RU2817423C1 - INTEGRATIVE PLASMID VECTOR pVEAL3-Lassa-Trim, PROVIDING SYNTHESIS AND SECRETION OF RECOMBINANT SURFACE GLYCOPROTEIN GPC OF Lassa VIRUS IN MAMMALIAN CELLS, RECOMBINANT CELL LINE STRAIN CHO-K1-GPC-LASV AND RECOMBINANT PROTEIN GPC-LASV PRODUCED BY SAID CELL LINE STRAIN CHO-K1-GPC-LASV - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is an integrative plasmid vector pVEAL3-Lassa-Trim, which provides expression and secretion of recombinant surface glycoprotein GPC LASV in mammalian cells, having size of 5738 bp. Also described is a recombinant Chinese hamster ovary cell line strain CHO-K1-GPC-LASV, which produces recombinant surface glycoprotein GPC LASV and containing integrative plasmid vector pVEAL3-Lassa-Trim and recombinant protein GPC LASV produced by the recombinant Chinese hamster ovary cell line strain CHO-K1-GPC-LASV.

EFFECT: creation of plasmid structure and new recombinant strain-producer of Chinese hamster ovary cells, producing surface glycoprotein GPC LASV in a more immunologically correct form, capable of interacting with antibodies of the immunized animal.

3 cl, 4 dwg, 1 tbl, 3 ex

Description

Field of technology

The invention relates to an integrative plasmid vector pVEAL3-Lassa-Trim, providing expression and secretion of the recombinant surface glycoprotein GPC of the Lassa virus in the eukaryotic system of mammalian cells, a strain of the recombinant cell line CHO-K1-GPC-LASV and the recombinant GPC-LASV protein produced by the specified cell line CHO-GPC-LASV and can be used in the field of genetic engineering and biotechnology.

The recombinant surface glycoprotein GPC of LASV can be used to create diagnostics for Lassa fever, as well as to solve laboratory problems, such as obtaining phage libraries.

State of the art

LASV infection is spreading beyond traditionally endemic areas of West Africa, posing a biothreat to the entire world [Warner B. M., Safronetz D., Stein D. R. Current research for a vaccine against Lassa hemorrhagic fever virus // Drug Des Devel Ther. - 2018. - No. 12. - P. 2519-2527. Bell-Kareem, A. R. & Smither, A. R. Epidemiology of Lassa fever. Cur. Top. Microbiol. Immunol. https://doi.org/10.1007/82_20 (2021)].

An effective preventive vaccine against Lassa fever has not yet been developed. So far, the only approved therapy for Lassa hemorrhagic fever is ribavirin, but this treatment has side effects and its effectiveness is questionable [Salam A.P. et al. Time to reconsider the role of ribavirin in Lassa fever // PLoS Negl. Trop. Dis. - 2021. - No. 15. - P. e0009522].

The main and important component of any candidate vaccine against LASV is the highly glycosylated GPC glycoprotein complex present on the surface of the virion [Watanabe Y. Structure of the Lassa virus glycan shield provides a model for immunological resistance. // Biochemistry. - 2018. - Vol. 115. - No. 28. - P. 7320-7325]. An important factor for generating LASV GPCs is the choice of expression system. Mammalian cells are the most promising because they make it possible to obtain the target protein in the closest form to its native form, i.e. has undergone the necessary post-translational modifications, which is very important for the immunogenicity of the recombinant protein.

Closest analogues

The development of American scientists is known (WO 2022232612, IPC A61K 39/12, 03.11.2022), which describes nanoantibodies that can neutralize a pseudotyped virus expressing LASV GPC and specifically bind to the stabilized LASV GPC trimer. The work presents the detailed design and characterization of the stabilized soluble GPC LASV trimer. Trimer stability was assessed as fractional binding reactivity with monoclonal antibody 37.7H. However, in the description of this international application there is no information about the technology for obtaining the GPC LASV trimer and in which system it was synthesized.

