Using the “RNA-Dependent RNA Polymerase Molecular Clock”, it was estimated that the common ancestor of coronavirus (CoV) appeared about 10,000 years ago. ^{2 , 3} The first human upper respiratory tract infection (URTI) caused by human CoV (H-CoV) was reported in 1965. ^{4 - 7} The first severe acute respiratory syndrome caused by CoV (SARS-CoV) was reported from Guangdong, China, in 2002, that ultimately spread over many countries causing an epidemic in the Americas, Europe, and Asia, infecting over 8,098 people and killing about 774 of the infected. ^{6 , 8} SARS-CoV has 99.6% genome sequence homology to CoV found in masked palm civets ( Paguma larvata) and 88% – 95% homology to CoV found in several horseshoe bats, Rhinolophus pussilius, R. macrotis, R. pearsoni and R. sinicus. ^{9- 11}

This was followed by another outbreak of a CoV epidemic in 2012 that started in Saudi Arabia, which is known as Middle East respiratory syndrome (MERS) and is caused by MERS-CoV. ^{12 , 13} It spread over 27 countries, reaching Western Africa to the west and South Korea to the east, infecting over 2,400 and killing over 850 people. ^{12 , 13} The survivors suffered from many diseases including heart, kidney, and multiorgan failures. ^{12 , 14 , 15} In November 2019, another CoV epidemic emerged in Wuhan, China, found to be caused by SARS-CoV-2 infections, leading to a global pandemic. That epidemic infected over 179 million people and killed over 3.8 million as of June 2021. ¹⁶

Coronavirus infections are not a new challenge to human survival. Some of those challenges, we know, others are unknown to us. However, it is clear that the recent pandemic of 2019 will not be the last, and we have to be alert and keep us ready to be safe for the future.

All types of viruses mutate and evolve as they replicate. Their prolonged presence in an uncontrolled environment favors development of new variants. That ability to generate de novo diversity in a short period of time, as well as the rate of spontaneous mutation, vary among viruses. Furthermore, mutation rates in RNA viruses are higher than DNA viruses, and are higher in single-stranded viruses than double-stranded viruses. ¹⁷

Hence, vaccinating a smaller segment of a population against SARS-CoV-2 may favor generation of new variants with new infectivity. In that scenario, even the vaccinated individuals would face risks from arrivals of new variants. This may only be brought under control by administering a safe and effective vaccine as soon as possible to a significantly large part of the population. Successes of such efforts will reduce the development of new variants and, thus, help the global human population attain herd immunity. ¹⁸

As cases of coronavirus disease 2019 (COVID-19) were growing globally, it brought together the efforts of worldwide biotechnologists, scientists, experts, pharmaceuticals, and investors to develop effective vaccines against SARS-CoV-2 as soon as possible. More than 50 such vaccine candidates were put into human trials in 2020, and a total of 250 vaccine candidates were in the process of being developed. ¹⁹ The safety and effectiveness of a vaccine are measured by the vaccine’s long-term antigenicity and immunogenicity for its therapeutic application as a vaccine. However, we still do not have direct evidence of the long-term efficacy of the short life mRNA and long-life cDNA antigenic vaccines.

SARS-CoV-2 mutates at a rate four times slower than the influenza virus, and the new variants have a minimal effect on antigenicity of the virus. ^{20 , 21} Although the coronaviruses mutate at a slower rate, some of the new variants of SARS-CoV-2, such as D 614 G, that mutated outside the receptor-binding domain (RBD) residue have a higher rate of infectivity by boosting viral replication in lung and respiratory tract tissues. ^{22 , 23} This suggests that the RBD residue is more conserved and, hence, the RBD protein vaccines will be effective against multiple SARS-CoV-2 variants.

The RBD is a small segment of the spike protein (S-protein) located on the outer membrane of the SARS-CoV-2 virion. It plays a critical role in binding the virus to the angiotensin-converting enzyme 2 (ACE-2) receptors on human mucosal cells, causing infection leading to COVID-19. Hence, an antibody generated against the RBD protein will be able to strongly prevent any SARS-CoV-2 infection.

