SARS coronavirus-2 envelope protein produced in the baculovirus expression system

Abstract

The invention relates to a SARS Coronavirus-2 (SARS-CoV-2) envelope protein produced in a baculovirus expression system. The inventive protein is a recombinant protein derived from the envelope gene of a local SARS-CoV-2 isolate. It is intended to be used as a candidate protein in recombinant vaccine platforms that can be prepared to protect against Covid-19 infection. Within the scope of the invention, a protein that can form the basis for the antibody ELISA method that can be prepared for the indirect diagnosis of SARS-CoV-2 infection has been produced.

Description

[0001] SARS CORONAVIRUS-2 ENVELOPE PROTEIN PRODUCED IN THE BACULOVIRUS EXPRESSION SYSTEM

[0002] Technical Field

[0003] The invention relates to a SARS Coronavirus-2 (SARS-CoV-2) envelope protein produced in a baculovirus expression system. The inventive protein is a recombinant protein derived from the envelope gene of a local SARS-CoV-2 isolate. It is intended to be used as a candidate protein in recombinant vaccine platforms that can be prepared to protect against Covid- 19 infection. Within the scope of the invention, a protein that can form the basis for the antibody enzyme- linked immunosorbent assay (ELISA) method that can be prepared for the indirect diagnosis of SARS-CoV-2 infection has been produced.

[0004] Prior Art

[0005] Existing systems in the art generally use proteins produced using mammalian cells. In this context, viral protein expression with baculovirus has advantages over other vector systems.

[0006] In the International patent document numbered WO22047316 in the state of the art, methods for treating a virus infection such as coronavirus are mentioned.

[0007] In the International patent document numbered WO22235663 in the state of the art, compositions and methods for inducing immune responses against both influenza and coronaviruses are mentioned.

[0008] In the International patent document numbered WO2122222222 in the state of the art, isolated polynucleotides, lentiviral vectors, virions and pharmaceutical compositions and methods of making and using them are mentioned. When the existing studies in the art were reviewed, there was a need to develop a SARS-Coronavirus-2 (SARS-CoV-2) envelope protein produced in a baculovirus expression system.

[0009] Objects of the Invention

[0010] The object of the present invention is to develop a SARS Coronavirus-2 (SARS- CoV-2) envelope protein produced in a baculovirus expression system.

[0011] A further object of the present invention is to develop a SARS Coronavirus-2 (SARS-CoV-2) envelope protein that stimulates the immune response to Covid- 19 infection and induces neutralizing antibody formation.

[0012] Detailed Description of the Invention

[0013] The invention relates to a SARS Coronavirus-2 (SARS-CoV-2) envelope protein produced in a baculovirus expression system and the protein structure is indicated on seq. ID no: 1.

[0014] Viral RNA Extraction and Reverse Transcription Polymerase Chain Reaction (RT- PCR): For this invention, SARS-CoV-2 Ank-1 field isolate (MT478018), which was isolated in Ankara University Faculty of Veterinary Medicine BSL3+ laboratories and whose complete genome was deposited in GenBank Basic Local Alignment Search Tool (BLAST) database, was used. [1] Viral RNA extraction of this isolate was performed using the Trizol LS method. Then, the envelope gene region was amplified by RT-PCR using the following primers designed within the scope of the invention.

[0015] Forward primer: 5'-GGGATCCGCTGATGAGTACGAACTTATG-3' amHI (Tm=60 °C)

[0016] Reverse primer: 5'-AACTGCAGGTTCGTTTAGACCAGAAGA-3' Pstl (Tm=54 °C)

[0017] * Bolded and underlined nucleotides indicate the start and end codons of the viral envelope gene. The Trizol Extraction Methodology applied within the scope of the invention is described below.

[0018] 250 pl of SARS-CoV-2 Ank-1 isolate previously inoculated into Vero cell culture was transferred to sterile 1.5 ml tubes, 750 pl of TRIzol® LS Reagent solution was added and kept at room temperature for 5 min.