The invention is known according to patent RU 2752858, IPC C12N 15/74, publ. 08/11/2021). This patent uses the closest integration vector pVEAL2 to create a stable producer of the SARS-CoV-2 RBD viral protein using the CHO-K1 cell line.

The closest analogue (prototype) for its intended purpose is the development of American scientists (US 20140377740, IPC C07K 14/005, publ. December 25, 2014), in which soluble forms of two subunits GP1, GP2, the precursor of the GPC glycoprotein and the nucleocapsid protein (NP) are obtained ) using pMAL vectors for expression in E. Coli.

The disadvantage of this invention is the use of an expression system in E. coli cells. It is known that prokaryotes lack some post-translational protein modification systems, such as glycosylation, necessary to obtain the correct form of LASV GPC. There is also a risk of incorrect molecular folding. In addition, the present invention produced two subunits GP1 and GP2 rather than a single LASV GPC protein.

Disclosure of the Invention

The technical result of the claimed invention is the creation of a plasmid construct and a new recombinant strain-producer of Chinese hamster ovary cells, producing the LASV surface glycoprotein GPC in a more immunologically correct form, capable of interacting with the antibodies of an immunized animal.

The technical result is achieved by creating an integrative plasmid vector pVEAL3-Lassa-Trim, which ensures the expression and secretion of the recombinant surface glycoprotein GPC LASV in mammalian cells, having a size of 5738 bp. and the nucleotide sequence of SEQ ID NO:1 and containing, in accordance with the physical and genetic map shown in FIG. 1, the following items:

- 5'ITR_SB, having coordinates from 88 to 317 bp;

- Integration arm 5'IFR, having coordinates from 318 to 368 bp;

- CMV enhancer, having coordinates from 369 to 733 bp;

- CmV promoter (coordinates from 734 to 937 bp)

- GPCL gene, which has coordinates from 1021 to 2292 bp. and encoding Lassa virus surface glycoprotein;

- Foldon, having coordinates from 2293 to 2373 bp;

- Sequence 10his, having coordinates from 2374 to 2403 bp. and being a polyhistidine tag for purification of recombinant protein using metal chelate chromatography;

- the internal landing site of the EMCV IRES ribosome, having coordinates from 2425 to 2999 bp;

- BleoR - resistance gene to the antibiotic bleomycin, having coordinates from 3004 to 3011 bp;

- PuroR, encoding the resistance factor to the antibiotic puromycin and having coordinates from 3012 to 3611 bp;

- T3 promoter, having coordinates from 3621 to 3628 bp;

- SV40 poly(A) signal, having coordinates from 3646 to 3767 bp;

- Integration arm 3'IFR, having coordinates from 3788 to 3835 bp;

- 3'ITR_SB, having coordinates from 3836 to 4065 bp;

- tonB terminator, having coordinates from 4117 to 4148 bp;

- KanR - resistance gene to the antibiotic kanamycin, having coordinates from 4252 to 5067 bp;

- The Ori replication origin site, which has coordinates from 5130 to 5717 bp.

The technical result was achieved by creating a strain-producer of Chinese hamster ovary cells CHO-K1-GPC-LASV, obtained by transfecting cells with the expression vector PVEAL3-Lassa-Trim according to claim 1 and producing the surface glycoprotein GPC LASV.

This technical result is also achieved by the fact that the created integrative plasmid vector PVEAL3-Lassa-Trim makes it possible to obtain the LASV GPC gene encoding the LASV surface glycoprotein in mammalian CHO cells as a result of expression.

The CHO-K1 cell line was chosen to develop the producer, since the expression system allows us to obtain the most correct form of the protein, providing all the necessary processes of post-translational modification. To obtain an effective producing crop in the claimed invention, the most productive clones were selected. This further increases the yield of the target product, since the polyclonal cell culture contains clones with different abilities to produce recombinant protein. A method is described for obtaining a strain of Chinese hamster ovary cells CHO-K1-GPC-LASV that produces recombinant surface glycoprotein LASV containing a genetic construct (SEQIDNO:1), introducing the said genetic construct into the cells by lipofection; Cell selection was performed using the antibiotic Puromycin B.