According to Huang, et al., ²⁴ the S-protein located on the SARS-CoV-2 virus envelope is composed of 1273 amino acids (aa). The S-protein consists of a 13 aa long signal peptide (1–13 residues), followed by a 672 aa long S1 domain (14-685 residues), and a 588 aa long S2 domain (686–1273 residues). The S1 domain contains a 292 aa long N—terminal domain (14–305 residues) and a 223 aa long RBD domain (319–541 residues). The SARS-CoV-2 encapsulating membrane holds many trimeric S-proteins, each containing three RBD protein monomers, that binds to the ACE-2 receptors present in human cells. ²⁵ Furthermore, Yang, et al., ²⁶ also demonstrated that the S1 domain of the SARS-CoV-2 spike protein directly binds to ACE-2 receptors expressed by the undifferentiated human alveolar cells (A549) facilitating the entry of SARS-CoV-2 into these cells. It was also reported that the SARS-CoV-2 receptor-blocking human antibody, HA001, attaches to amino acid residues A475 and F486 in the RBD of the SARS-CoV-2 spike protein. ²⁷ In this process, a 71 aa long segment of the RBD, known as receptor binding motif (RBM), tightly binds to the ACE-2 receptor. ²⁸ Furthermore, the RBD contains nine cysteine (Cys) residues making four pairs and keeping one free Cys. The three pairs are Cys336–Cys361, Cys379–Cys432, and Cys391–Cys525 residues that form the core RBD β sheet related to its 3D structure. The remaining pair, Cys480–Cys488 residue, binds to the N-terminal peptidase domain of ACE-2. ²⁹ All the above information supports that the RBD protein will be a highly effective antigen for vaccine production against SARS-CoV-2.

We propose to reconstruct an amp ^r plasmid expression vector carrying RBD and superfolder green fluorescent protein (sfGFP) cDNAs linked by an oligo DNA, coding for a fusion peptide (FP), Asp-Asp-Asp-Asp-Lys. ¹ The construct will be expressed using Escherichia coli, C2566H, producing the RBD-FP-sfGFP fusion protein. The RBD protein will be separated from the RBD-FP-sfGFP fusion protein by digestion with an enterokinase (specific for the FP) and isolated by hydrophobic interaction chromatography (HIC). The RBD eluate will be analyzed to determine its size by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), tested for its immuno-reactivity with SARS-CoV-2 S-protein antibody using a BioVision ELISA kit, and quantified by spectrophotometry. The purified RBD protein will be available to study its efficacy following approved vaccine formulation and clinical trials.

The following specialized reagents and materials (from the same or alternate sources) will be required in addition to regular materials and reagents available in a running biotechnology laboratory. 1.

RBD cDNA: Addgene plasmid, Cat #141184, pcDNA3-SARS-CoV-2-S-RBD-sfGFP (Amp ^R, Neo ^R/Kan ^R).

Bellow, we provide a unique study protocol for the production of SARS-CoV-2 RBD protein antigen using recombinant DNA technology. The RBD protein thus produced can be used as a COVID-19 vaccine after formulation, and evaluation for clinical efficacy and safety.

A sample of Escherichia coli from a stock carrying the Addgene plasmid, Cat #141184, will be grown overnight at 37°C in 10 ml LB-ampicillin broth. The plasmid DNA will be isolated from the cells using PureYield Plasmid Miniprep System I (Promega, Cat #A1222 and a Technical Bulletin #TB374). The purity of the plasmid DNA will be determined by the UV OD _260nm/OD _280nm (OD = optical density) absorption ratio. A ratio of ≥1.8 is generally accepted as a value for “pure” DNA. ³⁰ If the ratio is <1.8, we will add 0.1 μl (0.8 units) Proteinase K to the sample, incubate it at room temperature for 15 minutes, add two volumes of ice-cold 95% ethanol, mix well, centrifuge at 10,000 g at 4°C for five minutes, pour off the supernatant, bring the DNA into solution in TE, and determine the purity of the DNA following its OD _{2 60nm}/OD _280nm absorption ratio. The plasmid DNA concentration will be equal to, OD _260nm × the dilution factor × 50 = μg plasmid DNA/ml.

Linearizing the plasmid . The plasmid in solution will be digested with BamHI in BamHI buffer at 37°C for 2 hours, heat denatured at 65°C for one minute, and chilled at 4°C for 10 minutes. Then, two volumes of ice-cold 95% ethanol will be added into it, mixed well, chilled for 10 minutes at –20°C, and centrifuged at 10,000 g using a Sorvall SS34 Fixed Angle Rotor, at 4°C for five minutes. The supernatant containing the linearized plasmid will be transferred into a fresh microfuge tube. The linearized plasmid will carry the RBD cDNA at one end and the sfGFP cDNA at the other end, as shown in Figure 2.

