阅读说明：本技术 能够提高与SARS-CoV-2 S蛋白亲和力的ACE2突变组合及应用 (ACE2 mutant combination capable of improving affinity with SARS-CoV-2S protein and application thereof ) 是由 逯光文 叶飞 林曦 陈自敏 杨凡力 于 2021-09-16 设计创作，主要内容包括：能够提高与SARS-CoV-2S蛋白亲和力的ACE2突变组合及应用,所述能够提高与SARS-CoV-2S蛋白亲和力的ACE2突变具体的ACE2突变位点为,氨基酸序列第19位S突变为W,第27位T突变为W,第330位N突变为Y。(The ACE2 mutation combination capable of improving the affinity with SARS-CoV-2S protein and the application thereof, wherein the ACE2 mutation specific ACE2 mutation site capable of improving the affinity with SARS-CoV-2S protein is that the amino acid sequence 19 th site S mutation is W, the 27 th site T mutation is W, and the 330 th site N mutation is Y.)

1.ACE2 mutation capable of improving affinity with SARS-CoV-2S protein

The method is characterized in that specific ACE2 mutation sites are that the 19 th S mutation of an amino acid sequence is W, the 27 th T mutation is W, and the 330 th N mutation is Y.

2. The combination of any two or more of ACE2 mutation sites capable of improving affinity to SARS-CoV-2S protein according to claim 1.

3. ACE2 protein mutated at the positions according to claim 1, wherein the protein is ACE2/S19W, ACE2/T27W, ACE 2/N330Y.

4. The ACE2 protein based on the combination of site mutations of claim 2, wherein the protein is: ACE2[ W19/Y330], ACE2[ W27/Y330], ACE2[ W19/W27/Y330] proteins.

5. Use of the mutein according to any of claims 2 to 4 as a therapeutic agent for SARS-CoV-2.

6. The use of claim 5, wherein the mutein is capable of treating an existing strain of SARS-CoV-2 as a therapeutic agent for SARS-CoV-2 as well as partially mutated strains.

7. The use of claim 6, wherein the variant strain is: (ii) S-RBD variant strains capable of eliciting an existing immune escape.

8. The use of claim 6, wherein the S-RBD domain protein variant of the variant strain is specifically: S-RBD N439K, S-RBD L452R, S-RBDA475V, S-RBD V483A, S-RBD F490L, S-RBD Y508H, S-RBD S477N, S-RBD E484K, S-RBD G446V and S-RBD N450K on the basis of SEQ ID No. 6.

Technical Field

The invention relates to the research field of ACE2 mutation sites, in particular to an ACE2 mutation combination capable of improving the affinity with SARS-CoV-2S protein and application thereof.

Background

The novel coronavirus (SARS-CoV-2) is an important member of the genus beta coronavirus, has high similarity to the genome of atypical virus (SARS-CoV), and is one of three kinds of coronavirus which infect human and can cause serious symptoms at present. SARS-CoV-2 has higher infectivity than SARS-CoV and Middle East respiratory syndrome virus (MERS-CoV), and has caused more than 1.47 hundred million people to infect at present, 311 million people die, and has caused serious threat to the life health of people all over the world. Like SARS-CoV, SARS-CoV-2 also recognizes the membrane protein Angiotensin-converting enzyme II (ACE 2) on the surface of host cells via Spike protein (S), thereby causing viral infection. At present, a plurality of research institutions report the electron microscope structure of a complex of ACE2 and S full-length protein, and the crystal structure of a complex of an ACE2 extracellular domain and an S protein Receptor Binding Domain (RBD). The S protein is used as an important antiviral drug target and is the key of antibody treatment and vaccine development.

So far, various antibody medicines enter clinical experiments and obtain better curative effect. The world countries have also approved 15 vaccines for marketing, and hundreds of candidate vaccines are in clinical trials and preclinical studies, which make people hope for eliminating SARS-CoV-2. However, with the development of epidemic situation, the virus has continuous mutation, and among them, the S protein is one of the most easily mutated proteins in the virus. Therefore, a plurality of new variant strains (Brazilian strains, south Africa strains and the like) are generated, and the variant strains can resist the activity of a plurality of neutralizing antibodies and cause immune escape, so that the curative effect of antiviral methods such as convalescent plasma, polyclonal serum, monoclonal antibodies and vaccines is reduced and even completely lost. According to research reports, the recombinant expression human ACE2 protein can be used as a bait and competitively combined with host cell surface ACE2 protein to SARS-CoV-2, thereby achieving the antiviral effect and not generating immune escape. In addition, the recombinant human ACE2 protein passes clinical safety evaluation in various countries and is proved to be safe and reliable in vivo. In vivo and in vitro experiments show that the recombinant human ACE2 protein has better antiviral efficacy.

The recombinant expression ACE2 extracellular domain protein is used as a medicine with great potential for resisting SARS-CoV-2, improves the affinity of the medicine with SARS-CoV-2S-RBD and has important effect on enhancing the antiviral curative effect of the medicine. However, to date, the improvement of antiviral effects by modification of ACE2 has been less studied, and there has been no report of enhancing the antiviral ability of SARS-CoV-2 variant virus strains with immune escape. Therefore, the identification of ACE2 mutation and mutation combination capable of enhancing the binding activity with SARS-CoV-2S-RBD, and the improvement of ACE2 to realize the improvement of the affinity with SARS-CoV-2S-RBD and SARS-CoV-2 immune escape strain S-RBD protein, and further improve the antiviral ability to SARS-CoV-2 and its variant strain are the technical problems to be solved by the invention.

In-vivo and in-vitro experiments of ACE2 all prove that the novel coronavirus resistant effect is good, but wild type ACE2 ectodomain protein is still used in clinical experiments. The wild-type ACE2 extracellular domain protein has the defects of low affinity, general antiviral efficacy and the like. In view of the fact that ACE2 is one of the most potential antiviral drugs, the modification of ACE2 to make ACE have better antiviral therapeutic effect is of great significance. Therefore, the affinity of the ACE2 gene and S-RBD can be enhanced by mutating the ACE2 gene to find out three sites (S19W, T27W and N330Y), and the effect of resisting the new coronavirus is greatly improved. In addition, the resistance effect of the modified ACE2 protein on a new coronavirus variant strain with the immune escape capability is increased.

ACE2 is a potential therapeutic drug for COVID-19, and so far, modification research on ACE2 is relatively few, and relatively limited research is mainly focused on:

the C-terminal fusion Fc tag of ACE2 protein can enhance the affinity and antiviral ability to S-RBD.

2. Trimerization of ACE2 protein using protein engineering can enhance affinity to S-RBD and antiviral ability.

3. Mutations to the ACE2 protein may also enhance affinity for S-RBD and antiviral ability. For example: T27Y, H34A, H374N, etc.

