RU2774333C1 - RECOMBINANT PLASMID pET-GST-3CL-GPG PROVIDING SYNTHESIS OF SARS-CoV-2 3CL PROTEASE IN E. COLI CELLS IN SOLUBLE FORM - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. A recombinant plasmid pET-GST-3CL-GPG is described, which provides the synthesis of SARS-CoV-2 3CL protease in E. coli cells in a soluble form, having the nucleotide sequence of SEQ ID NO: 2. The plasmid has a map shown in Fig. 1 and contains the following elements: AmpR promoter; a genetic marker that determines ampicillin resistance of E. coli bacteria cells transformed with the recombinant AmpR plasmid; origin of replication site ori; lactose repressor lacI; DNA-binding protein that inhibits the expression of E. coli genes and ensures the expression of the target recombinant 3CL-GPG-6His protein after the addition of an isopropyl-β-D-1-thiogalactopyranoside (IPTG) inducer; lacI promoter; the T7 phage promoter, which ensures the expression of the GST-3CL-GPG gene sequence in E. coli cells; lac operator; RBS; gene GST-glutathione-S-transferase from Scihistosoma japonicum; Pep - region encoding the GSTSAVLQ peptide; the SARS-CoV-2 chimeric 3CL gene also including the GPG and 6His sequence, having the nucleotide sequence of SEQ ID NO: 1, and encoding the synthesis of the 3CL-GPG-6His protease with the amino acid sequence of SEQ ID NO: 3; T7 terminator.

EFFECT: invention can be used in biotechnology to obtain the 3CL enzyme, which is the main protease of SARS-CoV-2. This ensures the synthesis and transport of the chimeric variant of the 3CL protein into the periplasmic space of E. coli in a soluble form.

1 cl, 3 dwg, 1 tbl, 5 ex

Description

Technical field

The invention relates to a recombinant plasmid pET-GST-3CL-GPG that provides the synthesis of SARS-CoV-2 3CL protease in E. coli cells in a soluble form and can be used in biotechnology to obtain the 3CL enzyme (from English: 3C-like protease) , which is the main protease of SARS-CoV-2 sarbecovirus. The resulting 3CL protein can be used for the in vitro search for inhibitors of the main SARS-CoV-2 protease of potential therapeutic drugs (Froggatt, N.M., Heaton, B.E., Heaton, N.S. (2020). Development of a fluorescence-based, high-throughput SARS-CoV-2 3CLpro reporter assay Journal of virology, 94(22), e01265-20 Zhu, W., Xu, M., Chen, C.Z., Guo, H., Shen, M., Hu, X., … & Zheng, W. (2020). Identincation of SARS-CoV-2 3CL protease inhibitors by a quantitative high-throughput screening. ACS pharmacology & translational science, 3(5), 1008-1016.). The invention includes the design of genetic constructs, a method for transforming E. coli cells, purification and evaluation of target protein expression, a method for analyzing enzymatic activity.

State of the art

The SARS-CoV-2 genome encodes more than twenty proteins, among which two proteases are present - papain-like PLpro and 3-C like 3CLpro (3CL), (or Mpro, the main protease). Both proteases play an important role in the replication cycle of the virus, their function is the enzymatic hydrolysis of two synthesized viral polyproteins (1A and 1AB) into separate functional proteins.

Due to its small size, as well as high homology with similar proteins of SARS and MERS coronaviruses, the SARS-CoV-2 main protease is the most characterized target for potential antiviral drugs. The core protease cleaves the 1AB polyprotein at 11 specific sites. The site recognition sequence in most cases consists of a chain segment (L-Q)-(S-A-G), where the bond between glutamine and serine is cleaved. Inhibition of the activity of the main protease leads to a stop in the replication of the virus. Among human enzymes, no proteases with the same cleavage specificity are known, which may indicate the likely specific action and possible low toxicity of SARS-CoV-2 major protease inhibitors (Zhang L, Lin D, Sun X, et al. Crystal structure of SARS-CoV Dai W, Zhang B, Su H Jin Z, Du X, Xu Y, et al Structure of Mpro from COVID-19 virus and discovery of its inhibitors Nature April 2020:1-9 doi:10.1038/s41586-020-2223-y).

The closest analogue (prototype) to the claimed invention is the creation of the construction of the expression plasmid of the main SARS protease; transforming the expression plasmid of the main SARS protease to obtain a recombinant strain of Escherichia coli; Obtaining a target protein using an expression system by culturing a recombinant strain of Escherichia coli in a liquid nutrient medium (in LB culture), followed by obtaining a purified stable SARS-CoV-1 basic protease using a special purification method (China patent application CN111979214A, IPC C12N 15/ 70; C12N 9/50, published 08/31/2020).