Thus, the claimed group of inventions provides a high yield of recombinant LASV GPC protein in an immunologically correct form, which is a good platform for the creation of immunobiological drugs and vaccines. Technical solutions meet the criteria of “novelty” and “inventive step”.

Carrying out the invention

Description of the figures

The invention is illustrated by graphic materials presented in Fig. 1, 2 and table 1.

In fig. Figure 1 shows a physical and genetic map of the universal integrative plasmid vector pVEAL3-Lassa-Trim.

In fig. Figure 2 presents the results of an enzyme-linked immunosorbent assay for the interaction of the recombinant GPC-LASV protein with the monoclonal antibody 37.7H against LASV.

Note:

37.7H is a human monoclonal antibody against LASV, previously described by Robinson J.E., Hastie K.M., Cross R.W., et al. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits // Nature communications. -2016. - V.7. - No. 11544, received from the Department of Bioengineering.

iB20 is a human monoclonal antibody against SARS-CoV-2 (negative control), described in the article Kulemzin S.V., Sergeeva M.V., Baranov K.O., et al. VH3-53/66-Class RBD-Specific Human Monoclonal Antibody iB20 Displays Cross-Neutralizing Activity against Emerging SARS-CoV-2 Lineages // Journal of personalized medicine. - 2022. - V. 12. - No. 6. - P. 895.

MR191 is a human monoclonal antibody against MARV, previously described in the article by King L.B., Fusco M.L., Flyak A.I., et al. The Marburgvirus-Neutralizing Human Monoclonal Antibody MR191 Targets a Conserved Site to Block Virus Receptor Binding // Cell host & microbe. - 2018. - V. 23. - No. 1. - P. 101-109, obtained from the Department of Bioengineering (negative control).

In fig. Figure 3 shows the nucleotide sequence of the integrative plasmid vector pVEAL3-Lassa-Trim, and FIG. 4 - amino acid sequence of the LASV GPC surface glycoprotein.

Table 1 shows the sequences of the synthesized oligonucleotides used to obtain the pVEAL3-Lassa-Trim plasmid.

For a better understanding of the essence of the proposed invention, examples of its implementation are given below. All standard genetic engineering and microbiological manipulations, as well as DNA amplification and sequencing, were carried out according to known methods [Maniatis T., Fritsch E, Sambrook J. Molecular cloning, M.: Mir, 1984; DNA cloning. Methods. Ed. D. Glover, Trans. from English, Moscow, Mir, 1988; Saiki R.K. et al. Science. 1988, 239(4839):487-491; Sanger F. et al. Proc. Nat. Acad. Sci. 1977, 74:5463-5467].

Example 1. Construction of an integrative plasmid vector PVEAL3-Lassa-Trim, providing expression of the surface glycoprotein GPC of LASV.

To obtain the trimerized version of LASV GPC, the nucleotide sequence encoding the LASV GPC glycoprotein was taken from the GenBank database (AIT17266.1). Next, codon optimization was carried out for the Chinese hamster ovary cell line CHO-K1 in the online service https://www.thermofisher.com to increase the yield of the target protein. The synthesis of primers (Table 1) and the nucleotide sequence encoding the LASV GPC trimer was carried out in the commercial research and production company OOO DNA-Sintez (Moscow).

A synthesized sequence encoding a trimerized version of LASV GPC was cloned into the PVEAL3 vector (Fig. 1). The sequences of the synthesized primers used to assemble the integrative plasmid vector PVEAL3-Lassa-Trim are presented in Table 1.