It can be seen that, although the above methods can improve the antiviral ability of ACE2 by aiming at the modification of ACE2 protein at present, the affinity of ACE2 and SARS-CoV-2S-RBD can be improved by three mutations, namely S19W, T27W and N330Y, and no report is reported at present, and no effective combination of the three mutations and the inhibition effect of the corresponding ACE2 mutant recombinant protein on a mutant virus with immune escape are mentioned.

Disclosure of Invention

In order to solve the problems of the prior art, the invention aims to provide an ACE2 mutant combination capable of improving the affinity with SARS-CoV-2S protein and application thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

ACE2 mutation capable of improving affinity with SARS-CoV-2S protein

The specific ACE2 mutation site is that the 19 th S mutation of the amino acid sequence is W, the 27 th T mutation is W, and the 330 th N mutation is Y.

Further, any two or more of the ACE2 mutation sites that are capable of increasing the affinity to the SARS-CoV-2S protein are combined.

Further, the ACE2 protein with the site mutation is ACE2/S19W, ACE2/T27W and ACE 2/N330Y.

Further, the ACE2 protein obtained by combining the site mutations is: ACE2[ W19/Y330], ACE2[ W27/Y330], ACE2[ W19/W27/Y330] proteins.

Further, the mutant protein is applied as a SARS-CoV-2 therapeutic drug.

Furthermore, the mutant protein can be used as a SARS-CoV-2 therapeutic drug to treat the existing SARS-CoV-2 virus strain and partial variant strain.

Further, the variant strains are: (ii) S-RBD variant strains capable of eliciting an existing immune escape.

Further, the S-RBD region protein variation of the variant strain is specifically as follows: S-RBD N439K, S-RBD L452R, S-RBD A475V, S-RBD V483A, S-RBD F490L, S-RBD Y508H, S-RBD S477N, S-RBD E484K, S-RBD G446V and S-RBD N450K on the basis of SEQ ID No. 6.

ACE2 mutant combination capable of improving affinity with SARS-CoV-2S protein and application thereof

Through MOE software analysis and prediction, 7 ACE2 mutation sites are screened out, including S19W, T27W, H34E, G326W, G326K, N330Y and K353R; preparing high-purity proteins of SARS-CoV-2S-RBD, ACE2/WT, ACE2/S19W, ACE2/T27W, ACE2/H34E, ACE2/G326W, ACE2/N330Y and ACE2/K353R by using an insect expression system and a plurality of purification methods, and preparing ACE2 protein with mutated active sites (ACE 2/active-site-mutant); the affinity between SARS-CoV-2S-RBD and ACE2/WT, ACE2/S19W, ACE2/T27W, ACE2/H34E, ACE2/G326K, ACE2/G326W, ACE2/N330Y, ACE2/K353R, ACE2/active-site-mutant is determined by three independent repeated experiments through Surface Plasmon Resonance (SPR), and the affinity between ACE2/S19W, ACE2/T27W, ACE2/N330Y, ACE2/active-site-mutant and SARS-CoV-2S-RBD is higher than that of ACE 2/WT.

Based on active site mutation, S19W, T27W and N330Y are combined, and four high-purity ACE2[ W19/W27], ACE2[ W19/Y330], ACE2[ W27/Y330] and ACE2[ W19/W27/Y330] proteins are obtained by the same expression and purification method. And through a second SPR experiment, the affinity between SARS-CoV-2S-RBD and ACE2[ W19/Y330], ACE2[ W27/Y330] and ACE2[ W19/W27/Y330] proteins is improved by about 4-8 times. Then, high-purity ACE2/WT-Fc, ACE2[ W19/Y330] -Fc, ACE2[ W27/Y330] -Fc and ACE2[ W19/W27/Y330] -Fc protein are prepared by methods such as 293T cell expression and affinity chromatography. Through a pseudovirus inhibition experiment, the inhibition capability of the modified ACE2 protein on SARS-CoV-2 pseudovirus is verified to be improved by 30-173 times. Then, we analyzed the crystal structure of the complex of SARS-CoV-2S-RBD with ACE2[ W19/Y330], ACE2[ W27/Y330 ]. The structural alignment analysis structurally explains the reason that the mutations S19W, T27W and N330Y increase the ability of ACE2 to bind S-RBD.

In addition, we found 10 mutation sites capable of causing immune escape in SARS-CoV-2S-RBD, and expressed and purified to obtain high-purity S-RBD N439K, S-RBD L452R, S-RBD A475V, S-RBD V483A, S-RBD F490L, S-RBD Y508H, S-RBD S477N, S-RBD E484K, S-RBD G446V and S-RBD N450K proteins. SPR and pseudovirus inhibition experiments prove that the affinity of the modified ACE2 protein and the 10S-RBD mutant proteins is improved by 4-20 times, and the inhibition capability on SARS-CoV-2 variant strains is greatly enhanced.

Compared with the prior art, the invention has the beneficial effects that:

the invention finds 3 mutation sites (S19W, T27W and N330Y) of ACE2 for the first time, and the 3 sites are mutated individually or in combination, so that the affinity with SARS-CoV-2S-RBD and SARS-CoV-2 immune escape mutant strain S-RBD can be enhanced, and the invention proves that the recombinant human ACE2 protein containing the mutation or the mutation combination can obviously improve the inhibition capability on SARS-CoV-2 and SARS-CoV-2 immune escape mutant strains. Therefore, the recombinant human ACE2 protein containing one or more mutations in S19W, T27W and N330Y has important potential application value in treating COVID-19, especially in inhibiting infection of SARS-CoV-2 immune escape variant strain with antibody resistance.

1. Through MOE software prediction and experiments such as SPR, 3 single-site S19W, T27W and N330Y mutations of ACE2 are found to enhance the binding capacity with SARS-CoV-2S-RBD. In addition, we combined the above 3 sites and demonstrated that N330Y can synergize with S19W and T27W, but S19W and T27W cannot mutually promote each other.

2. Through structural biology means, the structural basis that the mutation of the three sites can enhance the combination is successfully revealed, and the molecular mechanisms that S19W, T27W and N330Y are synergistic and do not promote each other are decoded.

3. Through preparing 10S-RBD mutant (mutant site with antibody resistance) proteins and SPR experiments, the combination of the mutation and the mutation is verified that the binding capacity of the recombinant humanized ACE2 protein to the S-RBD protein of the SARS-CoV-2 immune escape mutant is also remarkably enhanced;

4. a pseudovirus inhibition experiment proves that the inhibition capability of the recombinant humanized ACE2 protein containing the mutation and the mutation combination on SARS-CoV-2 and SARS-CoV-2 immune escape variant virus strains with antibody resistance is greatly improved.