However, this invention has a number of disadvantages. Thus, the pET28a expression vector used does not contain the GST sequence, which provides more efficient transport of the target protein into the periplasmic space of Escherichia coli cells. In addition, the synthesized protein is the main protease of the SARS-CoV-1 coronavirus, which differs significantly in nucleotide and amino acid sequences from the main protease of the SARS-CoV-2 coronavirus.

Disclosure of invention

The objective of the present invention was to develop an expression system and structure of the GST-3CL-GPG SARS-CoV-2 chimeric gene that would provide a chimeric variant of the SARS-CoV-2 soluble 3CL protein.

The technical result of the invention is to obtain an expression genetic construct containing the nucleotide sequence of the 3CL protein (protease) with optimized codons and providing the synthesis and transport of the chimeric variant of the 3CL protein into the periplasmic space of E. coli.

This technical result is achieved by creating a recombinant plasmid pET-GST-3CL-GPG, which provides the synthesis and transport of the SARS-CoV-2 chimeric protein (protease) 3CL into the periplasmic space of E. coli cells in a soluble form, having the nucleotide sequence SEQ ID NO : 2 with a size of 7008 bp, and containing, in accordance with the physical and genetic map presented in Fig. 1, the following items:

- AmpR promoter (coordinates from 494 bp to 598 bp);

- a genetic marker that determines the resistance to ampicillin of E. coli bacterial cells transformed with the recombinant AmpR plasmid (coordinates from 599 to 1459 bp);

- site of origin of replication ori (coordinates from 1630 to 2218 bp);

- lacI - DNA-binding protein that inhibits the expression of E. coli genes and ensures the expression of the target recombinant protein 3CL-GPG-6His after the addition of the inducer isopropyl-β-D-1-thiogalactopyranoside (IPTG) (coordinates from 3648 to 4730 b.p. );

- lacI promoter (coordinates from 4731 to 4808 b.p.);

- T7 phage promoter (coordinates from 5117 to 5135 bp), which ensures the expression of the GST-3CL-GPG-6His gene sequence in E. coli cells;

- lac operator (coordinates from 5136 to 5160 bp);

- RBS - ribosome binding site (coordinates from 5191 to 5196 bp)

- GST gene (glutathione-S-transferase from Shistosoma japonium) (coordinates from 5205 to 5857 b.p.

- Pep - region encoding the GSTSAVLQ peptide, (coordinates from 5859 to 5882 bp)

chimeric SARS-CoV-2 3CL gene, which also includes the GPG and 6His sequence, having coordinates from 5883 to 6833 bp, the nucleotide sequence of SEQ ID NO: 1 and encoding the synthesis of the 3CL-GPG-6His protein (protease) with the amino acid sequence of SEQ ID NO: 3;

- T7 terminator (coordinates from 6936 to 6983 bp).

Thus, a codon-optimized nucleotide sequence having the sequence SEQ ID NO: 1 encoding the SARS-CoV-2 3CL protein, which also includes the GPG sequence and the 6×His sequence, was obtained, and the genetic plasmid construct pET-GST-3CL- GPG (Fig. 1), containing the nucleotide sequence 3CL-GPG SEQ ID NO: 2 and providing synthesis and transport into the periplasmic space of the 3CL-GPG-6His protein in E. coli cells.

In addition, a chimeric protein GST-3CL-GPG was obtained. The GST sequence at the N-terminus of the protein provides for transport of the fusion protein into the periplasmic space of E. coli cells. In the periplasmic space, GST is autocleaved due to the catalytic activity of the 3CL protease. The GSTSAVLQ sequence is the consensus sequence for the 3CL proteolysis site and is located between the GST protein and 3CL. At the C-terminus, the protein contains the sequence 6×His, which allows purification of the target protein using metal chelate affinity chromatography. The 3CL and 6xHis sequence is separated by the GPGSG amino acid sequence preventing auto-cleavage of the 6xHis sequence from 3CL.

The 3CL-GPG-6His protein with the amino acid sequence of SEQ ID NO: 3 is obtained by culturing E. coli cells, synthesizing the target protein, and isolating the soluble protein by metal chelate affinity chromatography.

The invention has a number of advantages compared to the closest analogue (prototype) in essence. The use of the GST protein makes it possible to obtain the 3CL-GPG-6His protein in a soluble form that retains enzymatic activity, which makes it possible to use it for the analysis of SARS-CoV-2 coronavirus inhibitor substances. The codon optimization of the DNA fragment encoding the 3CL protein ensures a high level of protein expression. The GSTSAVLQ sequence provides efficient autocleavage of the 3CL-GPG-6His protein. The use of the 6×His sequence in the target protein makes it possible to optimize the method for isolating and purifying the 3CL-GPG-6His protein.