Table 1 - Sequences of synthesized oligonucleotides used to obtain the integrative plasmid vector PVEAL3-Lassa-Trim Name Subsequence F-GPC 5’- aaaaaagctagCCATGGGCCAGATCGTGACATTCTTCCA -3’ R-GPC 5’-aaaaaaCACCATCATCACCATCACCACCACTAAGTCGAC -3’

The Lassa-Trim nucleotide sequence encoding the trimerized LASV GPC glycoprotein was amplified by PCR. PCR was carried out using the BIS PCR amplifier from BIS-N LLC (Russia). 50 µl reaction mixture containing 2 µg DNA (plasmid pGH-Lassa-Trim), 10 pM each primer (F-GPC and R-GPC) (Table 1), 10 µl 5x Q5 reaction buffer, 10 µl 5xQ5 High GC Enhancer , a mixture of deoxynucleotide triphosphates (dATP, dCTP, dTTP, dGTP 2.5 mM each) and 0.5 units. Q5 High-Fidelity DNA polymerase, the reaction was carried out at the following parameters: 10 s - 98°C (1 cycle), 10 s - 58°C, 70 s - 72°C (30 cycles). The finished PCR product was isolated and purified from the gel using the “Gel Extraction Kit” from Qiagen (Germany) in accordance with the manufacturer’s instructions.

The pre-generated and purified PVEAL3 plasmid and the PCR product were treated with restriction endonucleases AsuNHI and SalI. The hydrolysis reaction was carried out under the conditions recommended by the manufacturer. To purify the linearized vector, plasmid DNA was applied to a 1% agarose gel and isolated from the gel using the Gel Extraction Kit from Qiagen (Germany). The ligation reaction was carried out using DNA ligase from bacteriophage T4 (SibEnzyme, Novosibirsk). The reaction was carried out at +4°C overnight. The resulting ligase mixture was used to transform competent cells of E. coli strain Neb Stable.

Primary testing for the presence of the insert was carried out using colony PCR. Separation of amplification products was carried out in a 1% agarose gel, followed by staining with ethidium bromide (0.5 μg/ml).

Positive colonies were cultured in 5 ml of LB medium with ampicillin (50 μg/ml) overnight at 37°C at 170 rpm. Plasmid DNA was then isolated from bacterial cells using commercial DNAminikit kits from Qiagen according to the manufacturer's recommendations. The primary structure of the expression vector was confirmed by sequencing. Sequencing was carried out using the Sanger method at the Genomics Center for Common Use of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). As a result, an integrative plasmid vector PVEAL3-Lassa-Trim was obtained, having the nucleotide sequence SEQ ID NO: 1 (Fig. 3) and providing expression of the trimerized LASV GPC surface glycoprotein protein.

Sequencing of the plasmid DNA of positive clones in the insertion region made it possible to select clones with the absence of defects in the inserted genes (insertions, deletions, substitutions), after which the target plasmid DNA was produced and isolated from the selected clones.

Example 2. Obtaining a strain-producer of Chinese hamster ovary cells CHO-K1-GPC-LASV by transfecting CHO-K1 cells with the integrative plasmid vector PVEAL3-Lassa-Trim and producing a culture fluid containing trimerized surface glycoprotein GPC LASV.

The Chinese hamster ovary cell strain CHO-K1-GPC-LASV is derived from the Chinese hamster ovary cell line CHO-K1 using the developed PVEAL3-Lassa-Trim construct. CHO-K1 cell suspension, grown in an incubator at 5% CO₂, 80% humidity, temperature (37±1)°C in a humid atmosphere of 5%CO2 and a platform rotation speed of 185 rpm. When the suspension density reaches 5x10⁶ cells/ml, cells were transfected using polyethyleneamine (PEI 25K™) according to the manufacturer's instructions. Cells were transfected with a mixture of plasmids PVEAL3-Lassa-Trim and pCMV(CAT)T7-SB100, taken in a ratio of 1:10. 48 hours after transfection, the selective antibiotic puromycin was added at a concentration of 2 μg/ml. Selection was carried out for 12 days with a gradual increase in the antibiotic concentration from 2 to 10 μg/ml. The resulting pool of cells was cryopreserved, after which cloning was carried out in order to select individual clones with high production. Cloning was performed using the limiting dilution method in 96-well plates. After 14 days, wells containing a single clone were visually selected, for which a culture liquid was selected to assess the productivity of the clones using ELISA, from which the claimed recombinant strain CHO-K1-GPC-LASV was obtained.