[0019] Then 200 pl of chloroform was added to the tube and shaken by hand for approximately 10 seconds (s) and allowed to stand for 2-15 min at room temperature.

[0020] After incubation, the tubes were centrifuged at 12000 xg for 15 min at +4 °C.

[0021] After centrifugation, approximately 500 pl of the upper aqueous phase was transferred to a new 1.5 ml tube, 100% isopropanol was added and allowed to stand at room temperature for 10 min.

[0022] It was then centrifuged at 12000 xg for 10 min at +4 °C.

[0023] After centrifugation, the isopropanol in the tube was completely removed, leaving only the pellet in the tube, 75% ethanol was added into the tube at -20 °C and vortexed.

[0024] Then centrifuged at 7500 xg for 5 min at +4 °C.

[0025] After centrifugation, the ethanol in the tubes was completely removed and the pellet remaining in the tubes was left to dry.

[0026] The dried pellet was reconstituted with 20-50 pl RNAse / DNAse-free (nuclease free) water and incubated in a heat block at 55 °C for 10-15 min.

[0027] - Finally, the tubes were stored at - 80 °C for use in RT-PCR.

[0028] RT-PCR application:

[0029] The RT step was performed using a single stranded complementary cDNA synthesis kit (First stranded cDNA synthesis kit) (Thermo) according to the protocol reported by the company.

[0030] Total viral RNA of SARS-CoV-2 Ank 1 isolate extracted by Trizol method was used. 3 pl of viral RNA was added to 3 pl of molecular grade RNase / DNase free deionized distilled water and 0.5 pl of Random hexamer primer and kept in a thermal cycler at 70 °C for 5 minutes. The tubes were cooled at +4 °C for 1 minute.

[0031] To this mixture, 2 pl 5X RT buffer, 1 pl 10 mM dNTP mix and 0.5 pl reverse transcriptase (RT) enzyme were added. Complementary DNA (cDNA) was obtained by applying a thermal cycler program at 25 °C for 10 minutes, 37 °C for 1 hour and 70 °C for 5 minutes.

[0032] Taq DNA Polymerase, 10 X Reaction buffer, 25 mM MgCh, 10 mM dNTP mix, Forward and Reverse primers, RNAse / DNAse free molecular grade distilled water were used as master mix components. 3 pl of cDNA was added to the mixture (master mix) and reacted in a thermal cycler program at 96 °C for 6 minutes, 48 °C for 1 minute-72 °C for 2 minutes-94 °C for 1 minute (35 cycles), 50 °C for 1 minute and 72 °C for 10 minutes.

[0033] The reaction result was visualized on 1% agarose gel under ultraviolet light. The 228 bp PCR products obtained were subjected to sequence analysis and confirmed by comparison with the envelope gene region of SARS-CoV-2 virus sequences available in GenBank.

[0034] Cloning of viral envelope protein into transfer plasmid pVL1393 : The PCR product amplifying the envelope gene of SARS-CoV-2 Ank-1 and the stock plasmid (pVL93, BD Baculo Gold™ Transfection Kit, USA, Cat. No: 560129) were prepared by a commercial kit for the formation of compatible ends in the ligation step.

[0035] The PCR product and plasmid were cut with the help of restriction enzymes. Two restriction endonucleases, TG / i / HI and PstI, were used for this process. Restriction fragment length polymorphism (RFLP) analysis was performed using the amounts and conditions given in Table 1.

[0036] Table 1

[0037] Both ends of the products were brought together and ligation was performed. The information is given in table 2.

[0038] Table 2 Transformation:

[0039] 1. 100 pl of competent cells (DH 5 alpha) and all of the ligation product were incubated on ice for 30 min.

[0040] 2. After incubation, they were kept on ice at 42 °C for 2 min and 5 min consecutively. 3. The whole mixture was transferred into 900 pl of Luria Bertoni (LB) broth and incubated on a shaker at 37 °C, 250 rpm for 1 hour.