Implementation of the invention

The invention is illustrated by the following graphics.

In FIG. 1. The physical and genetic map of the 7008 bp pET-GST-3CL-GPG plasmid vector is presented.

In FIG. 2 shows the electrophoregram of the separation of protein preparations in 15% SDS-PAGE:

1 - E. coli biomass before adding the IPTG inducer;

2 - E. coli biomass after adding the IPTG inducer;

3 - fraction of periplasmic proteins (soluble);

4 - insoluble protein fraction;

M - molecular weight marker, 10-200 kDa

In FIG. 3 shows the electropherogram of the separation of chromatographic fractions in 15% SDS-PAGE:

1 - lysate of E. coli cells containing the plasmid pET-GST-3CL-GPG before chromatographic purification;

2 - ballast (impurity) proteins that have not contacted the sorbent (application breakthrough);

3 - fraction of impurity proteins after washing the column with three volumes of washing buffer;

4 - target protein fraction after chromatographic purification;

M - molecular weight marker, 10-200 kDa.

Below are examples 1-3 of the specific implementation of the invention.

Example 1 Construction of a Genetic Sequence for the Synthesis of the 3CL-GPG-6His Protein

The sequence of the gene encoding the 3CL protein (3264S - 3569Q) of the SARS-CoV-2 coronavirus (GenBank MZ149976) was borrowed from the GenBank database and the codon frequencies for mammals were optimized using the JCat service (http://www.jcat.de/) , while the CAI codon adaptation index reached 0.87.

Based on the pET-GST vector, the pET-GST-3CL-GPG construct (FIG. 1) containing the 3CL-GPG-6His nucleotide sequence (SEQ ID NO: 1) was obtained.

The nucleotide sequence encoding the Pep peptide (GSTSAVLQ), 6His, the GPG sequence and the BamHI and CciNI restriction sites were introduced into the 3CL sequence simultaneously using the NDP. Primer pairs 3CL-GPG-F and 3CL-GPG-R were used (Table 1).

Example 2 Creation of the plasmid construct pET-GST-3CL-GPG

2.1. PCR amplification of the nucleotide sequence encoding SARS-CoV-2 3CL

Fragment amplification was carried out according to the standard protocol. The reaction mixture, with a volume of 50 μl, contained 10 × buffer, 25 mm dNTP, oligonucleotide primers (table 1) in the amount of 15 pmol each, 5 units. Q5 polymerase, 1 µl of plasmid DNA solution. The reaction was carried out in a Veriti™ 96-Well Thermal Cycler. Temperature-time profile of PCR: melting - 95°C - 5 min. (1 cycle). 95°C - 30 seconds. Annealing - 58°C - 30 seconds, elongation - 72°C - 30 seconds (35 cycles).

2.2. Isolation of DNA from agarose gel

After visualization under UV radiation, DNA fragments of the required length, separated in an agarose gel, were excised from the gel and eluted using the Evrogen Cleanup Standard kit (Russia) in accordance with the manufacturer's recommendations.

2.3. Enzymatic hydrolysis of pET-GST vector and PCR product (Pep-3CL-GPG sequence)

For cloning of the 3CL-GPG gene in the pET-GST vector, restriction endonucleases BamHI and CciNI (Sibenzym, Russia) were used with buffers attached to them. The reaction mixture was prepared in accordance with the activity of the enzyme (2-5 u per 1 μg of DNA) and the concentration of plasmid DNA. Reaction conditions: temperature, buffer composition, and duration of DNA enzymatic hydrolysis were selected in accordance with the instructions of the Sibenzym manufacturer.

2.4. Ligation of PCR product (Pep-3CL-GPG sequence) and pET-GST vector

The ligation reaction was carried out for 30 min. at room temperature using a mixture of 2 µg of DNA template amplicons, 1 µg of vector plasmid, and 20 u.a. T4 DNA ligases in the reaction buffer supplied with the commercial kit. The resulting ligation mixture was used to transform a competent cell culture of E. coli strain Neb Stable.

2.5. Heat shock transformation

To the "competent" cells of E. coli strain Neb Stable were added 10 μl of the ligation mixture (ratio 1:10), incubated on ice for 30 min. After that, the cells were subjected to "temperature shock" at 42°C for 45 sec. The cells were cooled on ice for 2 min, then 200 µl of SOB medium was added and incubated at 37°C for 60 min. At the end of the incubation, the transformed cells were seeded on a Petri dish with a solid LB nutrient medium containing an antibiotic.

2.6. Selection of clones for restriction analysis. E. coli cells of the Neb Stable strain transformed with the plasmid were selectively cultivated in 10 ml of LB liquid nutrient medium supplemented with an antibiotic at a working concentration of 25 μg/ml.