Drawings

FIG. 1 is a graph showing the prediction of the mutation site of ACE2 by MOE software.

FIG. 2 construction scheme of ACE2 protein.

FIG. 3: molecular sieve chromatographic purification and SDS-PAGE identification of ACE2/WT and its muteins.

FIG. 4 is a schematic diagram of SARS-CoV-2S and S-Trx-RBD proteins.

FIG. 5: molecular sieve chromatographic purification and SDS-PAGE identification of S-RBD protein.

FIG. 6 SPR technique measures the affinity of S-RBD for ACE2 and its muteins.

FIG. 7: ACE2 combined with molecular sieve chromatographic purification and SDS-PAGE identification of muteins.

FIG. 8 SPR technique for determining the affinity between SARS-CoV-2S-RBD and ACE2 combination muteins.

FIG. 9 alignment of the S19W, T27W and N330Y mutations before and after and with details of S-RBD action.

FIG. 10 SDS-PAGE patterns of purification of ACE2/WT-Fc, ACE2[ W19/W330] -Fc, ACE2[ W27/W330] -Fc and ACE2[ W19/W27/Y330] -Fc proteins.

FIG. 11 ACE2/WT, ACE2[ W19/W330], ACE2[ W27/W330] and ACE2[ W19/W27/Y330] proteins inhibit new coronavirus entry.

FIG. 12 ACE2/WT-Fc, ACE2[ W19/W330] -Fc, ACE2[ W27/W330] -Fc and ACE2[ W19/W27/Y330] -Fc inhibit new crown pseudovirus invasion.

FIG. 13: molecular sieve chromatographic purification and SDS-PAGE identification of S-RBD mutant protein.

FIG. 14 SPR technique for determining the affinity of the S-RBD mutein for the enhanced ACE2 protein.

FIG. 15 is a drawing for inhibiting invasion of SARS-CoV-2 mutant pseudovirus by ACE2/WT, ACE2[ W19/W330], ACE2[ W27/W330] and ACE2[ W19/W27/Y330 ].

Detailed Description

The technical scheme of the invention is further described in detail by combining the drawings and the detailed implementation mode:

as shown in figure 1 of the drawings, in which,

receptor Binding Domain (RBD): the region which consists of amino acids 320-537 of the novel coronavirus S protein and binds to the cell surface receptor ACE 2.

Angiotensin converting enzyme II (Angiotensin converting enzyme2, ACE 2): is a homologue of angiotensin converting enzyme, can efficiently hydrolyze angiotensin II to form vasodilator angiotensin 1-7, and is related to cardiovascular diseases, kidney diseases, lung diseases and the like. This is primarily a functional receptor for the new coronavirus, mediating its invasion.

The extracellular domain: the ACE2 is used as a single transmembrane protein, and the 19 th to 615 th amino acids at the N terminal are positioned outside a cell membrane to form a large extracellular domain which can be recognized by a new coronavirus.

Immune escape: immunosuppressive pathogens antagonize, block and suppress the body's immune response through their structural and nonstructural products. The S protein of the new coronavirus is mutated, so that the immune response of an organism can be avoided, and the effects of a neutralizing antibody and a vaccine are reduced or completely lost.

The enzyme activity center is as follows: the enzyme molecule is a site that can directly bind to a substrate molecule and catalyze a chemical reaction of the substrate, and this site becomes the active center of the enzyme. Herein, the binding region of ACE2 and angiotensin II substrate is referred to.

Trx tag: the thioredoxin tag, which has a molecular weight of about 13kD, is a tagged protein that is widely used to help correct folding of recombinant proteins.

Combinatorial mutant proteins: the S19W, T27W and N330Y mutations of ACE2 ectodomain protein are combined to form 4 kinds of multi-mutant protein, including ACE2[ W19/W27], ACE2[ W19/Y330], ACE2[ W27/Y330] and ACE2[ W19/W27/Y330 ].

Enhanced ACE2 protein: namely 3 kinds of multi-protamine with stronger affinity with S-RBD in the ACE2 combined mutant protein, including ACE2[ W19/Y330], ACE2[ W27/Y330] and ACE2[ W19/W27/Y330 ].

1 prediction of binding force enhancing ACE2 mutation site of S protein of novel coronavirus

By searching PDB database, downloading SARS-CoV-2S-RBD/ACE 2(PDB number is 6LZG) complex crystal structure, analyzing the complex structure in Pymol software, and finding out key amino acid (distance is less than or equal to that of the binding interface of SARS-CoV-2S-RBD and ACE2)). The calculation of these key amino acid sites in the complex structure by MOE software predicts a series of ACE2 single mutation sites, which may be related to the enhancement of SARS-CoV-2S-RBD affinity, the specific steps are shown in FIG. 1.

FIG. 1 is a graph of prediction of the mutation site of ACE2 by the MOE software. Based on the structure of SARS-CoV-2S-RBD/ACE 2 complex, ACE2 mutation capable of enhancing S-RBD binding ability is predicted, and the mutation site is listed.

And (3) calculating a plurality of ACE2 mutation sites by MOE software, and finally selecting 7 single mutation sites with the highest possibility by repeatedly analyzing the structure, wherein the single mutation sites comprise ACE2/S19W, ACE2/T27W, ACE2/H34E, ACE2/G326W, ACE2/G326K, ACE2/N330Y and ACE 2/K353R.

Preparation of 2ACE2 ectodomain and muteins thereof

2.1 acquisition of the Gene encoding the extracellular Domain protein of ACE2

According to the amino acid sequence of the ACE2(GenBank sequence number is AB193259.1) protein in human lung tissues reported in NCBI (http:// www.ncbi.nlm.nih.gov), we obtained the ACE2 whole gene sequence (see sequence table SEQ ID NO.1) by a gene synthesis method.

2.2 construction of recombinant plasmids of the extracellular domain of ACE2 and its mutants

We designed and synthesized the following primers for amplification and engineering of ACE2 ectodomain and muteins:

in order to inactivate the enzyme activity of ACE2, primers F total ACE-His, R total ACE-His, FH374/378A, RH374/378A, FE402A and RE402A are used for mutating enzyme activity central sites, namely H374A, H378A and E402A, so that P-ACE2-active-site-mutant is obtained; obtaining P-ACE2/WT by using primers F total ACE-His and R total ACE-His; primer FS19W and R total ACE-His were used to obtain P-ACE 2/S19W; primer FT27W and R Total ACE-His were used to obtain P-ACE 2/T27W; obtaining P-ACE2/H34E by using primers FH34E, F total ACE-His and R total ACE-His; P-ACE2/G326W was obtained using primers FG326W, RG326W, F total ACE-His and R total ACE-His; P-ACE2/G326K was obtained using primers FG326K, RG326K, F total ACE-His and R total ACE-His; P-ACE2/N330Y was obtained using primers FN330Y, RN330Y, F total ACE-His and R total ACE-His; primers FK353R, RK353R, F total ACE-His and R total ACE-His were used to obtain P-ACE 2/K353R. All constructs were used for purification by introducing a 6xHis tag at the C-terminus via the most downstream primer R total ACE-His.

