectively, centrifuging at 300g, and discarding the supernatant; blocking the cells with 1% BSA in PBS for 30min at room temperature; respectively taking 90ul or PBS of phage clone supernatant, and adding the phage clone supernatant into a U-shaped 96-well plate; 20 XTF (Sigma, T3309) (final concentration 5ug/ml) and 20 Xanti-M13 antibody (abcam, ab9225) were prepared at a final concentration of 1:100, diluting; respectively adding 5 ul/well Tf and 5 ul/well anti-M13 antibodies or 5 ul/well PBS and 5 ul/well anti-M13 antibodies into a U-shaped 96-well plate, fully mixing, and incubating for 1 hour at room temperature; centrifuging the cells, removing supernatant, respectively adding the mixture into the U-shaped 96-well plate, and incubating for 1 hour at 4 ℃ in a shaking table; centrifuging at 300g, and discarding the supernatant; precooling PBS to wash the cells for 3 times; goat anti-mouse IgG-Alexa Fluor647(abcam, ab150115) 1: diluting at 2000 deg.c and incubating in dark at 4 deg.c for 30 min; precooling PBS to wash the cells for 3 times; FACS buffer 200ul resuspended cells and tested on the machine. CytExpert software analyzes data. The experimental results are shown in the attached figure 6: the double positive rate of GFP/M13 of phage clones A2, E11, G9, D5, A5 and F10 under the condition of Tf is more than 5%, which indicates that the clones can be mediated by Tf to bind to TfR 1.
Example 2: screening for Tf binding proteins using stably expressing HEK293-hTfR1 cells: collecting HEK293F-hTfR1 cells, centrifuging at 300g, and discarding the supernatant; blocking the cells with 1% BSA in PBS for 30min at room temperature; preparing 4 XTF (Sigma, T3309) (final concentration 5ug/ml), 4 XTF-OVA antibody (abcam, ab181688) (final concentration 1:100 dilution) and 2 XTF binding protein (final concentration 10ug/ml), adding 25 ul/well Tf, 25 ul/well anti-OVA antibody and 50ul Tf binding protein or 25 ul/well PBS, 25 ul/well anti-OVA antibody and 50ul Tf binding protein into U-shaped 96-well plate, mixing thoroughly, and incubating at room temperature for 1 hr; centrifuging the cells, removing supernatant, respectively adding the mixture into the U-shaped 96-well plate, and incubating for 1 hour at 4 ℃ in a shaking table; centrifuging at 300g, and discarding the supernatant; precooling PBS to wash the cells for 3 times; alexa
Figure BDA0002803446180000033
647donkey anti-rabbit IgG antibody (biolegend,406414)1: diluting with 400 ℃, and incubating for 30 minutes at 4 ℃ in a dark place; precooling PBS to wash the cells for 3 times; FACS buffer 200ul resuspended cells and tested on the machine. CytExpert software analyzes data. The experimental results are shown in the attached figure 7: the double positive rates of Tf-binding proteins SLN0042, SLN0044 and SLN0049GFP/OVA are respectively as follows: 7.12%, 11.34% and 20.95%, can bind to Tf and bind to the cell surface via Tf-TfR 1.
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Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val
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Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys
195 200 205
Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr
210 215 220
Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 3
<211> 21
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu
1 5 10 15
Glu Asn Pro Gly Pro
20

Claims (8)

1. A cell line stably expressing the functionally active transferrin receptor 1, TfR 1.
2. The method of claim 1 for establishing a cell line and the use thereof.
3. The cell line of claim 1, which is obtained by inserting a foreign gene fragment into the genome of the cell by genetic engineering means.
4. The cell line of claim 1 which expresses TfR1 of human origin.
5. The cell line of claim 1 which is HEK293F or other mammalian cell.
6. The use of a cell line according to claim 2 comprising testing the transferrin (Tf) and TfR1 mediated binding capacity of a drug molecule to a cell.
7. The assay of claim 6 comprising an ELISA, FACS or like based assay.
8. A drug molecule of claim 6, which is a Tf-binding or TfR1 targeting drug, comprising a small molecule compound, a short peptide, a genetically engineered protein or antibody, or the like.
CN202011367027.6A 2020-11-27 2020-11-27 Preparation method and application of cell strain for stably expressing human transferrin receptor 1 Pending CN112501124A (en)

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Cited By (1)

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Citations (2)

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WO2018152285A1 (en) * 2017-02-17 2018-08-23 Denali Therapeutics Inc. Transferrin receptor transgenic models
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WO2018152285A1 (en) * 2017-02-17 2018-08-23 Denali Therapeutics Inc. Transferrin receptor transgenic models
WO2020104479A1 (en) * 2018-11-20 2020-05-28 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and compositions for treating cancers and resistant cancers with anti transferrin receptor 1 antibodies

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张斌 等: "人转铁蛋白受体基因载体的构建与鉴定及在Hela细胞中的表达", 《中华消化病与影像杂志(电子版)》, vol. 3, no. 2 *
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115785251A (en) * 2021-07-16 2023-03-14 百奥赛图(北京)医药科技股份有限公司 TFR1 gene humanized non-human animal and construction method and application thereof

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