2.7. Determination of the DNA nucleotide sequence

Sequencing was performed according to the Sanger method. A CEQ2000Dye Terminator Cycle Sequencing Kit and an ABI 3130xl 16-capillary automatic sequencer were used.

Example 3 Production of recombinant 3CL-GPG-6His protein

The resulting plasmid pET-GST-3CL-GPG (Fig. 1) was transformed into E. coli BL21(DE3) cells. Individual E. coli colonies containing recombinant plasmids were cultured overnight on an orbital shaker in LB medium containing 100 μg/ml ampicillin at 37°C and 180 rpm. The inoculum at a ratio of 1/100 was transferred to an Erlenmeyer flask containing LB medium and grown to an absorbance of 0.8 (at λ=600 nm). The inducer isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. The culture was additionally cultivated on a shaker for 5 h at 37°C and 180 rpm. The biomass was precipitated by centrifugation for 20 min at 5000 g and 4°C. The resulting pellet was dissolved in lysis buffer: 12 mM Tris; 120 mM NaCl; 0.1 mM EDTA; 1mM DTT. To destroy the cell wall, ultrasonic treatment of the resulting solution was performed. After the suspension was centrifuged at 16000g for 20 minutes at 4°C to separate the insoluble fraction. The resulting fractions were analyzed by electrophoresis under denaturing conditions in 15% PAAG. The protein was in the soluble fraction (Fig. 2).

Example 4 Purification of Recombinant Protein

The supernatant containing the target protein was subjected to metal chelate chromatography. To do this, a chromatographic column with a Ni Sepharose 6 Fast Flow sorbent was equilibrated with a wash buffer containing 15 mM imidazole in PBS. The supernatant was applied to the column at a ratio of 30 mg of protein per 1 ml of sorbent and washed with 10 volumes of wash buffer. The protein was eluted with 3 volumes of elution buffer (350 mM imidazole in PBS). The resulting eluate was dialyzed against 100 volumes of 50 mm Tris pH 7.5. The resulting fractions were analyzed by electrophoresis under denaturing conditions in 15% PAAG (Fig. 3).

Example 5. Analysis of the enzymatic activity of the recombinant protein. Analysis of the enzymatic activity of the resulting protein was carried out by fluorimetry using a plate fluorimeter CLARIOstar Plus, excitation wavelength - 495 nm, emission - 520 nm. The synthetic peptide Dabcyl-VNSTLQSGLR-Lys(5FAM)-MA was used as a substrate. Substrate hydrolysis with chymosin leads to fluorescence enhancement due to removal of the fluorophore and quencher. The rate of increase in fluorescence reflects the rate of the enzymatic reaction.

Reaction mixtures were prepared in 384 well plates and then incubated at room temperature for 5 min. All measurements were carried out at 25°C. Each well contained one reaction mixture. The instrument was calibrated using a peptide solution subjected to complete hydrolysis. The fluorescence value of this mixture was taken as 80%. The reaction mixtures contained 5 µl of Tris-HCl buffer (pH 8.0; 50 mM Tris; 50 mM NaCl), 2 µl of substrate (C=33 µg/ml). The reaction was started by adding 3 μl of 3CL-GPG-6His (C=0.025 mg/ml). Enzymatic activity was indicated by an increase in the fluorescence signal. When a specific peptide substrate (Dabcyl-VNSTLQSGLR-Lys(5FAM)-MA) was incubated with an enzyme (protease 3CL-GPG-6His), the fluorescence level increased to 200,000 RFU (relative fluorescence units), which makes it possible to judge the specific enzymatic activity of the protein 3CL-GPG-6His.

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APPENDIX

Sequence listing

<110> Federal budgetary institution of science "State Scientific Center

virology and biotechnology "Vector" of the Federal Service for Supervision in the

protection of consumer rights and human well-being (FBUN SSC VB "Vector"

Rospotrebnadzor)

<120> Recombinant plasmid pET-GST-3CL-GPG providing protease synthesis

3CL SARS-CoV-2 in E. coli cells in soluble form.