The PCR reaction conditions are pre-denaturation at 96 ℃ for 2min (denaturation at 96 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 0.5-2min) for 30 cycles and final extension at 72 ℃ for 10 min. Constructing fragments obtained by PCR into pFastBac1-GP67 vectors respectively by using a homologous recombination method, wherein a recombination system contains 5 mu L of homologous recombinase and 0.1 mu g of vectors pFastBac1-GP67 (after the enzyme digestion of EcoR I and Hind III), each fragment is 0.3 mu g, and finally ddH2O is used for supplementing to 10 mu L; the reaction conditions were 50 ℃ for 50 min. The above 9 constructs obtained by recombination were sequenced to confirm that the inserted foreign fragment was completely correct.

The vector used for construction is pFastBac1 vector (pFastBac1-GP67) containing GP67 signal peptide, which is constructed for the laboratory, namely, a coding sequence of GP67 signal peptide is inserted between restriction enzyme sites BamH I and EcoR I on a commercial pFastBac1 vector (Invitrogen company) (the coding nucleic acid sequence of GP67 signal peptide is shown in SEQ ID NO.2, and the amino acid sequence of GP67 signal peptide is shown in SEQ ID NO. 3).

The protein construction sequence after the transformation comprises an extracellular segment (amino acids 19-615) of ACE2 protein, and the obtained construction is ACE2/active-site-mutant, ACE2/WT, ACE2/S19W, ACE2/T27W, ACE2/H34E, ACE2/G326W, ACE2/G326K, ACE2/N330Y and ACE 2/K353R. The protein coding nucleic acid sequence of ACE2/WT is shown in SEQ ID NO.4, the amino acid sequence thereof is shown in SEQ ID NO.5, and other constructions are mutated on the basis of ACE 2/WT.

The ACE2/WT expression constructs a strategy pattern diagram as shown in FIG. 2.

2.3 preparation of recombinant ACE2 extracellular domain and muteins thereof

ACE2/active-site-mutant, ACE2/WT, ACE2/S19W, ACE2/T27W, ACE2/H34E, ACE2/G326W, ACE2/G326K, ACE2/N330Y and ACE2/K353R were transformed into competent cells DH10Bac, respectively, and then plated on LB plates containing 50. mu.g/ml kanamycin, 7. mu.g/ml gentamicin, 10. mu.g/ml tetracycline, and 100. mu.g/ml X-gal and 40. mu.g/ml IPTG, and after 48H incubation at 37 ℃, white colonies were picked up and after incubation, the corresponding recombinant Bacmid was extracted using a plasmid extraction kit.

And (3) transfecting the recombinant Bacmid extracted above to sf9 insect cells, and culturing for 72h to obtain the P1 generation recombinant baculovirus. The recombinant baculovirus was expanded to P3 passages by subculturing in sf9 cells. The recombinant baculovirus of the generation P3 was inoculated into sf9 cells for expression. Cell supernatants were harvested 96h after inoculation with virus. The crude purification by affinity chromatography was carried out using HisTrap affinity chromatography column (GE Healthcare), followed by further fine purification by molecular sieve chromatography.

In affinity chromatography purification, for example, the ACE2/WT protein was purified by first passing the harvested culture supernatant containing ACE2/WT protein through a HisTrap affinity column, then washing the column with 10 column volumes of buffer B (10mM Imidazole, 20mM Tris-HCl, 150mM NaCl, pH 8.0), and then eluting the desired protein from the affinity column with buffer C (20mM Tris-HCl, 150mM NaCl, pH 8.0, 300mM Imidazole), thereby obtaining crude ACE2/WT protein. The protein was further purified finely on a Superdex 200 Increate 10/300(GE Healthcare) column and finally the ACE2/WT protein was exchanged into buffer A (20mM Tris-HCl, 150mM NaCl, pH 8.0). SDS-PAGE identification shows that the purity of the protein reaches more than 95%, and the elution performance of the protein in molecular sieve chromatography shows that the protein has good uniformity in solution. ACE2/active-site-mutant, ACE2/S19W, ACE2/T27W, ACE2/H34E, ACE2/G326W, ACE2/G326K, ACE2/N330Y, ACE2/K353R are the same as the purification method of ACE2/WT protein. The results are shown in FIG. 3.

Therefore, the proteins of ACE2/active-site-mutant, ACE2/WT, ACE2/S19W, ACE2/T27W, ACE2/H34E, ACE2/G326W, ACE2/G326K, ACE2/N330Y and ACE2/K353R with high purity and uniform properties are obtained.

Preparation of 3 SARS-CoV-2S-RBD protein

3.1 acquisition and construction of S-RBD protein coding Gene

The new coronavirus S-RBD protein is obtained by selecting 320-537 amino acids (sequence shown in SEQ ID NO.6) of the S protein of a new coronavirus strain (GenBank: MN908947.3) sequenced by a first whole genome, and the gene sequence is synthesized by Changzhou Jiuzhou Biotechnology Limited company (nucleic acid sequence shown in SEQ ID NO. 7). The Trx gene (to assist S-RBD folding) and the 6XHis tag (to assist subsequent purification) are added to the upstream of the S-RBD of the new coronavirus, an EK enzyme cutting site is added after the Trx gene (to remove the tag and all redundant amino acids of the non-S-RBD by EK enzyme cutting), and the Trx gene is constructed into a pFastBac1-gp67 vector (the same vector as used for ACE2 construction), and the construction is completed by Anhui general Biotechnology, Inc. The nucleotide sequence of the Trx tag, the His tag and the EK restriction site is shown in SEQ ID NO.8, and the amino acid sequence is shown in SEQ ID NO. 9.

The strategy pattern diagram constructed by the expression of the S-RBD is shown in FIG. 4.

3.2 preparation of S-RBD protein

The plasmid of Trx-RBD is transformed into a competent cell DH10Bac, then is coated on an LB plate containing 50 mu g/ml kanamycin, 7 mu g/ml gentamicin, 10 mu g/ml tetracycline, 100 mu g/ml X-gal and 40 mu g/ml IPTG, and after culturing for 48 hours at 37 ℃, white colonies are picked up, and after culturing, a plasmid extraction kit is used for extracting corresponding recombinant Bacmid.