[0041] 4. After incubation, inoculation was performed by smear inoculation method on Luria Bertoni (LB) agar containing 0.1% ampicillin. It was incubated at 37 °C in an oven overnight.

[0042] 5. The next day, the petri dishes were checked for colony growth and colonies with growth were numbered and selected.

[0043] 6. Colony PCR was performed simultaneously during the selection process.

[0044] 7. The selected colonies were inoculated into Luria Bertoni (LB) broth containing 0.1% ampicillin and incubated in a shaker at 37 °C, 250 rpm for 1 night.

[0045] 8. After incubation, plasmid extraction (plasmid mini prep, Fermentas) was performed for each tube with growth.

[0046] 9. RT-PCR test using SARS-CoV-2 envelope gene primers designed to check for transformation was repeated for each tube found positive by colony PCR. Transformation was confirmed by sequence analysis of the PCR products.

[0047] 10. In addition, RFLP test was repeated on the purified plasmids for confirmation. A confirmed and transformed positive product was inoculated into 250 ml of LB broth containing 0.1% ampicillin to prepare maxiprep and incubated at 37 °C, 250 rpm on a shaker for 1 night.

[0048] 11. After growth was observed on the next day, plasmid extraction (Plasmid extraction maxiprep, Sigma) was performed and controlled on 1% agarose gel and then portioned and stored at -80 °C.

[0049] Cloning of SARS-CoV-2 envelope gene into an intermediate vector: The envelope (env) gene of SARS-CoV-2 Ank 1 isolate obtained within the scope of the previous work package was separated from the transfer plasmid in which it was cloned. For this process, restriction enzymes were used at the ends of the designed primers used in the production of this gene region.

[0050] Forward primer: 5'-GGGATCCGCTGATGAGTACGAACTTATG-3' amHI

[0051] Reverse primer: 5'-AACTGCAGGTTCGTTTAGACCAGAAGA-3' Pstl Bac-to-Bac baculovirus vector system (Invitrogen) was used for cloning of SARS- CoV-2 envelope gene into an intermediate vector. [2] This system is a system used for recombinant baculovirus creation, replication and gene expression. The system basically consists of cloning a gene of interest into a commercial recombinant plasmid and transferring it into specially prepared competent Escherichia coli cells, followed by amplification of the resulting baculovirus DNA in insect cell culture.

[0052] In the first step of the study, env gene DNA obtained from SARS-CoV-2 Ank 1 isolate was ligated to pJET (CloneJet PCR Cloning Kit - Thermo) vector at a ratio of 3 : 1 by volume. For this, 10 pl 2x ligation buffer, 1 pl DNA, 6 pl purified water and 1 pl DNA blunt end enzyme were combined and kept at 70 °C for 5 minutes. Thus, the pendulous end PCR product was converted into a blunt end and made available for cloning into the pJET vector. After cooling to complete the blunt end reaction, 1 pl T4 DNA ligase and 1 pl pJET vector were added to the tube. Thus, the mixture with a final volume of 20 pl was kept for 15 minutes at room temperature. After the holding process, transformation of the plasmid containing the ligated envelope gene was performed.

[0053] E. coli DHP cells and CaCh method were used for transformation of the ligation product. Plasmids were isolated from the colonies formed in the transformation petri dish. In order to determine whether the obtained plasmid DNAs contained the desired DNA region, the Bgl II enzyme, which has a sequence at the beginning and end of the multiple cloning site of this plasmid, was used. The presence of the expected 228 bp band for the envelope DNA after enzyme digestion confirmed that the SARS-CoV-2 env gene was cloned into the pJET vector. The number 1 of the confirmed plasmids was encoded as pJET-e / ?v and reserved for further study.