<160> SEQ ID NO:3

<210> SEQ ID NO:1

<211> 951

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence of the chimeric 3CL-GPG-6His gene

<400> 1

1 AGCGGTTTTC GTAAAATGGC ATTTCCGAGC GGTAAAGTTG AAGGTTGTAT GGTTCAGGTT

61 ACCTGTGGCA CCACGACACT GAATGGTCTG TGGCTGGATG ATGTTGTTTA TTGTCCGCGT

121 CATGTTATTT GTACCAGCGA AGATATGCTG AACCCGAATT ATGAAGATCT GCTGATTCGC

181 AAAAGCAACC ATAATTTTCT GGTTCAGGCA GGTAATGTTC AGTGCGTGT TATTGGTCAT

241 AGCATGCAGA ATTGTGTGCT GAAACTGAAA GTTGATACCG CCAATCCGAA AACGCCGAAA

301 TATAAGTTTG TTCGTATTCA GCCTGGTCAG ACCTTTAGCG TTCTGGCATG TTATAATGGT

361 AGCCCGAGCG GTGTTTATCA GTGTGCAATG CGTCCGAATT TTACCATTAA AGGCAGCTTT

421 CTGAATGGTA GCTGTGGTAG CGTTGGTTTC AACATTGATT ATGATTGCGT GAGCTTCTGC

481 TATATGCATC ATATGGAACT GCCGACCGGT GTTCATGCAG GCACCGATCT GGAAGGTAAC

541 TTTTATGGTC CGTTTGTTGA TCGTCAGACC GCACAGGCAG CAGGTACAGA TACCACCATT

601 ACCGTTAATG TTCTGGCCTG GCTGTATGCA GCAGTTATTA ATGGTGATCG CTGGTTTCTG

661 AATCGTTTTTA CAACAACCCT GAACGATTTT AATCTGGTGG CCATGAAATA TAACTATGAA

721 CCGCTGACAC AGGATCATGT TGATATTCTG GGTCCGCTGA GCGCACAGAC CGGTATTGCA

781 GTTCTGGATA TGTGTGCAAG CCTGAAAGAA CTGTTACAGA ATGGTATGAA TGGTCGTACA

841 ATTCTGGGTA GCGCACTGCT GGAAGATGAA TTCACCCCGT TTGATGTTGT GCGTCAGTGT

901 AGCGGTGTTA CCTTTCAGGG ACCAGGTAGC GGTCATCATC ACCATCACCA C

<210> SEQ ID NO:2

<211> 7008 b.p.

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence of recombinant plasmid pET-GST-3CL-GPG.