Ligating covalently the linker oligo DNA (FP-DNA) into the linearized plasmid . We will add 1 μl of the linker oligo to the linearized plasmid in the microfuge tube. Then, we will add 1 μl T4 DNA (NEB, M0202) ligase containing 400 – 500 units in 1 × T4 DNA ligase buffer, incubate at 16°C for 2 hours, deactivate the ligase by heating at 65°C for 10 minutes, chill at 4°C for five minutes, add two volumes of ice-cold 95% ethanol, mix well, and centrifuge at 10,000 g at 4°C for five minutes. The supernatant containing the ligated plasmid will be transferred into a fresh microfuge tube and the purity of the plasmid DNA will be determined following UV absorption ratio at OD _260nm/OD _280nm reaching ≥ 1.8. If the ratio is <1.8, add 0.1 μl (0.8 units) Proteinase K, incubate at room temperature for 15 minutes, add two volumes of ice-cold 95% ethanol, mix well, centrifuge at 10,000 g at 4°C for five minutes, pour off the supernatant, bring the DNA into solution in TE, and then again determine the purity of the DNA following its OD _260nm/OD _280nm absorption ratio. The plasmid DNA concentration will be equal to: OD _260nm × the dilution factor × 50 in μg plasmid DNA/ml.

The incorporation of the oligo DNA ( Figure 1), into the plasmid (Addgene, 141184) will be accomplished following a standard protocol for plasmid linearization, insertion of the oligo DNA, and ligation using T4 DNA ligase. The insertion of the oligo DNA will take place at the BamHI site located terminally at the RBD cDNA, and 21 bp upstream to the sfGFP cDNA as in Figure 3.

The oligo DNA insert will code for a heptapeptide, DDDDKRS, fused in-between the RBD-sfGFP fusion protein, coding for a novel RBD-FP-sfGFP fusion protein ( Figure 4).

The original plasmid carries RBD cDNA-sfGFP cDNA is shown in Figure 5A, and the ligated plasmid reconstruct will carry the RBD cDNA-FP DNA-sfGFP cDNA linked in order within one orf, as shown in Figure 5B.

The Escherichia coli, C2566H, carrying T7 RNA polymerase gene will be used as a host. The host, transformed with the recombined plasmid, will express the orf producing RBD-FP-sfGFP fusion protein.

Transformation. We will thaw a sample of competent Escherichia coli, C2566H, cells for 10 minutes in ice at 4°C, mix well gently, pipette out 50 μl of the cells in suspension into an ice-cold fresh 1.5 ml microfuge tube in ice, add 100 pg reconstructed plasmid DNA, mix well gently without vortexing, chill the microfuge tube in ice for 30 minutes, place the microfuge tube into a Styrofoam holder, transfer the holder into a 42°C water bath, wait for 15 seconds, take out the microfuge tube, and chill it in ice for five minutes without mixing.

Then, we will add 950 μl SOC, ³¹ maintained at room temperature, into the microfuge tube containing the transformed cells, incubate at 37°C for 60 minutes, and shake vigorously while under incubation using a rotator. This will complete the transformation process.

Plating the transformed Escherichia coli . We will warm up prepared petri dishes/plates (100 mm × 15 mm) containing 20 ml LB-Amp-Agar (1.5%) at room temperature for 15 minutes. Using a sterile pipette, we will add aseptically 250 μl SOC medium containing the transformed E. coli cells into the petri dish and add 80 μl filter sterilized 100 mM IPTG solution into the dish over the SOC medium. Then, we will spread the SOC medium with the transformed cells evenly over the agar throughout the dish/plate with a sterilized spreader and incubate it overnight at 37°C. The E. coli cells transformed with the reconstructed plasmid will produce green fluorescent cfu, when observed using a 300 nm UV lamp.