Characteristics of the recombinant strain CHO-K1-GPC-LASV.

Morphology: spindle-shaped and epithelial-like cells with round nuclei containing 1 to 2 nucleoli.

Cultivation method: suspension.

Culture medium: 97% HyCell CHO, 2% Glutamax, 1% Pluronic-F68.

Cultivation temperature: 37°C.

Inoculation concentration: 1x10 ⁶ cells in 1 ml.

Passaging frequency: 3-4 days.

Cryopreservation conditions: nutrient medium DMEM/F-12 (1:1) - 50%, fetal blood serum of a cow - 40%, DMSO - 10%.

Freezing mode: at a temperature of 4°C - 1 hour, minus 80°C - 12 hours, minus 196°C.

Storage conditions: in cryovials in the amount of 5 pcs. stored in liquid nitrogen at minus 196°C.

Passage number in liquid nitrogen: 3.

Viability after cryopreservation: 85-90%.

The producer strain CHO-K1-GPC-LASV was cultured on roller machines and a culture liquid containing trimerized surface glycoprotein GPC LASV protein having the amino acid sequence SEQ ID NO:1 shown in FIG. 4.

Example 3. Enzyme-linked immunosorbent assay for the interaction of monoclonal trimerized LASV GPC with anti-LASV monoclonal antibody 37.7H

An enzyme-linked immunosorbent assay was used to identify the trimerized LASV GPC surface glycoprotein protein. The antibody used was the neutralizing monoclonal antibody 37.7H against LASV. The culture liquid containing trimerized LASV GPC was diluted at a ratio of 1:100 in sodium carbonate solution (pH 8.0) and 150 μl was added to the wells of an ELISA plate. The antigen was incubated at 4°C for 16 hours and then removed from the wells by shaking. Blocking occurred by adding 200 μl of blocking buffer (phosphate-buffered saline (PBS) + 1% BSA) to the wells of sorbed antigens and incubating at 37°C for 2 hours. After removing the solutions, the wells were washed four times with wash buffer PBS-0.5% Tween20 (PBS-T). To test the specificity of the trimer, recombinant human monoclonal antibodies 37.7H, iB20 (negative control) and MR191 (negative control) were used. Antibodies are described above. Antibodies were diluted in blocking buffer to a concentration of 4 μg/ml and 150 μl were added to wells. The plates with solutions were incubated for 2 hours at 37°C. Then the wells were washed four times with washing buffer and 150 Goat anti-human IgG-HRP conjugate (Sigma-Aldrich, USA) at a dilution of 1:30,000 was added to the wells. They incubated for 2 hours at 37°C and washed the wells four times with PBS-T. To visualize the results, 100 μl of a 0.02% solution of TMB chromogen in citrate-phosphate buffer with hydrogen peroxide (BRS) was added to the wells. After 15 min of incubation at 37°C, 50 μl of a 0.9 M sulfuric acid solution was added to the wells as a stop reagent. The results were recorded in a plate reader at a wavelength of 450 nm. The results of the enzyme immunoassay confirm the interaction of the recombinant GPC-LASV protein with the anti-LASV monoclonal antibody 37.7H, which are presented in Fig. 2.

Thus, the group of claimed inventions provides the production of an immunologically correct form of the recombinant protein of the trimerized surface glycoprotein GPC of LASV, which can be used as a platform for creating Lassa fever diagnostics, as well as for solving laboratory problems, such as obtaining phage libraries.