<400> 2

1 TGGCGAATGG GACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG TGGTTACGCG

61 CAGCGTGACC GCTACACTTG CCAGCGCCCT AGCGCCCGCT CCTTTCGCTT TCTTCCCTTC

121 CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG TCAAGCTCTA AATCGGGGGC TCCCTTTAGG

181 GTTCCGATTT AGTGCTTTAC GGCACCTCGA CCCCAAAAAA CTTGATTAGG GTGATGGTTC

241 ACGTAGTGGG CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG AGTCCACGTT

301 CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC AACCCTATCT CGGTCTATTC

361 TTTTGATTTA TAAGGGATTT TGCCGATTTC GGCCTATTGG TTAAAAAATG AGCTGATTTA

421 ACAAAAATTT AACGCGAATT TTAACAAAAT ATTAACGTTT ACAATTTCAG GTGGCACTTT

481 TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACATT CAAATATGTA

541 TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA GGAAGAGTAT

601 GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTTTTT GCGGCATTTT GCCTTCCTGT

661 TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT TGGGTGCACG

721 AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGTT TTCGCCCCGA

781 AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA TGTGGCGCGG TATTATCCCG

841 TATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA ATGACTTGGT

901 TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA GAGAATTATG

961 CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC TTACTTCTGA CAACGATCGG

1021 AGGACCGAAG GAGCTAACCG CTTTTTTGCA CAACATGGGG GATCATGTAA CTCGCCTTGA

1081 TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGTGACA CCACGATGCC

1141 TGCAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA CTCTAGCTTC

1201 CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC TTCTGCGCTC

1261 GGCCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC GTGGGTCTCG

1321 CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG TTATCTACAC

1381 GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG ATCGCTGAGA TAGGTGCCTC

1441 ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT AGATTGATTT

1501 AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA ATCTCATGAC

1561 CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG AAAAGATCAA

1621 AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA CAAAAAAACC

1681 ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT TTCCGAAGGT

1741 AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC CGTAGTTAGG

1801 CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA TCCTGTTACC

1861 AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA GACGATAGTT

1921 ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC CCAGCTTGGA

1981 GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA GCGCCACGCT

2041 TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG

2101 CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG GGTTTCGCCA

2161 CCTCTGACTT GACGTCGAT TTTTTGTGATG CTCGTCAGGG GGGCGGAGCC TATGGAAAAA

2221 CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG CTCACATGTT

2281 CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTTTG AGTGAGCTGA

2341 TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG AAGCGGAAGA

2401 GCGCCTGATG CGGTATTTTC TCCTTACGCA TCTGTGCGGT ATTTCACACC GCATATATGG

2461 TGCACTCTCA GTACAATCTG CTCTGATGCC GCATAGTTAA GCCAGTATAC ACTCCGCTAT

2521 CGCTACGTGA CTGGGTCATG GCTGCGCCCC GACACCCGCC AACACCCGCT GACGCGCCCT

2581 GACGGGCTTG TCTGCTCCCG GCATCCGCTT ACAGACAAGC TGTGACCGTC TCCGGGAGCT

2641 GCATGTGTCA GAGGTTTTCA CCGTCATCAC CGAAACGCGC GAGGCAGCTG CGGTAAAGCT

2701 CATCAGCGTG GTCGTGAAGC GATTCACAGA TGTCTGCCTG TTCATCCGCG TCCAGCTCGT

2761 TGAGTTTCTC CAGAAGCGTT AATGTCTGGC TTCTGATAAA GCGGGCCATG TTAAGGGCGG

2821 TTTTTTCCTG TTTGGTCACT GATGCCTCCG TGTAAGGGGG ATTTTCTGTTC ATGGGGGTAA

2881 TGATACCGAT GAAACGAGAG AGGATGCTCA CGATACGGGT TACTGATGAT GAACATGCCC