Growing cells from green fluorescent cfu to produce the RBD-FP-sfGFP fusion protein. We will select a 15 ml (16 mm × 125 mm) microbial culture tube containing 5 ml sterile LB-Amp broth, warm it up at 37°C for five minutes and add 16 μl filter sterilized 100 mM IPTG solution into the tube reaching a concentration of 0.4 mM IPTG. Then, we will select a green fluorescent cfu from the culture plate, add the cells into the tube, mix gently and grow the cells overnight at 37°C.

Transcription of the SARS-CoV-2 RBD-FP-sfGFP containing ORF from the recombined plasmid will be stimulated by the T7 promoter and regulated by the T7 RNA polymerase produced by E. coli, C2566H, that carries a genomic copy of the T7 RNA polymerase gene inducible by IPTG. Hence, the transformed E. coli, C2566H, will produce SARS-CoV-2 RBD-FP-sfGFP fusion protein from the reconstructed plasmid. ^{32 - 34}

Similar to the RBD-sfGFP fusion protein, the RBD-FP-sfGFP fusion protein will retain its green fluorescence at UV 300 nm. This is supported by the fact that the RBD-sfGFP fusion protein produced by the Addgene plasmid, 141184, expresses green fluorescence in E. coli carrying T7 RNA polymerase. ^{35 - 37}

A comparison of the recombinant RBD protein with the Addgene, 141184, RBD protein sequences are presented in the results section.

Pipette out 1.5 ml cell culture from the tube into a 1.5 ml microfuge tube, centrifuge it at 5,000 g for 10 minutes at 4°C, pour off the supernatant, add 250 μl 1X TE buffer into the cell pellet, gently resuspend the cells in the pellet by pipetting up and down the buffer along with the cell pellet, add 50 μl lysozyme solution (Millipore-Sigma, Cat # L3790) reaching a final concentration of 0.2 mg lysozyme/ml, mix well, and incubate in a shaker at room temperature for 15 minutes. This will break open the cells and release the RBD-FP-sfGFP fusion protein into the buffer, centrifuge the solution at 2000 g for 10 minutes at 4°C, collect the supernatant containing the RBD-FP-sfGFP fusion protein, and observe the solution using UV 300 nm. The presence of RBD-FP-sfGFP fusion protein in solution will be indicated by a fluorescent green colour in UV 300 nm. Hold the tube containing the extract at 4°C for further use.

The RBD-FP-sfGFP fusion protein will be separated by HIC (hydrophobic interaction chromatography) using a BIO-RAD protein extraction kit (Cat #166-0005EDU), https://www.bio-rad.com/webroot/web/pdf/lse/literature/4006099.pdf, using the following steps:

1.

Using a pair of scissors, cut off the bottom of the hydrophobic resin prefilled HIC column.

Separation of the RBD protein from the RBD-FP-sfGFP fusion protein after the enterokinase (NEB Cat #P8070) digestion of the FP will be completed by HIC. This is a routine technique to isolate non-hydrophobic proteins from hydrophobic proteins. In this digestion, the enterokinase will digest the DDDDKRS fusion peptide in between DDDDK and RS, leaving DDDDK fused with the RBD protein at its C-terminal. The remaining RS dipeptide will remain fused with the GGSGSG, forming RSGGSGSG. This residue will remain fused with the sfGFP at its N-terminal. Since the sfGFP present in the RBD-FP-sfGFP fusion protein is strongly hydrophobic, the RBD-FP-sfGFP fusion protein will bind to the resins in the HIC column. Once the enterokinase completes the digestion, the RBD protein will become separated from the sfGFP. The separated RBD protein will be eluted by 1.3 M (NH ₄) ₂SO ₄ buffer from the hydrophobic resins in the column, leaving the sfGFP protein bound to the resins ( Figure 6).

The RBD protein eluate from the hydrophobic column will contain approximately 1.3 M (NH ₄) ₂SO ₄ and stray molecules of enterokinase. The enterokinase molecules will be removed by using an enterokinase removal kit (Sigma-Aldrich, Cat # PRKE) followed by the removal of (NH ₄) ₂SO ₄ as follows: 1.

Add 50 μl anti-enterokinase–agarose conjugate pellet (following Sigma-Aldrich, PRKE protocol) to the RBD eluate, mix gently; centrifuge at 1000 g for 2 minutes at 4°C; collect the supernatant containing RBD and <1.3 M (NH ₄) ₂SO ₄. Add v/v Tris-buffer containing 20 mM Tris, 200 mM NaCl, pH 8.0 to the RBD eluate.

