RU2746162C2 - Method of transfection and cultivation of cells synthesizing recombinant protein - glutamic acid decarboxylase - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of molecular genetics and medical biotechnology and can be used in medicine for designing technology of differential diagnosis of autoimmune diabetes, stiff-person syndrome and other diseases. A method has been developed for transfection and cultivation of cells synthesizing recombinant protein - glutamate decarboxylase GAD65, based on transfection of HEK293 cell culture with a specially constructed plasmid containing human protein GAD65 gene under the eukaryotic promotor. Stable gene expression and synthesis of GAD65 protein in transfected HEK293 cells is achieved through regular additional enrichment of the culture medium with glutamine.

EFFECT: proposed method for cultivation of GAD65-producer cells in a medium with increased glutamine content allows ensuring stable synthesis of glutamate decarboxylase under laboratory conditions with simultaneous increased transfection efficiency.

1 cl, 2 dwg, 3 tbl, 3 ex

Description

The invention relates to the field of molecular genetics and medical biotechnology and can be used in medicine to create technologies for the differential diagnosis of autoimmune diabetes mellitus, muscle stiffness syndrome and other diseases.

Glutamate decarboxylase (GAD, GAD65, GAD67) is an enzyme that catalyzes the conversion of glutamic acid to gamma-aminobutyric acid (GABA) through a decarboxylation reaction as shown in FIG. one.

In mammals, glutamate decarboxylase exists in two isoforms, GAD67 and GAD65, encoded by two genes, GAD1 and GAD2. The numbers 67 and 65 indicate the molecular weight of the isoforms - 67 kDa and 65 kDa, respectively. Both genes are expressed in brain cells and in the pancreas, with GAD65 found mainly in synaptic terminals, and GAD67 in synaptic terminals, in the neuron body and in axons (Kaufman DL, Houser CR, Tobin AJ Two forms of the gamma-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions. - J. Neurochem. 1991; 56: 720-723). The emergence of autoantibodies to GAD65 precedes the development of insulin-dependent diabetes mellitus (Baekkeskov S., Aanstoot HJ, Christgau S., Reetz A., Solimena M, Cascalho M, Folli F, Richter-Olesen H, De Camilli P., Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase Nature 1990, 347 (6289): 151-6).

Also, antibodies to GAD are often found in patients with muscle stiffness syndrome (Lohmann T., Hawa M., Leslie RD, Lane R., Picard J., Londei M. “Immune reactivity to glutamic acid decarboxylase 65 in stiffman syndrome and type 1 diabetes mellitus. "Lancet, July 2000, 356 (9223): 31-5. DOI: 10.1016 / S0140-6736 (00) 02431-4). In fact, in these cases, autoantibodies to GAD65 are markers of the corresponding autoimmune disease. In healthy people, autoantibodies to GAD65 are absent. The detection of autoantibodies to GAD65 in the blood of patients is an important step in the differential diagnosis of autoimmune diabetes mellitus, muscle stiffness syndrome and other diseases.

For laboratory measurement of the concentration of autoantibodies to GAD65 in human biological fluids, an antigen is required that is GAD65, having the same amino acid sequence and preferably the same conformation as GAD65 present in human cells.

Cultivation of the glutamate decarboxylase protein or the use of cultures expressing the glutamate decarboxylase gene has already become the subject of research. A known method of adding vitamin B6 to improve the yield of glutamate decarboxylase in E. coli cell culture ((Pat. No CN 104531652) Method for adding vitamin B6 to improve yield of glutamate decarboxylase, and application thereof). However, this method is not suitable for working with glutamate decarboxylase in eukaryotic cells. The cultivation of glutamate decarboxylase is preferably carried out in eukaryotic cell cultures, since this protein naturally exists in mammalian cells.

The aim of the present invention was to develop a method for producing such an antigen in a laboratory setting. To solve this problem, we created a test system consisting of a HEK293 cell culture obtained from an aborted human embryo adrenal gland cell expressing the GAD65 protein gene. It is assumed that autoantibodies present in the blood of patients will interact with the antigen synthesized in this way. For this, a plasmid was constructed containing the gene for the human protein GAD65 under the eukaryotic promoter. After transfection of the HEK293 cell culture with the resulting plasmid, stable gene expression and GAD65 protein synthesis were achieved in transfected HEK293 cells. Such cell culture can be used to detect autoantibodies to GAD65 in patient sera by standard immunocytochemical assays (Bosman FT. Monoclonal antibodies in immunocytochemistry. Acta Histochem Suppl. 1988; 35: 27-32). Patients here mean people who have been reliably diagnosed with type 1 diabetes due to the appearance of autoantibodies to GAD65. The blood of the patients was obtained with their consent, and after centrifugation, the plasma was separated from the formed elements, and then frozen at -20 ° C for further analysis.