2941 GGTTACTGGA ACGTTGTGAG GGTAAACAAC TGGCGGTATG GATGCGGCGG GACCAGAGAA

3001 AAATCACTCA GGGTCAATGC CAGCGCTTCG TTAATACAGA TGTAGGTGTT CCACAGGGTA

3061 GCCAGCAGCA TCCTGCGATG CAGATCCGGA ACATAATGGT GCAGGGCGCT GACTTCCGCG

3121 TTTCCAGACT TTACGAAACA CGGAAACCGA AGACCATTCA TGTTGTTGCT CAGGTCGCAG

3181 ACGTTTTGCA GCAGCAGTCG CTTCACGTTC GCTCGCGTAT CGGTGATTCA TTCTGCTAAC

3241 CAGTAAGGCA ACCCCGCCAG CCTAGCCGGG TCCTCAACGA CAGGAGCACG ATCATGCCGCA

3301 CCCGTGGGGC CGCCATGCCG GCGATAATGG CCTGCTTCTC GCCGAAACGT TTGGTGGCGG

3361 GACCAGTGAC GAAGGCTTGA GCGAGGGCGT GCAAGATTCC GAATACCGCA AGCGACAGGC

3421 CGATCATCGT CGCGCTCCAG CGAAAGCGGT CCTCGCCGAA AATGACCCAG AGCGCTGCCG

3481 GCACTGTCC TACGAGTTGC ATGATAAAGA AGACAGTCAT AAGTGCGGCG ACGATAGTCA

3541 TGCCCCGCGC CCACCGGAAG GAGCTGACTG GGTTGAAGGC TCTCAAGGGC ATCGGTCGAG

3601 ATCCCGGTGC CTAATGAGTG AGCTAACTTA CATTAATTGC GTTGCGCTCA CTGCCCGCTT

3661 TCCAGTCGGG AAACTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC GCGGGGAGAG

3721 GCGGTTTGCG TATTGGGCGC CAGGGTGGTT TTTCTTTTCA CCAGTGAGAC GGGCAACAGC

3781 TGATTGCCCT TCACCGCCTG GCCCTGAGAG AGTTGCAGCA AGCGGTCCAC GCTGGTTTGC

3841 CCCAGCAGGC GAAAATCCTG TTTGATGGTG GTTAACGGCG GGATATAACA TGAGCTGTCT

3901 TCGGTATCGT CGTATCCCAC TACCGAGATA TCCGCACCAA CGCGCAGCCC GGACTCGGTA

3961 ATGGCGCGCA TTGCGCCCAG CGCCATCTGA TCGTTGGCAA CCAGCATCGC AGTGGGAACG

4021 ATGCCCTCAT TCAGCATTTG CATGGTTTGT TGAAAACCGG ACATGGCACT CCAGTCGCCT

4081 TCCCCGTTCCG CTATCGGCTG AATTTGATTG CGAGTGAGAT ATTTATGCCA GCCAGCCAGA

4141 CGCAGACGCG CCGAGACAGA ACTTAATGGG CCCGCTAACA GCGCGATTTG CTGGTGACCC

4201 AATGCGACCA GATGCTCCAC GCCCAGTCGC GTACCGTCTT CATGGGAGAA AATAATACTG

4261 TTGATGGGTG TCTGGTCAGA GACATCAAGA AATAACGCCG GAACATTAGT GCAGGCAGCT

4321 TCCACAGCAA TGGCATCCTG GTCATCCAGC GGATAGTTAA TGATCAGCCC ACTGACGCGT

4381 TGCGCGAGAA GATTGTGCAC CGCCGCTTTA CAGGCTTCGA CGCCGCTTCG TTCTACCATC

4441 GACACCACCA CGCTGGCACC CAGTTGATCG GCGCGAGATT TAATCGCCGC GACAATTTGC

4501 GACGGCGCGT GCAGGGCCAG ACTGGAGGTG GCAACGCCAA TCAGCAACGA CTGTTTGCCC

4561 GCCAGTTGTT GTGCCACGCG GTTGGGAATG TAATTCAGCT CCGCCATCGC CGCTTCCACT

4621 TTTTCCCGCG TTTTCGCAGA AACGTGGCTG GCCTGGTTCA CCACGCGGGA AACGGTCTGA

4681 TAAGAGACAC CGGCATACTC TGCGACATCG TATAACGTTA CTGGTTTCAC ATTCACCACC

4741 CTGAATTGAC TCCTTCTCCGG GCGCTATCAT GCCATACCGC GAAAGGTTTT GCGCCATTCG

4801 ATGGTGTCCG GGATCTCGAC GCTCTCCCTT ATGCGACTCC TGCATTAGGA AGCAGCCCAG

4861 TAGTAGGTTG AGGCCGTTGA GCACCGCCGC CGCAAGGAAT GGTGCATGCA AGGAGATGGC

4921 GCCCAACAGT CCCCCGGCCA CGGGGCCTGC CACCATACCC ACGCCGAAAC AAGCGCTCAT

4981 GAGCCCGAAG TGGCGAGCCC GATCTTCCCC ATCGGTGATG TCGGCGATAT AGGCGCCAGC

5041 AACCGCACCT GTGGCGCCGG TGATGCCGGC CACGATCGCGT CCGGCGTAGA GGATCGAGAT

5101 CTCGATCCCG CGAAATTAAT ACGACTCACT ATAGGGGAAT TGTGAGCGGA TAACAATTCC

5161 CCTTAGAAA TAATTTTGTT TAACTTTAAG AAGGAGATAT ACATATGTCC CCTATACTAG

5221 GTTATTGGAA AATTAAGGGC CTTGTGCAAC CCACTCGACT TCTTTTGGAA TATCTTGAAG

5281 AAAAATATGA AGAGCATTTG TATGAGCGCG ATGAAGGTGA TAAATGGCGA AACAAAAAGT

5341 TTGAATTGGG TTTGGAGTTT CCCAATCTTC CTTATTATAT TGATGGTGAT GTTAAATTAA

5401 CACAGTCTAT GGCCATCATA CGTTATATAG CTGACAAGCA CAACATGTTG GGTGGTTGTC

5461 CAAAAGAGCG TGCAGAGATT TCAATGCTTG AAGGAGCGGT TTTGGATATT AGATACGGTG

5521 TTTCGAGAAT TGCATATAGT AAAGACTTTG AAACTCTCAA AGTTGATTTT CTTAGCAAGC

5581 TACCTGAAAT GCTGAAAATG TTCGAAGATC GTTTATGTCA TAAAACATAT TTAAATGGTG

5641 ATCATGTAAC CCATCCTGAC TTCATGTTGT ATGACGCTCT TGATGTTGTT TTATACATGG

5701 ACCCAATGTG CCTGGATGCG TTCCCAAAAT TAGTTTGTTT TAAAAAACGT ATTGAAGCTA

5761 TCCCACAAAT TGATAAGTAC TTGAAATCCA GCAAGTATAT AGCATGGCCT TTGCAGGGCT

5821 GGCAAGCCAC GTTTGGTGGT GGCGACCATC CTCCAAAAGG ATCCACCTCA GCTGTTTTGC

5881 AGAGCGGTTT TCGTAAAATG GCATTTCCGA GCGGTAAAGT TGAAGGTTGT ATGGTTCAGG