And (3) transfecting the recombinant Bacmid extracted above to sf9 insect cells, and culturing for 72h to obtain the P1 generation recombinant baculovirus. The recombinant baculovirus was expanded to P3 passages by subculturing in sf9 cells. The recombinant baculovirus of the generation P3 was inoculated into sf9 cells for expression. Cell supernatants were harvested 96h after inoculation with virus. Purification is carried out by affinity chromatography using HisTrap affinity chromatography column (GE Healthcare), EK digestion, molecular sieve chromatography, etc. In the affinity chromatography purification, the harvested supernatant of the medium containing the Trx-tagged S-RBD protein (Trx-S-RBD) is first passed through a HisTrap affinity chromatography column, and then the column is washed with 10 column volumes of buffer B (10mM Imidazole, 20mM Tris-HCl, 150mM NaCl, pH 8.0), and then the objective protein is eluted from the affinity chromatography column with buffer C (20mM Tris-HCl, 150mM NaCl, pH 8.0, 300mM Imidazole), thereby obtaining a crude Trx-RBD protein; adding EK enzyme into Trx-RBD protein for enzyme digestion (enzyme and substrate are 1U: 0.5mg), and performing cleavage at 18 ℃ for 12 h; adding the cut protein into a HisTrap affinity chromatography column, and binding at 4 ℃ for 15min to obtain flow-through protein, namely S-RBD protein;

the protein was further purified finely on a Superdex 200 Incrase 10/300(GE Healthcare) column and finally the S-RBD protein was pipetted into buffer A (20mM Tris-HCl, 150mM NaCl, pH 8.0). SDS-PAGE identification shows that the purity of the protein reaches more than 99 percent, and the elution performance of the protein in molecular sieve chromatography shows that the protein has good uniformity in solution. Therefore, the S-RBD protein with high purity and uniform properties is obtained. The results are shown in FIG. 5.

Affinity determination of 4S-RBD with ACE2/WT and muteins thereof

The affinity between SARS-CoV-2S-RBD and ACE2/WT, ACE2/S19W, ACE2/T27W, ACE2/H34E, ACE2/G326W, ACE2/G326K, ACE2/N330Y, ACE2/K353R, ACE2/active-site-mutant protein was determined by BIACORE 8K instrument, respectively, and the results are shown in FIG. 6.

In the experiment, SARS-CoV-2S-RBD is used as a stationary phase, the fixed value is about 700RUs, and ACE/WT and mutants thereof are used as a mobile phase. The concentration gradient of ACE2/WT, ACE2/S19W, ACE2/T27W, ACE2/H34E, ACE2/G326W, ACE2/N330Y and ACE2/active-site-mutant is 1.5-192nM, the concentration gradient of ACE2/G326K is 3.125-400nM, and the concentration gradient of ACE2/K353R is 31.25-4000 nM. From the results analysis, all the data above were calculated using a kinetic model of slow binding/slow dissociation. Wherein the affinity of ACE2/S19W (48.6 + -12.8 nM), ACE2/T27W (38.5 + -8.5 nM), ACE2/N330Y (29.3 + -10.4 nM) and ACE2/active-site-mutant (74.5 + -13.9 nM) mutants is higher than that of ACE2/WT (81.8 + -4.7 nM). Therefore, we will combine based on these four mutations to further design higher affinity combinatorial mutations.

Preparation of 5ACE2 combination mutant protein

The SPR results of the previous step show that the mutation of S19W, T27W, N330Y and active-site-mutant site increases the affinity of ACE2 and SARS-CoV-2S-RBD. To further improve the affinity, we combined these four mutation sites. On the basis of ACE2/active-site-mutant, the combination of S19W/T27W, S19W/N330Y, T27W/N330Y and S19W/T27W/N330Y are respectively carried out to obtain combined mutant construction which is respectively named as ACE2[ W19/W27], ACE2[ W19/Y330], ACE2[ W27/Y330] and ACE2[ W19/W27/Y330 ]. The combined mutation of ACE2 was consistent with the construction and expression of ACE 2/WT. Finally, the ACE2[ W19/W27], ACE2[ W19/Y330], ACE2[ W27/Y330] and ACE2[ W19/W27/Y330] proteins with high purity and uniform properties are obtained. The result is shown in fig. 7.

Affinity assay for 6S-RBD in combination with ACE2 mutant proteins

The affinity between SARS-CoV-2S-RBD and ACE2[ W19/W27], ACE2[ W19/Y330], ACE2[ W27/Y330], ACE2[ W19/W27/Y330] proteins was determined by BIACORE 8K instrument, and the results are shown in FIG. 8.

In the experiment, SARS-CoV-2S-RBD is taken as a stationary phase, the fixed value is about 700RUs, ACE2[ W19/W27], ACE2[ W19/Y330], ACE2[ W27/Y330] and ACE2[ W19/W27/Y330] proteins are taken as mobile phases, and the concentration gradients are all 1.5-192 nM. From the above graph analysis, all data were calculated using a kinetic model of slow binding/slow dissociation. The results showed that, compared with ACE2/WT, the affinity of ACE2[ W19/W27] to SARS-CoV-2S-RBD was not increased, the affinity of ACE2[ W19/Y330] to SARS-CoV-2S-RBD was increased by about 8 times, the affinity of ACE2[ W27/Y330] to SARS-CoV-2S-RBD was increased by about 6 times, and the affinity of ACE2[ W19/W27/Y330] to SARS-CoV-2S-RBD was increased by about 4 times. Finally, 3 kinds of ACE2 mutant proteins with enhanced binding capacity (ACE2[ W19/Y330], ACE2[ W27/Y330], ACE2[ W19/W27/Y330]) are obtained and named as enhanced ACE 2.

7 SARS-CoV-2S-RBD/ACE 2[ W19/Y330] and SARS-CoV-2S-RBD/ACE 2[ W27/Y330] compound crystal screening and structure analysis

A gas-phase diffusion sitting drop method (Vapor-diffusion sitting drop) is adopted to carry out crystal screening on compound protein under the condition of 18 ℃ by utilizing a commercial crystal screening kit (Hampton and Molecular Dimensions), and finally, compound protein crystals with better quality of SARS-CoV-2S-RBD/ACE 2[ W19/Y330] are obtained under the condition of a Molecular Dimensions kit Structure Screen 2MD1-0223# (1.6 MAmmonia sulfate, 0.1M MES pH 6.5 and 10% v/v 1,4-Dioxane), and the compound protein crystals with better quality of SARS-CoV-2S-RBD/ACE 2[ W19/Y330] are obtained under the condition of a Structure Screen 1MD 54-0130 # (2.0 MAmmonia sulfate, 0.1M Sodium HEPES pH 7.5 and 2% v/v PEG 400) and the compound protein crystals with better quality of SARS-CoV-2S-RBD/ACE 2[ W36330 ] are selected under the condition of Structure Screen 1MD1-0130# (2.0.0.0% v/v PEG 400).