Removal of (NH ₄) ₂SO ₄ from a protein extract using dialysis is a routine procedure. ^{40– 42} Berndt, et al., ⁴³ ligated cDNAs of an RBD protein of SARS-CoV-2 with a cDNA coding for a 5’mClover green fluorescent protein gene, which was expressed by a transformed Chlamydomonas reinhardtii. The RBD-mClover fusion protein, expressed by C. reinhardtii, was separated by HIC. ⁴³ The eluate RBD molecules retained its full immunogenic activity, as observed by its ability to bind to ACE-2 receptor proteins. ⁴³ This supports that (NH ₄) ₂SO ₄ does not affect the structural integrity of the RBD proteins. Furthermore, (NH ₄) ₂SO ₄ is known to stabilize the 3D structure of proteins. ⁴⁴ Park, et al., ⁴⁵ isolated recombinant colorectal cancer vaccine protein, GA733-FcK, using 50% (5.05M) (NH ₄) ₂SO ₄ in its active form. Hence, we predict 1.3 M (NH ₄) ₂SO ₄ solution used in this protocol will have no impact on the 3D structure of the RBD protein.

Tan, et al., ⁴² dialyzed RBD-SpyVLP eluate for 16 hours in Tris-buffered saline (TBS). In our protocol, we propose to use a Tris-buffer (20 mM Tris, 200 mM NaCl, pH 8.0) to the RBD eluate, v/v, and put it into a SnakeSkin dialysis tube (Thermo Fisher Scientific, Cat # 88243) with a 10 kDa cut-off, following Tai, et al. ³⁹ Since the molecular weight of RBD protein monomer is 25 kDa, the porosity of the SnakeSkin dialysis tube will save the RBD protein inside the tube while allowing (NH ₄) ₂SO ₄ to leach out. Upon completion of the dialysis, storing RBD protein in Tris-buffer, pH 8.0, at 4°C will save the RBD protein from bacterial and enzymatic degradation.

We will be using SDS-PAGE NuPAGE 4-12% gradient gel for the electrophoresis as follows: 1.

Place the NuPAGE into a Mini Gel Tank, Thermo Fisher Scientific, Cat # A25977.

The SDS-PAGE procedure separates proteins primarily by mass, since SDS denatures and binds to proteins to make them negatively charged. Hence, in an electric field, the SDS-bound RBD proteins will migrate through the gel toward the positively charged electrode based on its mass. A protein molecule of a lower mass size will have higher mobility in comparison to a protein molecule of higher mass size.

This procedure will help us determine the molecular sizes of the RBD proteins in the eluate and compare them with known values.

We will test 1 μl sample of the dialyzed RBD protein for immunoactivity using a SARS-CoV-2 RBD Elisa kit, BioVision, Cat # E4877, for a qualitative determination of the RBD protein, following the supplier’s protocol.

This procedure follows the ELISA principle. It contains SARS-CoV-2 RBD protein samples in solutions, detection solutions, pre-coated RBD antibodies, and all necessary ingredients. This technique is known to be highly sensitive, detecting <10 pg RBD/ml.

In this protocol, a standard RBD concentration graph will be plotted using OD _{450 nm} of RBD samples of known concentrations. This will be accomplished by a tagged RBD-antibody-RBD-antigen binding, followed by a chromogenic reaction. An RBD sample from the dialyzed eluate will be tested using the same RBD-antibody-RBD-antigen binding followed by the chromogenic reaction and OD _{450 nm} measurement. Based on the OD _{450 nm} of the dialyzed RBD protein sample, its concentration will be determined from the standard graph. This procedure will help vaccine development in two ways: it will detect the presence of RBD protein in the HIC eluate that is purified by dialysis, and it will measure the concentration of RBD protein antigen present in the dialyzed sample.