[0054] Cloning of SARS-CoV-2 envelope Gene into Expression System Vector: The isolated pJET-e / ?v and expression system vector pFastBac HTB plasmids were subjected to cutting for further study. For this, both vectors were cut separately with BamHUPstl enzyme pairs. The reaction mixture was prepared as follows and the mixture was kept at 37 °C for 2 hours. Cutting reaction:

[0055] 0,4 JJ.1 TG / i / HI (10 u / JJ.1)

[0056] 0,4 pl Vl (10 u / pl)

[0057] 2 JJ.1 Tango buffer (10X)

[0058] 12 JJ.1 Plasmid DNA (pFastBac HTB or pJET-env)

[0059] 5,2 pl H2O

[0060] Cutting products were electrophoresed in 1% agarose gel. The pFastBac HTB vector linearised by enzymes and the env region cut from the pJET-ew vector were excised from the agarose gel. The bands extracted from the gel were cleaned using DNA clean-up kit (Nucleospin, Cat. No: 11457).

[0061] The DNA of the env gene, which was cut with restriction enzymes and cleaned from the gel, and the pFastBacHTB vector, which was linearised by treatment with the same restriction enzymes, were ligated at a ratio of 3 : 1 (DNA:vector). The reaction was prepared as 2 pl 10X T4 DNA ligase enzyme buffer (Promega), 1 pl T4 DNA ligase enzyme (Promega), 2 pl pFastBacHTB vector, 6 pl DNA fragment and 9 pl dH2O and the reaction was kept at 16 °C overnight and ligation was performed.

[0062] The recombinant plasmid obtained was transformed into E. coli DH10P cells. The presence of recombinant plasmids (pFBHT-c / n’) in these cells was analysed and visualised by 1% agarose gel electrophoresis by restriction enzymes after plasmid DNA isolation. The number 1 of the confirmed plasmids was coded as pFBHT-ew and reserved for further study.

[0063] Transfer of SARS-CoV-2 env Gene into Bac-to-Bac® Baculovirus Vector System: In order to transfer the pFBHT-ew vector prepared in the previous steps into the baculovirus vector system, E. coli DHIOBac bacterial cells must be transformed. These cells are specialised cells and have a helper plasmid producing the enzyme required for transposition and Autographa californica nucleopolyhedrovirus (AcNPV) viral DNA (bacmid) with bacterial replication origin.

[0064] For transformation, competent cells were prepared using DHIOBac cells. Transformation was performed by adding 5 pl (1 ng) pFBHT-c / n’ to 100 pl of prepared DHIOBac competent cells. These cells were grown for 48 hours in medium containing gentamicin (for selection of pFastBacHTb donor plasmid containing the gene region), kanamycin (for bacmid selection), tetracycline (for selection of helper plasmid producing the enzyme required for transposition), IPTG and X-gal. Empty bacmids contain the LacZ gene, transposition takes place within this LacZ gene and the result is determined by the formation of white colonies of the transposed ones. After transformation, transposition between the pFBHT-c / n’ vector and bacmid DNA is required due to the homologous regions that both have. Recombinant bacmid DNA was isolated from the white colonies formed as a result of transformation. Whether the bacmid DNAs contained the env gene was analysed by PCR using the primer pair pUC / M13F - pUC / M13R. These primers give a 300 bp product in the case of empty bacmids. However, in the case of full bacmids, they give a total product of 2658 bp, including the env region of 228 bp in addition to the gentamicin gene (2430 bp), which can come from the pFastBac HT vector by transposition of the gene. The confirmed clone was selected, encoded as Bacmid- env and used in the transfection process.

[0065] Transfection of Recombinant Bacillus DNA into Insect Cells and Replication of Recombinant Virus: One day before, Spodoptera frugiperda (Sf9) insect cells were placed in 6-well cell culture dishes with 2xl06cells per well and kept at 28 °C for attachment to the bottom. For the preparation of the transfection mixture, 1 pg of recombinant bacmid DNA (~5pl) was mixed with 100 pl of unamended Grace's Insect medium (Gibco®) (Solution A).

[0066] On the other hand, 8 pl Cellfectin® II Reagent (Invitrogen) and 100 pl Grace's Insect medium without additives were mixed homogenously (Solution B).