Diffraction data of the two protein crystals are completed at Shanghai synchrotron radiation light source BL18U1 line station. The collected diffraction data were integrated and averaged by HKL 2000; the structure analysis firstly carries out molecular replacement by taking the SARS-CoV-2S-RBD/ACE 2 structure as a model (PDB number: 6LZG), then carries out multi-round refinement and optimization through Coot, REFMAC5 and PHASER, and finally obtains the crystal structures of SARS-CoV-2S-RBD/ACE 2[ W19/Y330] and SARS-CoV-2S-RBD/2 [ W27/Y330] through analysis.

8S19W, T27W and N330Y mutations enhance the molecular mechanism of SARS-CoV-2S-RBD binding

In the previously reported SARS-CoV-2S-RBD/ACE 2(PDB:6LZG) structure, S19 of ACE2 had only 2 amino acids interacting with S-RBD, whereas in the structure of SARS-CoV-2S-RBD/ACE 2[ W19/W330] complex, S19 was mutated to W19, and 5 amino acids were observed to interact with S-RBD. S19 interacts with S-RBD A475 and G476 through hydrogen bonding, van der Waals forces, and hydrophobic interactions, and CCP4i calculates that there are only 7 of their interatomic interactions. When S19 is mutated to W19, a macrocyclic indolyl is introduced into the amino acid side chain to make it combine closely with S-RBD. Thus, we observed that W19 interacts with K458, Y473, Q474, a475, and G476 of S-RBD through hydrogen bonding, van der waals forces, and hydrophobic interactions, and up to 52 interatomic interactions were calculated for CCP4 i. The details are shown in fig. 9 b.

By comparing SARS-CoV-2S-RBD/ACE 2(PDB:6LZG) and SARS-CoV-2S-RBD/ACE 2[ W27/W330]]We observed that T27 and W27 are distant from S-RBDThe amino acids in the above are all 4, and are respectively F456, Y473, A475 and Y489. However, by calculation with CCP4i, we found that when T27 was mutated to W27, the interatomic interaction changed from 15 to 35. The details are shown in fig. 9 c.

As shown in FIG. 9d, N330 of ACE2 has 2 amino acids interacting with S-RBD. N330 interacts with T500 and N330 of S-RBD through hydrogen bonds, van der Waals forces and hydrophobic interactions, and 8 interatomic interactions between them were calculated. After mutation of N330 to Y330, it was observed that the distance between 3 amino acids and S-RBD was not more than Y330 also interacted with P499, T500 and N330 of S-RBD through hydrogen bonding, van der waals forces and hydrophobic interactions, and 16 interatomic interactions were calculated.

Taken together, the S19W, T27W, and N330Y mutations increased the affinity of ACE2 for SARS-CoV-2S-RBD, consistent with an interaction assay in the structure.

Inhibition of 9 enhanced ACE2 protein on SARS-CoV-2 pseudovirus

Due to the nature of the transmission of new coronaviruses by droplets and contact, the high infectivity of these coronaviruses requires that the handling of live viruses be done in a biosafety laboratory of the P3 grade, a high biosafety level not available in this laboratory. The pseudovirus system can well simulate the virus invasion process, is used for inhibition experiments of the targeted virus invasion process, and can avoid the limitation of high-biosafety level operation. Based on a pseudovirus packaging system which is in existence in a laboratory and takes lentivirus as a framework, the plasmid pNL4.3-Luc-R-E and the plasmid SARS-CoV-2-S are co-transfected to 293T cells to be packaged to obtain the new coronaviruses, and the invasion characteristics of the pseudoviruses to susceptible cells are quantitatively analyzed by infecting 293T cells which stably transfer ACE2 and measuring the luciferase activity by a microplate reader.

9.1 construction, expression and purification of ACE2-Fc

It is reported that the Fc tag can play a role in prolonging the half-life of functional proteins in plasma (27), and the ACE2 protein is used as a potential therapeutic drug of new coronavirus, and the Fc tag is introduced into the C terminal of the novel coronavirus, so that the virus inhibition capability of the ACE2 protein can be improved. Thus, we will construct ACE2/WT-Fc, ACE2[ W19/W330] -Fc, ACE2[ W27/W330] -Fc and ACE2[ W19/W27/Y330] -Fc proteins. ACE2 (amino acids 19 to 615) regions in ACE2/WT, ACE2[ W19/W330], ACE2[ W27/W330] and ACE2[ W19/W27/Y330] constructions were amplified by primers F total ACE-His and R total ACE-His, and then fragments were homologously recombined into pCAGGS vectors containing Fc tag sequences. The IL2 signal peptide on the pCAGGS vector secretes the protein at the N-terminus of the ACE2 sequence and the Fc tag at the C-terminus of the ACE2 sequence. The IL2 and Fc gene sequences are respectively shown in SEQ ID NO.10 and SEQ ID NO.11, and the amino acid sequences are respectively shown in SEQ ID NO.12 and SEQ ID NO. 13.

These constructs were transfected into 293T cells, and cell supernatants were collected after 72 hours and purified by protein A column to obtain high purity ACE2/WT-Fc, ACE2[ W19/W330] -Fc, ACE2[ W27/W330] -Fc, and ACE2[ W19/W27/Y330] -Fc proteins as shown in FIG. 10.

9.2 inhibition of SARS-CoV-2 pseudovirus by enhanced ACE2 protein

Through a pseudovirus inhibition experiment, the inhibition capacity of ACE2/WT, ACE2[ W19/W330], ACE2[ W27/W330] and ACE2[ W19/W27/Y330] on SARS-CoV-2 pseudovirus is determined. As shown in FIG. 11, the IC50 of ACE2/WT was 15.44. mu.g/mL, the IC50 of ACE2[ W19/W330] was 1.21. mu.g/mL, the IC50 of ACE2[ W27/W330] was 1.524. mu.g/mL, the IC50 of ACE2[ W19/W27/Y330] was 2.036. mu.g/mL, and the pseudovirus inhibitory ability was improved by about 8-13 times.