5941 TTACCTGTGG CACCACGACA CTGAATGGTC TGTGGCTGGA TGATGTTGTT TATTGTCCGC

6001 GTCATGTTAT TTGTACCAGC GAAGATATGC TGAACCCGAA TTATGAAGAT CTGCTGATTC

6061 GCAAAAGCAA CCATAATTTT CTGGTTCAGG CAGGTAATGT TCAGCTGCGT GTTATTGGTC

6121 ATAGCATGCA GAATTGTGTG CTGAAACTGA AAGTTGATAC CGCCAATCCG AAAACGCCGA

6181 AATATAAGTT TGTTCGTATT CAGCCTGGTC AGACCTTTAG CGTTCTGGCA TGTTATAATG

6241 GTAGCCCGAG CGGTGTTTAT CAGTGTGCAA TGCGTCCGAA TTTTACCATT AAAGGCAGCT

6301 TTCTGAATGG TAGCTGTGGT AGCGTTGGTT TCAACATTGA TTATGATTGC GTGAGCTTCT

6361 GCTATATGCA TCATATGGAA CTGCCGACCG GTGTTCATGC AGGCACCGAT CTGGAAGGTA

6421 ACTTTTATGG TCCGTTTGTT GATCGTCAGA CCGCACAGGC AGCAGGTACA GATACCACCA

6481 TTACCGTTAA TGTTCTGGCC TGGCTGTATG CAGCAGTTAT TAATGGTGAT CGCTGGTTTC

6541 TGAATCGTTT TACAACAACC CTGAACGATT TTAATCTGGT GGCCATGAAA TATAACTATG

6601 AACCGCTGAC ACAGGATCAT GTTGATATTC TGGGTCCGCT GAGCGCACAG ACCGGTATTG

6661 CAGTTCTGGA TATGTGTGCA AGCCTGAAAG AACTGTTACA GAATGGTATG AATGGTCGTA

6721 CAATTCTGGG TAGCGCACTG CTGGAAGATG AATTCACCCC GTTTGATGTT GTGCGTCAGT

6781 GTAGCGGTGT TACCTTTCAG GGACCAGGTA GCGGTCATCA TCACCATCAC CACTAAAGCGG

6841 CCGCACTCGA GCACCACCAC CACCACCACT GAGATCCGGC TGCTAACAAA GCCCGAAAGG

6901 AAGCTGAGTT GGCTGCTGCC ACCGCTGAGC AATAACTAGC ATAACCCCTT GGGGCCTCTA

6961 AACGGGTCTT GAGGGGTTTT TTGCTGAAAG GAGGAACTAT ATCCGGAT

<210> SEQ ID NO:3

<211> 317 a.a.

<212> DNA

<213> Artificial Sequence

<220>

<223> Amino acid sequence of recombinant protein 3CL-GPG-6His

<400> 3

1 SGFRKMAFPS GKVEGCMVQV TCGTTTLNGL WLDDVVYCPR HVICTSEDML NPNYEDLLIR

61 KSNHNFLVQA GNVQLRVIGH SMQNCVLKLK VDTANPKTPK YKFVRIQPGQ TFSVLACYNG

121 SPSGVYQCAM RPNFTIKGSF LNGSCGSVGF NIDYDCVSFC YMHHMELPTG VHAGTDLEGN

181 FYGPFVDRQT AQAAGTDTTI TVNVLAWLYA AVINGDRWFL NRFTTTLNDF NLVAMKYNYE

241 PLTQDHVDIL GPLSAQTGIA VLDMCASLKE LLQNGMNGRT ILGSALLEDE FTPFDVVRQC

301 SGVTFQGPGS GHHHHHH

<---

Claims (13)

Recombinant plasmid pET-GST-3CL-GPG, which provides the synthesis and transport of SARS-CoV-2 3CL protease into the periplasmic space of E. coli cells in a soluble form, having a 7008 bp nucleotide sequence of SEQ ID NO: 2. and containing in accordance with the physical and genetic map presented in Fig. 1, the following items: - AmpR promoter, having coordinates from 494 to 598 bp; - a genetic marker that determines the resistance to ampicillin of E. coli bacteria cells transformed with the recombinant AmpR plasmid, having coordinates from 599 to 1459 bp; - site of origin of replication ori, having coordinates from 1630 to 2218 bp; - lacI - DNA-binding protein that inhibits the expression of E. coli genes, which ensures the expression of the target recombinant protein 3CL-GPG-6His after the addition of the inducer isopropyl-β-D-1-thiogalactopyranoside (IPTG), having coordinates from 3648 to 4730 bp .; - lacI promoter, having coordinates from 4731 to 4808 bp; - T7 phage promoter, having coordinates from 5117 to 5135 bp. and providing expression of the GST-3CL-GPG gene sequence in E. coli cells; - lac operator, having coordinates from 5136 to 5160 bp; - RBS - ribosome binding site, having coordinates from 5191 to 5196 bp; - GST gene - glutathione-S-transferase from Shistosoma japonicum, having coordinates from 5205 to 5857 bp; - Pep - region encoding the GSTSAVLQ peptide, having coordinates from 5859 to 5882 bp; - chimeric gene 3CL SARS-CoV-2, which also includes the sequence of GPG and 6His, having the nucleotide sequence of SEQ ID NO: 1, coordinates from 5883 to 6833 p. and encoding the synthesis of the protease 3CL-GPG-6His with the amino acid sequence of SEQ ID NO: 3; - T7 terminator, having coordinates from 6936 to 6983 b.p.

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