The modification of the RBD protein will not affect its immunogenic ability as demonstrated by Keng, et al. ⁴⁷ Keng, et al., ⁴⁷ in an antibody neutralization experiment demonstrated that fragmented spike protein DNA containing variable lengths of RBD proteins, expressed by transformed E. coli, retained the immunogenic ability against SARS-CoV-2 virus. Furthermore, this modification will not affect the internally located RBM, the epitope of the RBD protein. ²⁹ The RBD antigen thus produced can be applied as a safer vaccine after formulation for trials as described by Batty, et al. ⁴⁸

We will take a 10 μl sample of the dialyzed RBD protein and determine its concentration by measuring OD at UV 280 _nm using a UV-Vis spectrophotometer following Arbeitman, et al. ⁴⁹ The total amount of RBD protein in the dialyzed sample will be equal to M = (OD ÷ L) x D. (Where, M = amount of RBD protein present in mg per ml in the original sample; OD = Optical density at 280 _nm; L = the length of cuvette light path in cm; D = dilution factor.) ^{50 , 51}

The RBD protein sequences, before ( Figure 7) and after modification ( Figure 8) will be tested for their alignments with known 2019-nCoV RBD protein sequence available through a computerized program, UniProt Protein Blast (UniProtKB: P00750): https://www.uniprot.org/blast/.

The details of the alignment are presented in the results section following Figure 9 and Figure 10, below:

Add a measured volume of glycerol to the dialyzed RBD solution, reaching a final concentration of 10% (v/v) glycerol in the solution and mix well. Make aliquots of the RBD solution in separate pre-laveled micro-vials, freeze them quickly in liquid nitrogen, and store them in a –80°C freezer following Tee, et al. ⁴⁶

The aliquots of RBD can be reused for vaccine formulations as needed. Edwards, et al., ⁵² have observed under electron microscopy that RBD molecules stored at 22°C or 37°C in a buffer (2 mM Tris, pH8.0, 200 mM NaCl, 0.02% sodium azide) for one week displayed well-ordered trimeric structure. ⁵² Hence, we anticipate that the quick-frozen RBD monomers, isolated in the protocol, will form dimers and trimers after thawing at 22°C or 37°C, as a natural phenomenon. Since both the RBD dimers and trimers have higher ACE-2 binding activity than its monomers, the dimerization and trimerization will increase vaccination efficacy of the isolated RBD proteins. ^{53 , 54}

The original plasmid, Addgene, 141184, carries a 639 bp long RBD cDNA linked with a 714 bp long sfGFP cDNA ( Figure 5A). The RBD cDNA will code for a 221 amino acid long RBD protein as shown in Figure 7.

The modified RBD protein encoded by the recombined plasmid is shown in Figure 8. As mentioned earlier, the modified RBD protein will have a five amino acid long FP (DDDDK) replacing six amino acids (GGSGSG) at its C-terminal ( Figure 8).

The RBD protein β sheet, critical for its 3D structure, will also remain fully stable since the modification of the RBD protein at the C-terminal has no effect on the pairing of the sulfur containing amino acid Cys336–Cys361, Cys379–Cys432, and Cys391–Cys525. ²⁹ We proved this by aligning the RBD protein sequences, before and after modification, with the RBD sequence produced by the 2019-nCoV genome using a computerized program, UniProt Protein Blast (UniProtKB: P00750), https://www.uniprot.org/blast/as), as shown in Figure 9 and Figure 10.

As shown in Figure 9, the RBD protein encoded by the RBD cDNA, Addgene, 141183, matches perfectly with the respective RBD protein sequence produced by 2019-nCoV strain of the SARS-CoV-2 coronavirus. Three pairs of Cys-Cys residues, Cys336-Cys361, Cys379-Cys432 and Cys391-Cys525, encoded by the RBD cDNA, responsible for the core RBD protein β sheet formation, match perfectly with the respective RBD protein sequence produced by the 2019-nCoV strain. The total amino acid length of the RBD protein encoded by the RBD cDNA, Addgene, 141183, is 221, which has 99.0% identity match and 99.5% positivity match with the RBD protein sequence produced by the 2019-nCoV ( Figure 9).

Similarly, as shown in Figure 10, the RBD protein encoded by the modified RBD cDNA matches perfectly with the respective RBD protein sequence generated by 2019-nCoV strain of the SARS-CoV-2 coronavirus. Three pairs of Cys-Cys residues, Cys336-Cys361, Cys379-Cys432 and Cys391-Cys525, encored by the modified RBD cDNA, that form the core RBD protein β sheet, match perfectly with the respective RBD protein sequence produced by the 2019-nCoV strain. The total amino acid length of RBD protein encoded by the modified RBD cDNA is 220, which has 98.5% identity match and 99.5% positivity match with the RBD protein sequence produced by the 2019-nCoV.