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tgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgcca

ttctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaat

taataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaa

ctgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgat

atgaataaattgcagtttcacttgatgctcgatgagtttttctaatgagggcccaaatgtaatcacctgg

ctcaccttcgggtgggcctttctgcgttgctggcgtttttccataggctccgcccccctgacgagcatca

caaaaatcgatgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccct

ggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctccctt

cgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaa

gctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgag

tccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggt

atgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttgg

tatctgcgctctgctgaagccagttacctcggaaaaagagttggtagctcttgatccggcaaacaaacca

ccgctggtagcggtggttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaaga

tcctttgattttctac</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="2">

<INSDSeq>

<INSDSeq_length>491</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..491</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q10">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MGQIVTFFQEVPHVIEEVMNIVLIALSILAVLKGLYNLATCGLVGLVTFLLL

CGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIMVGNETGLELTLTNTSLINHKFCNLSD

AHKKNLYDHALMSIISTFHLSIPNFNQYEAMSCDFNGGKISVQYNLSHSYAVDAANHCGTVANGVLQTFM

RMAWGGSYIALDSGRGGWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTRDIYISRRLLGTF

TWTLSDSEGKDTPGGYCLTRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAIQRLKAEAQM

SIQLINKAVNALINDQLIMKNHLRDIMGIPYCNYSKYWYLNHTTTGRTSLPRCWLVSNGSYLNETHFSDD

IEQQADNMITEMLQKEYMERQGKTPLGLVDLFVFSTSFYLISIFLHLVKIPTHRHIVGKSCPKPHRLNHM

GICSCGLYKQPGVPVKWKR</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (20)

1. Integrative plasmid vector pVEAL3-Lassa-Trim, providing expression and secretion of the recombinant surface glycoprotein GPC LASV in mammalian cells, having a size of 5738 bp. and the nucleotide sequence of SEQ ID NO: 1 and containing, in accordance with the physical and genetic map shown in FIG. 1, the following items: - 5'ITR_SB, having coordinates from 88 to 317 bp; - integration arm 5'IFR, having coordinates from 318 to 368 bp; - MV enhancer, having coordinates from 369 to 733 bp; - CmV promoter (coordinates from 734 to 937 bp); - GPCL gene, which has coordinates from 1021 to 2292 bp. and encoding Lassa virus surface glycoprotein; - Foldon, having coordinates from 2293 to 2373 bp; - sequence 10his, having coordinates from 2374 to 2403 bp. and being a polyhistidine tag for purification of recombinant protein using metal chelate chromatography; - the internal landing site of the EMCV IRES ribosome, having coordinates from 2425 to 2999 bp; - BleoR - resistance gene to the antibiotic bleomycin, having coordinates from 3004 to 3011 bp; - PuroR, encoding the resistance factor to the antibiotic puromycin and having coordinates from 3012 to 3611 bp; - T3 promoter, having coordinates from 3621 to 3628 bp; - SV40 poly(A) signal, having coordinates from 3646 to 3767 bp; - integration arm 3'IFR, having coordinates from 3788 to 3835 bp; - 3'ITR_SB, having coordinates from 3836 to 4065 bp; - tonB terminator, having coordinates from 4117 to 4148 bp; - KanR - resistance gene to the antibiotic kanamycin, having coordinates from 4252 to 5067 bp; - the Ori replication origin site, which has coordinates from 5130 to 5717 bp. 2. Recombinant strain of Chinese hamster ovary cell line CHO-K1-GPC-LASV, producing recombinant surface glycoprotein GPC LASV and containing the integrative plasmid vector pVEAL3-Lassa-Trim according to claim 1. 3. Recombinant protein GPC LASV, produced by the recombinant strain of the Chinese hamster ovary cell line CHO-K1-GPC-LASV according to claim 2, having the amino acid sequence SEQ ID NO: 2 and intended for the creation of immunobiological drugs.

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