However, in the course of work with the HEK293 cell culture, it was revealed that the rate of division of HEK293 cells synthesizing the recombinant protein glutamate decarboxylase (GAD65) (hereinafter referred to as HEK293-GAD) is reduced in comparison with cells that do not synthesize this protein (Table 1). This effect is due to the fact that glutamate decarboxylase synthesized in transfected cells converts glutamic acid coming from the culture medium into gamma-aminobutyric acid, as a result of which the transfected cells experience a deficiency of glutamic acid, which reduces the rate of synthesis of proteins necessary for cell division.

As a result of any transfection, a mixture of transfected and non-transfected cells is obtained. With further cultivation, all cells divide or die. Since untransfected cells divide faster, over time they practically displace transfected cells from culture (Table 1).

This makes it difficult to use cultured cells to detect autoantibodies to GAD65 in patient sera using immunocytochemical studies. The same factor, in addition, is an obstacle to the creation of a cell line that stably synthesizes the recombinant glutamate decarboxylase GAD65.

The technical problem of the present invention is to develop a method for transfection and cultivation of producer cells, providing stable synthesis of glutamate decarboxylase GAD65.

Proposed technical solution

Standard media for the cultivation of animal cells (for example, Eagle's medium, Eagle's MEM, 199, DMEM, DMEM / F12) contain, among other components, glutamine (0.3-0.6 mg / ml) and glutamic acid (from 0.007). During storage of the medium, glutamine is gradually converted to glutamic acid. In animal organisms, this process is catalyzed by glutaminase (Fig. 2).

In living organisms, glutamic acid is present in proteins, a number of low molecular weight substances and in free form. It plays an important role in nitrogen metabolism. Glutamic acid belongs to the group of nonessential amino acids and plays an important role in the body, its content in the body is up to 25% of all amino acids.

The use of transfected cells to detect autoantibodies to GAD65 in patient sera by immunocytochemistry suggests overexpression of GAD65 in these cells. That is, transfected cells useful in diagnostics must synthesize much more GAD65 than it is synthesized under natural conditions. If there is no overexpression, then the sensitivity of such test systems will be too low. It is quite expected that a large amount of synthesized GAD65 metabolizes a significant amount of glutamic acid, thereby depleting the cell with this amino acid.

We have found that regular additional enrichment with glutamine of the medium in which HEK293-GAD cells are cultivated solves the above technical problem (Table 1). In addition, it was found that glutamine enrichment of the medium in which cells are transfected with a plasmid containing the GAD65 gene significantly increases the efficiency of transfection (Table 3). Moreover, to achieve these effects, the final concentration of glutamine in the culture medium should significantly exceed the concentration of glutamic acid in standard culture media such as Igla, Igla MEM, 199, DMEM, F12, DMEM / F12 and others.

Example 1. Detection of GAD65 using Western blot analysis.

HEK293 cells were cultured at + 37 ° C in a CO ₂ incubator (5% CO ₂ , humidity 100) in DMEM / F12 medium containing fetal bovine serum (FBS) (10%), antibiotics (5000 U / ml penicillin G, and 50 μg / ml streptomycin), amphotericin B (25 μg / ml) and glutamine (up to 9.6 mg / ml). When the cells formed a monolayer, it was disintegrated with a Versene solution and the cells were washed twice with PBS. To 10 ⁷ cells, 500 μL of buffer solutions I and II were added for protein extraction (buffer I: 50 mM Tris-HCl, pH 7.4, 250 mM sucrose, 25 mM NaCl, 0.1 mg / ml trypsin inhibitor from soy, 2 mM EDTA, 0.1% NP-40; buffer II: 50 mM Tris-HCl, pH 8.0, 1% Triton X-100, 150 mM NaCl). The cells in the buffer were pipetted, resuspended with a vortex, incubated on ice for 30 min, and centrifuged at 13000 g for 10 min at 4 ° C. In the supernatant containing solubilized membrane proteins, the protein concentration was determined. Proteins of the supernatant were separated by electrophoresis (50 μg total protein per lane) in a polyacrylamide gel under denaturing conditions, and then transferred (semi-dry electrotransfer) to a nitrocellulose membrane (Anderson et al., 1982) for subsequent Western blot analysis. Protein-free membrane areas were blocked using a 3% skim milk solution prepared in PBS (Fluka, Switzerland) for 30 min, then the membrane was incubated with sera containing antibodies to GAD65 (dilution from 1:10 to 1: 100 ) for 1 hour, then washed and incubated with antibodies to human antibodies conjugated with horseradish peroxidase for 1 hour (dilution 1: 5000). 1 μg of recombinant GAD65 was applied as a positive control. 4-chloro-1-naphthol was used as a chromogenic substrate, or visualization was performed by the chemiluminescence method using ECL reagents (GE Healthcare, UK).

Example 2. Construction of plasmid pGAD65-RFP encoding a chimeric protein.

The coding sequence of the GAD65 gene (cDNA) was inserted into the pRFP plasmid (Novagen, USA) with MCS before RFP. PCR was used to amplify the desired fragment of GAD65 using two specially designed primers.