[0067] Then these two solutions were mixed together in a tube and kept at room temperature for 30 minutes by overturning every 5 minutes. The medium was removed from the adherent cells and the cells were washed with 2 ml of undoped Grace's Insect medium. To the lipid-DNA mixture, 800 pl of unamended Grace's Insect medium was added, gently mixed and slowly added to the cells. Cells were incubated in this way for 5 hours in an oven at 28 °C. The transfection solution was removed from the cells and 2 ml of Sf-900™ II SFM (Gibco®) medium containing 5% Fetal Bovine Serum (FBS) was added, incubated at 28 °C for 72 hours and transfection was performed. After incubation, cells containing recombinant viruses were collected and centrifuged at 500xg for 5 minutes. The supernatant obtained was called viral stock. 9,0xl06cells left in T75 flasks were infected using 1 ml of viral stock. Cells were harvested 5 days after infection and the supernatant containing baculovirus harbouring recombinant baculovirus DNAs was stored at 4 °C for subsequent infections.

[0068] Production of Recombinant Env Protein in Cell Culture: In order to produce Env protein of SARS-CoV-2 env gene in cell culture, Sf9 cells grown in T25 cell culture flasks were firstly adapted to growth in suspension culture. For this purpose, Sf9 cells grown by clinging to the bottom in T25 flasks were suspended in 6 ml of growth medium. 5 ml of this suspension was added to 15 ml of medium in a 250 ml flask. The cells in the flask were grown in a shaker at 100 rpm for 3 days at 28 °C. At the end of the period, 20 ml of medium was added to the flask and grown for another 3 days. At the end of 6 days, cells were counted and infection was initiated in a final volume of 100 ml with a multiplicity of infection (MOI) of 2xl06cells / ml for Sf9 and multiplicity of infection (MOT) of 0.1 for Bacmid-e / ?v. Infection was continued in the shaker for 3 days. At the end of the period, the medium in which the cells were present was also precipitated at 4 °C for 5 minutes at 500xg. The pellet of cells was used for protein purification or stored at -80 °C for further use.

[0069] Purification of the Produced Protein: Isolation and purification of the obtained cell pellet was performed to remove other proteins. For this, a commercial protein isolation kit (Promega, Cat. No: V8500) compatible with the purification of recombinant proteins produced with a tail consisting of histidine (His) amino acids as a feature of the expression system was used. The kit procedure was followed for isolation. The pure protein was stored at -80 °C until use.

[0070] Determination of Protein Concentration: Protein concentration was determined according to the method developed by Bradford. [3] Bovine serum albumin (BSA) was used as a standard in the calibration curve. For the calibration curve, solutions containing 0, 5, 10, 25, 50, 50, 100, 150, 200, 250, 300 pg BSA were prepared by adding dlfcO to 500 pl. Ten pl each of these solutions and the samples whose concentrations were to be determined were taken and 200 pl of the prepared dye solution (Protein Reagent, Sigma) was added and vortexed. The prepared standards and samples were transferred to 96-well dishes with 3 replicates. After 15 min incubation time, measurements were performed at 595 nm using SpectraMax M2 (Molecular Devices). The concentration of Env protein was determined as 433 pg / ml.

[0071] Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE): SDS-PAGE analysis was performed to observe protein expression. Each sample was prepared to contain equal amounts of protein and the samples were kept at 95 °C for 5 minutes after adding treatment buffer (60 mM Tris Hydrochloride (Tris- HC1) (pH 6.8), 25% Glycerol, 2% SDS, 5% P-mercaptoethanol, 0.1% bromophenol blue). The samples were then loaded onto a 10% SDS-PAGE gel as described by Laemmli (1970). Separation was performed by applying a constant current of 80 Volts to the gel. [4]

[0072] Protein Transfer to Membrane and Western Blot Analysis:

[0073] Transfer of the prepared SDS-PAGE gel to the membrane for Western Blot analysis was performed using a semi-dry blotting device (Bio-Rad). For this, the membrane and Whatman papers were cut to the size of the gel. Membranes and papers were wetted in blotting buffer (48 mM Tris, 39 mM Glycine, 0.0375% w / v SDS) and placed in the device as 2 whatman papers, membrane, gel and 2 whatman papers respectively. The device was operated at 0.04 Amperes for 1 hour and thus blotting was performed. The membrane was then washed in washing buffer (0.5% milk powder and 0.05% Tween20 in PBS) for 2 minutes and blocked in PBS-T (IX PBS, 0.05% Tween20) containing 5% milk powder also in PBS for 1 hour at room temperature. Subsequently, the membrane was washed 3 times for 10 minutes each with washing buffer. The membrane was then incubated for 1 night in the washing solution containing Anti-poly Histidine antibody (1 : 1000 dilution) (Sigma, H- 1029) at room temperature with gentle shaking and washed again 3 times for 10 minutes. Then incubated in wash buffer containing Anti-Mouse IgG (Alkaline Phosphatase antibody (1 : 1000 dilution) (Sigma, A-3562) for 1 hour at room temperature with gentle shaking. The membrane was then washed 3 times for 10 min each in wash buffer and 2 times for 10 min each in AP buffer (0.1 M Tris-HCl, 0.1 M NaCl, 5 mM MgCh, pH 9.5, IX). Finally, the membrane was stained in 10 ml AP buffer containing 100 pl nitroblue tetrazolium and bromochi oroindolyl phosphate (NBT / BCIP stock solution, Roche, 11681451001). The reaction was stopped by washing the membrane copiously with dH2O after detection of protein bands. The membrane was dried and scanned and computerised.

[0074] As a result, a total protein band of ~14 kDa with His amino acid tail (3 kDa) was identified on the membrane.

[0075] Antibody screening in experimental animal serum samples by ELISA:

[0076] In order to investigate the specific antibody response in mouse sera vaccinated with the protein developed within the scope of the invention and in the control group, ELISA was performed with the following method steps.

[0077] 1. 96-well ELISA plates with detachable strips and high protein coverage were coated with recombinant protein and incubated overnight at +4°C.

[0078] 2. 5-10 pg / eye of the inventive SARS-CoV-2 recombinant protein was reconstituted with 1XPBS and 100 pl was added to each eye.

[0079] 3. After incubation, 100 pl of washing solution was added to each eye and washed once. Blocking solution prepared with 100 pl BSA was added to each eye.

[0080] 4. Pellets were incubated for 1 hour at room temperature. After incubation, the eyes were washed 2 times with washing solution.

[0081] 5. After washing, serum samples reconstituted in 8 vertical rows of 1 / 50 initial dilution and 1 / 6400 final dilution in a clean pellet were added to 100 pl in each well of the ELISA pellet.

[0082] 6. Pellets were incubated for 2 hours at room temperature. After incubation, the eyes were washed 3 times with washing solution. 7. After washing, 100 pl of anti-mouse IgG conjugate diluted 1 / 10000 with IX PBS was added to each eye. The pellets were incubated for 1 hour at room temperature.

[0083] 8. After incubation, the eyes were washed 3 times with washing solution. 3, 3', 5,5'- Tetramethylbenzidine (TMB) substrate was added 100 pl to each eye.

[0084] 9. After incubation at room temperature for 20 min, 100 pl of stop solution was added to each pellet and the results were read at 450 nm on an ELISA reader (Table 3).

[0085] Table 3

[0086] Detection of virus-specific neutralising antibody response:

[0087] In order to test the presence of neutralising antibodies in the blood sera obtained from experimental animals, virus neutralisation test was performed by the following method. 1. Logs based dilutions of serum samples inactivated at 56 °C for 30 min were prepared.

[0088] 2. 80 pl of serum-free DMEM (Dulbecco's Minimal Essential Medium) medium was added to the first row of sterile 96-well cell culture tablets and 50 pl of serum-free DMEM (Dulbecco's Minimal Essential Medium) medium was added to the cells to be reconstituted.