In addition, we also determined the inhibitory ability of ACE2/WT-Fc, ACE2[ W19/W330] -Fc, ACE2[ W27/W330] -Fc and ACE2[ W19/W27/Y330] -Fc proteins against SARS-CoV-2 pseudovirus. As shown in FIG. 12, the IC50 of ACE2/WT-Fc was 1.435. mu.g/mL, the IC50 of ACE2[ W19/W330] -Fc was 0.089. mu.g/mL, the IC50 of ACE2[ W27/W330] -Fc was 0.254. mu.g/mL, and the IC50 of ACE2[ W19/W27/Y330] -Fc was 0.532. mu.g/mL. Compared with ACE2/WT, the ACE2 protein introduced with Fc tag has about 11-173 times higher pseudovirus inhibiting ability.

Affinity determination of 10-enhanced ACE2 protein and S-RBD mutant

10.1 recombinant plasmid construction of S-RBD mutants

In the new coronavirus variant strain, part of mutations can generate antibody resistance and immune escape, and many of the mutation sites are positioned in the S-RBD region of the new coronavirus. Therefore, we selected 10 mutations in the S-RBD region for construction, including N439K, L452R, A475V, V483A, F490L, Y508H, S477N, E484K, G446V and N450K mutation sites. We designed and synthesized the following primers for amplification and mutation of S-RBD protein:

primer name	Primer sequence (5 '-3')
		F(N439K)	GTGATCGCTTGGAACTCAAAGAACCTGGACTCCAAGGTC
R(N439K)	GACCTTGGAGTCCAGGTTCTTTGAGTTCCAAGCGATCAC
		F(L452R)	GGTGGCAACTACAACTACAGGTACAGGCTGTTCAGAAAG
R(L452R)	CTTTCTGAACAGCCTGTACCTGTAGTTGTAGTTGCCACC
		F(A475V)	TCAACCGAAATCTACCAGGTCGGTTCCACTCCCTGCAAC
R(A475V)	GTTGCAGGGAGTGGAACCGACCTGGTAGATTTCGGTTGA
		F(F490L)	GAGGGCTTCAACTGCTACCTGCCCCTGCAGTCCTACGGT
R(F490L)	ACCGTAGGACTGCAGGGGCAGGTAGCAGTTGAAGCCCTC
		F(Y508H)	GGAGTCGGTTACCAGCCTCACCGTGTGGTCGTGCTGAGC
R(Y508H)	GCTCAGCACGACCACACGGTGAGGCTGGTAACCGACTCC
		(S477N)-F	GAAATCTACCAGGCTGGTAACACTCCCTGCAACGGTGTG
(S477N)-R	CACACCGTTGCAGGGAGTGTTACCAGCCTGGTAGATTTC
		(E484K)-F	ACTCCCTGCAACGGTGTGAAGGGCTTCAACTGCTACTTC
(E484K)-R	GAAGTAGCAGTTGAAGCCCTTCACACCGTTGCAGGGAGT
		(G446V)-F	AACCTGGACTCCAAGGTCGTCGGCAACTACAACTACCTG
(G446V)-R	CAGGTAGTTGTAGTTGCCGACGACCTTGGAGTCCAGGTT
		(N450K)-F	AAGGTCGGTGGCAACTACAAGTACCTGTACAGGCTGTTC
(N450K)-R	GAACAGCCTGTACAGGTACTTGTAGTTGCCACCGACCTT

The construction strategy is the same as that of the S-RBD, and all the S-RBD mutants are obtained by site-directed mutagenesis on the basis of the construction of the wild type S-RBD. The synthesized wild type S-RBD plasmid is amplified by a PCR method, and mutation sites are introduced into the primers to finally obtain the 10S-RBD mutants. Primers F (N439K) and R (N439K) were used to obtain S-RBD N439K; primers F (L452R) and R (L452R) were used to give S-RBD L452R; S-RBD A475V was obtained using primers F (A475V) and R (A475V); S-RBD F490L was obtained using primers F (F490L) and R (F490L); primers F (Y508H) and R (Y508H) were used to give S-RBD Y508H; S-RBD E484K was obtained using primers (E484K) -F and (E484K) -R; S-RBD G446V was obtained using primers (G446V) -F and (G446V) -R; S-RBD G446V was obtained using primers (N450K) -F and (N450K) -R.

10.2 preparation of S-RBD and muteins thereof

S-RBD N439K, S-RBD L452R, S-RBD A475V, S-RBD V483A, S-RBD F490L, S-RBD Y508H, S-RBD S477N, S-RBD E484K, S-RBD G446V and S-RBD N450K are the same as the purification method of the S-RBD protein. The results are shown in FIG. 13.

Thus, high-purity and homogeneous proteins S-RBD N439K, S-RBD L452R, S-RBD A475V, S-RBD V483A, S-RBD F490L, S-RBD Y508H, S-RBD S477N, S-RBD E484K, S-RBD 446V and S-RBD N450K were obtained.

10.3 affinity assay of S-RBD muteins with enhanced ACE2 protein

The affinity between S-RBD N439K, S-RBD L452R, S-RBD A475V, S-RBD V483A, S-RBD F490L, S-RBD Y508H, S-RBD S477N, S-RBD E484K, S-RBD G446V, S-RBD N450K and ACE2/WT, ACE2[ W19/Y330], ACE2[ W27/Y330], ACE2[ W19/W27/Y330], respectively, was determined by the BIACORE 8K instrument. The results of the measurement methods are shown in FIG. 14 (FIG. 14a, FIG. 14b and FIG. 14 c).

In the experiment, S-RBD mutant protein is used as a stationary phase, the fixed values are about 700RUs, ACE2[ W19/W27], ACE2[ W19/Y330], ACE2[ W27/Y330] and ACE2[ W19/W27/Y330] protein are used as a mobile phase, and the concentration gradient is 1.5-192 nM. From the above graph analysis, all data were calculated using a kinetic model of slow binding/slow dissociation. The results show that the affinities of ACE2[ W19/Y330], ACE2[ W27/Y330] and ACE2[ W19/W27/Y330] for all mutants of S-RDB are significantly increased compared to ACE 2/WT.

Inhibition of wild type 11 and enhanced ACE2 on SARS-CoV-2 and its variant strain

In order to obtain SARS-CoV-2 and its variant pseudovirus, we constructed the S gene of new crown virus and its variant into PCAGGS plasmid, the mutation site is identical to the site of S-RBD, including N439K, L452R, A475V, V483A, F490L, Y508H, S477N, E484K, G446V and N450K, then co-transfected with pNL4.3-Luc-R-E plasmid into 293T cells to obtain new crown variant pseudovirus. The invasion characteristics of the pseudovirus to susceptible cells were quantitatively analyzed by infecting HEK293 cell line stably transfected with ACE2 and measuring luciferase activity with a microplate reader, and the results are shown in FIG. 15.