The forward primer is characterized by the sequence SEQ ID No. 1, namely, 5'- ctg AAGCTT caATGGGGCCCTGGGGCTGGA, its calculated annealing temperature (Ta) is 60 ° C; the part of the oligonucleotide complementary to GAD65 is shown in bold; the recognition site for the restriction endonuclease Hind III is underlined.

The reverse primer is characterized by the sequence SEQ ID No. 2, namely 5'- ctg GTCGAC CGCCACGTCATCCTCCAGACT, its calculated annealing temperature (Ta) is 58 ° C, the part of the oligonucleotide complementary to the 3'-end of the coding portion of GAD65 is highlighted in bold; the recognition site for the restriction endonuclease Sal I is underlined. To calculate the melting and annealing temperatures of the primers, the Oligos v.7.1 program was used.

PCR was carried out in 30 μl of the reaction mixture. Plasmid pEGFP-N3 and the resulting PCR product were treated with restriction endonucleases Hind III and Sal I (Fermentas, Lithuania) and then purified. DNA isolation from agarose gel was carried out using a kit from Promega (WizardSV Gel and PCR Clean-Up System, USA) according to the manufacturer's instructions. Ligation was performed using T4 DNA ligase (Fermentas, Lithuania). Competent cells of Escherichia coli strain DH5 (alpha) were transformed with DNA obtained after ligation and transferred to a selective medium containing 100 μg / ml of kanamycin. Plasmid DNA from grown clones was isolated by alkaline lysis followed by treatment with RNAse, deproteinization during DNA extraction with a mixture of phenol and chloroform, and DNA precipitation with alcohol. The quality of plasmid DNA was assessed by electrophoresis in 0.8% agarose gel. To identify plasmids with the GAD65 cDNA insert, sequencing was performed on a 3500 genetic analyzer (Applied Biosystems, USA). For further work, we used clones in whose plasmid DNA an insert was present and there were no mutations in the coding region of GAD65.

Example 3. Transfection of cells with plasmid DNA.

HEK293 cells were cultured at + 37 ° C in a CO ₂ incubator (5% CO ₂ , humidity 100) in DMEM / F12 medium containing fetal bovine serum (FBS) (10%), antibiotics (5000 U / ml penicillin G, and 50 μg / ml streptomycin), amphotericin B (25 μg / ml) and glutamine (300 μg / ml). The day before transfection, the cells were transferred to the wells of plates with coverslips at the bottom. The number of cells introduced into the well was selected so that the next day they occupied 20-30% of the glass surface. The HEK293 cell line was transfected with circular forms of the parent pEGFP-N3 and pLDLR-EGFP purified from agarose gel. Transfection was performed according to a standard method (Chen C., Okayama H 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 7: 2745-2752) using 100 ng of plasmid DNA per well of a 96-well plate. 24 h after transfection, the culture medium was replaced with DMEM / F12 medium containing antibiotics and antimycotics, as well as 10% FBS and glutamine at a concentration of 4.8 mg / ml. up to 9.6 mg / ml. After incubation for 1 to 9 days, the cells were carefully washed with PBS and fixed with 4% paraformaldehyde in PBS for 10 min. The preparations were washed with PBS for at least 1 h, incubated for 1 h at room temperature in a TNB blocking solution (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5% blocking reagent (Perkin Elmer, FP1020, USA)), then 1 h with antibodies against GAD65 at 37 ° C in a humid chamber. The preparations were washed 3 times in PBS solution for 5 min. Then incubated for 1 hour with fluorescently-labeled secondary antibodies at 37 ° C. After washing the preparations in PBS (3 times for 5 min), cover slips were mounted on the slides using a polymerizing resin for fluorescence microscopy (DAKO, USA).

The proposed method for culturing GAD65-producing cells in a medium with an increased glutamine content provided a stable synthesis of glutamate decarboxylase under laboratory conditions with a simultaneous increase in the efficiency of transfection.

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LIST OF SEQUENCES

<110> FSBSI "IEM" FSBSI "IEM"

<120> Cell transfection and culture method,

synthesizing the recombinant protein glutamate decarboxylase

<140>

<141>

<160> 2

<210> 1

<211> 30

<212> Nucleotide sequence

<213> Artificial sequence

<220>

<223> Gene Amplification Forward Primer Nucleotide Sequence

GAD65

<400> 1

ctgaagcttc aatggggccc tggggctgga

<210> 2

<211> 30

<212> Nucleotide sequence

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the reverse primer for gene amplification

GAD65

<400> 2

ctggtcgacc gccacgtcat cctccagact

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Claims (1)

A method for transfection and cultivation of cells synthesizing recombinant glutamate decarboxylase GAD65, including the creation of a plasmid containing a eukaryotic promoter and a nucleotide sequence encoding the protein glutamate decarboxylase GAD65, transfection of an animal cell culture with the obtained plasmid and their cultivation on a nutrient medium, characterized by the fact that containing the GAD65 protein gene, is carried out for at least 3 days in a nutrient medium with an increased content of glutamine at a concentration of 2.4 mg / ml to 9.6 mg / ml.

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