[0089] 3. Using 2 wells for each serum, 20 pl of serum to be tested was added to the first row of wells. Serial dilutions up to 1 / 320 were made with a multichannel pipette.

[0090] 4. An equal volume (50 pl) of virus suspension diluted at lOODKIDso was added to the diluted serum samples.

[0091] 5. At the same time, virus control (using the back titration method) and cell control wells were prepared.

[0092] 6. The culture tablets were then incubated in an oven containing 5% CO2 at 37°C for 1 hour.

[0093] 7. At the end of the period, Vero cell suspension was added to each well at 300.000 cells / ml and the tablets were incubated in an oven at 37°C with 5% CO2.

[0094] 8. Tablets were controlled by inverted microscope for 3-5 days. When the result of back titration was evaluated as cytopathic effect (cpe) in four of four eyes in DKID50 dilution, the test was terminated, SN50 amounts in sera were determined. (Table 4)

[0095] According to these results, it was determined that the SARS-CoV-2 envelope recombinant protein produced by using baculovirus expression system in Ifnar^^ mice stimulated the immune response and induced neutralising antibody formation at rates ranging from 1 / 60-1 / 120.

[0096] Table 4

[0097] The immune response was also checked using different adjuvants (Alum, CpG, Alum+CpG) (Table 5). The highest antibody response was detected at 1 / 6400 dilution in animals in the group using complete / incomplete adjuvant, while positivity was detected at 1 / 1600 to 1 / 3200 dilutions in other adjuvant groups.

[0098] Table 5

[0099] Virus Neutralisation Test:

[0100] The control study of humoral immune response in Ifnar(' / _)mice was determined by virus neutralisation test (VNT) and the results are given in table 6.

[0101] Table 6 Neutralising antibody positivity was in parallel with ELISA results. Among the experimental groups, SNso values were determined at 1 / 60 dilution in the animals in the complete / incomplete adjuvant group, while SNso antibody titres were detected at varying rates at dilutions of 1 / 20 to 1 / 120 in the other adjuvant groups. In the negative control groups that received only adjuvant, no neutralising antibody response was detected as expected.

[0102] References:

[0103] [1] Alireza Hanifehnezhad, Ebru §ahin Kehribar, Sidika Oztop, Ali Sheraz, Serkan Kasirga, Koray Ergiinay, Sevgen Onder, Erkan Yilmaz, Doruk Engin, T. igdem Oguzoglu, Urartu Ozgiir §afak §eker, Engin Yilmaz, Aykut Ozkul. Characterization of local SARS-CoV-2 isolates and pathogenicity in IFNAR" / _mice. Heliyon, Volume 6, Issue 9, September 2020, e05116.

[0104] [2] Anderson, D., Harris, R., Polayes, D., Ciccarone, V., Donahue, R., Gerard, G., and Jessee, J. 1996. “Rapid Generation of Recombinant Baculoviruses and Expression of Foreign Genes Using the Bac-ToBac® Baculovirus Expression System”. Focus 17, 53-58.

[0105] [3] Bradford, M.M. 1976. “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.” Analytical biochemistry, 72 (1-2), 248-254.

[0106] [4] Laemmli, U.K. 1970. “Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4.” Nature, 227, 680-685.

Claims

CLAIMS1. A SARS Coronavirus-2 (CoV-2) envelope protein produced in a baculovirus expression system, characterised in that the protein structure is as indicated in seq. ID no:

1.

2. A SARS Coronavirus-2 (CoV-2) envelope protein according to claim 1, characterised in that the Forward primer structure in the envelope gene region is 5'-GGGATCCGCTGATGAGTACGAACTTATG-3'.

3. A SARS Coronavirus-2 (CoV-2) envelope protein according to in claim 1, which is related to the envelope protein and is characterised in that a Reverse primer structure of 5'-AACTGCAGGTTCGTTTTAGACCAGAAGA-3' in the envelope gene region.

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