TABLE 1ACE2 (wild type and enhanced) list of pseudoviruses IC50 inhibiting SARS-CoV-2 wild type and its mutant respectively

The inhibitory capacity of the different mutant ACE2 proteins against different pseudoviruses is indicated in the table, calculated using GraphPad Prism 5.

The results show that the inhibitory effect of the enhanced ACE2 proteins (ACE2[ W19/W330], ACE2[ W27/W330] and ACE2[ W19/W27/Y330]) on SARS-CoV-2 and the pseudovirus with antibody resistant variant strains is also obviously enhanced.

In conclusion, the single or multiple site mutations of S19W, T27W and N330Y in the ACE2 protein can increase the affinity of the ACE2 protein and S-RBD and mutants thereof, and greatly enhance the antiviral effect on SARS-CoV-2 and variant strains thereof.

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.

Sequence listing

<110> Sichuan university

<120> ACE2 mutant combination capable of improving affinity with SARS-CoV-2S protein and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

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atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60

attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120

tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180

aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240

caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300

cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360

aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420

tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480

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gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600

gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660

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catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780

tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840

ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900

gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960

cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020

aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080

tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140

tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200

catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260

attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320

aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380

tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440

aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500

gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560

taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620

aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680

ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740

gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800

aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860

aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920

tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980

cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040

tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100

gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160

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Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr

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Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala

20 25 30

Ala His Ser Ala Phe Ala Ala Asp

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gtccaaaaca tgaataatgc tggggacaaa tggtctgcct ttttaaagga acagtccaca 180

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aatgagaggc tctgggcttg ggaaagctgg agatctgagg tcggcaagca gctgaggcca 480

ttatatgaag agtatgtggt cttgaaaaat gagatggcaa gagcaaatca ttatgaggac 540

tatggggatt attggagagg agactatgaa gtaaatgggg tagatggcta tgactacagc 600

cgcggccagt tgattgaaga tgtggaacat acctttgaag agattaaacc attatatgaa 660

catcttcatg cctatgtgag ggcaaagttg atgaatgcct atccttccta tatcagtcca 720

attggatgcc tccctgctca tttgcttggt gatatgtggg gtagattttg gacaaatctg 780

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gaccaggcct gggatgcaca gagaatattc aaggaggccg agaagttctt tgtatctgtt 900

ggtcttccta atatgactca aggattctgg gaaaattcca tgctaacgga cccaggaaat 960

gttcagaaag cagtctgcca tcccacagct tgggacctgg ggaagggcga cttcaggatc 1020

cttatgtgca caaaggtgac aatggacgac ttcctgacag ctcatcatga gatggggcat 1080

atccagtatg atatggcata tgctgcacaa ccttttctgc taagaaatgg agctaatgaa 1140

ggattccatg aagctgttgg ggaaatcatg tcactttctg cagccacacc taagcattta 1200

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ctgctcaaac aagcactcac gattgttggg actctgccat ttacttacat gttagagaag 1320

tggaggtgga tggtctttaa aggggaaatt cccaaagacc agtggatgaa aaagtggtgg 1380

gagatgaagc gagagatagt tggggtggtg gaacctgtgc cccatgatga aacatactgt 1440

gaccccgcat ctctgttcca tgtttctaat gattactcat tcattcgata ttacacaagg 1500

accctttacc aattccagtt tcaagaagca ctttgtcaag cagctaaaca tgaaggccct 1560

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aggcttggaa aatcagaacc ctggacccta gcattggaaa atgttgtagg agcaaagaac 1680

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg Pro

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn

305 310 315 320

Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys Asn

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala Asp

595

<210> 6

<211> 218

<212> PRT

<213> novel coronavirus (Severe acid respiratory syndrome coronavirus 2)

<400> 6

Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu

1 5 10 15

Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr

20 25 30

Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val

35 40 45

Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser

50 55 60

Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser

65 70 75 80

Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr

85 90 95

Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly

100 105 110

Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly

115 120 125

Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro

130 135 140

Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro

145 150 155 160

Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr

165 170 175

Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val

180 185 190

Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro

195 200 205

Lys Lys Ser Thr Asn Leu Val Lys Asn Lys

210 215

<210> 7

<211> 657

<212> DNA

<213> novel coronavirus (Severe acid respiratory syndrome coronavirus 2)

<400> 7

gtgcagccaa ccgaatctat cgtcagattc ccaaacatca ctaacctgtg ccctttcgga 60

gaggtgttca acgctaccag gttcgccagc gtctacgctt ggaaccgcaa gcgtatcagc 120

aactgcgtcg ccgactactc tgtgctgtac aactccgcta gcttctctac tttcaagtgc 180

tacggcgtgt cacctaccaa gctgaacgac ctgtgcttca ctaacgtcta cgccgactcc 240

ttcgtgatcc gcggagacga agtccgtcag atcgctcctg gacagaccgg aaagatcgct 300

gactacaact acaagctgcc agacgacttc actggctgcg tgatcgcttg gaactcaaac 360

aacctggact ccaaggtcgg tggcaactac aactacctgt acaggctgtt cagaaagtca 420

aacctgaagc ctttcgagcg cgacatctca accgaaatct accaggctgg ttccactccc 480

tgcaacggtg tggagggctt caactgctac ttccccctgc agtcctacgg tttccagcca 540

accaacggag tcggttacca gccttaccgt gtggtcgtgc tgagcttcga actgctccac 600

gctcctgcta ctgtgtgcgg tcccaagaag tctactaacc tggtcaaaaa caaataa 657

<210> 8

<211> 387

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agcgacaaga tcatccacct gactgacgac agcttcgaca ctgacgtgct gaaggctgac 60

ggtgctatcc tggtcgactt ctgggccgag tggtgcggcc cttgcaagat gatcgctccc 120

atcctggacg agatcgccga cgagtaccag ggtaaactga ctgtggccaa gctgaacatc 180

gaccagaacc ccggtactgc tcctaagtac ggcatccgtg gtatccccac tctgctgctg 240

ttcaagaacg gtgaggtggc cgctaccaag gtcggtgctc tgagcaaggg ccagctgaag 300

gagttcctgg acgctaacct ggctggttcc ggcagcggcc acatgcacca ccaccaccat 360

cacagcagcg gcgacgacga cgacaag 387

<210> 9

<211> 129

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val

1 5 10 15

Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys

20 25 30

Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu

35 40 45

Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro

50 55 60

Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu

65 70 75 80

Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys

85 90 95

Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly Ser

100 105 110

Gly His Met His His His His His His Ser Ser Gly Asp Asp Asp Asp

115 120 125

Lys

<210> 10

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt caccaattcg 60

<210> 11

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser

20

<210> 12

<211> 684

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa ataa 684

<210> 13

<211> 227

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225