CN114949175A - Application of new coronavirus S protein in preparation of medicine for treating tumors - Google Patents

Abstract

The invention relates to a new coronavirus S protein and application of related products thereof in preparing a medicament for treating tumors. The inventor finds that S protein of SARS-CoV-2 virus can obviously inhibit the activity of tumor cells in vitro through the interaction with ACE2 on the surface of the tumor cells, and can obviously inhibit the formation of tumor bodies and the growth of tumors in vivo, so the S protein has good clinical application prospect in the aspect of tumor treatment. The invention provides a brand-new treatment strategy for treating tumors, which is completely different from the existing treatment method.

Description

Application of new coronavirus S protein in preparation of medicine for treating tumor

Technical Field

The invention relates to the field of biological medicine, in particular to application of a novel coronavirus S protein and related products thereof in preparation of a medicine for treating tumors.

Background

The new crown pneumonia epidemic (COVID-19) poses a significant threat to global public health and human health. SARS-CoV-2 virus is the pathogen of new coronary pneumonia epidemic situation, and is the third coronavirus causing human epidemic situation since 21 century.

Spike glycoprotein (also known as Spike protein, or S protein) is the most important surface membrane protein of coronaviruses, and contains two subunits, S1 and S2. Wherein S1 mainly contains Receptor Binding Domain (RBD) responsible for recognizing cell receptor. S2 contains the domains required for the membrane fusion process. The S protein has the functions of binding virus and host cell membrane receptor and membrane fusion, and is an important action site of host neutralizing antibody and a key target of vaccine design. SARS-CoV-2 virus belongs to positive strand RNA virus, and the mode of infecting cells is that after S protein on the surface of virus is combined with angiotensin converting enzyme 2(ACE2) as host cell surface receptor, the virus RNA enters into cells through the fusion path of virus envelope and cell membrane or receptor mediated endocytosis path. After RNA enters a host cell, RNA polymerase required by viral RNA replication and structural proteins required by viral assembly are expressed, the assembly is completed in endoplasmic reticulum, and the RNA is released to the outside of cells in an exocytosis mode. During virus assembly, part of S protein which is not assembled is finally integrated on host cell membrane to form cell with S protein on cell membrane.

During the synthesis of the S protein of SARS-CoV-2, the S1/S2(R685) site is cleaved by serine protease, exposing the N-terminus of the S2 subunit, and when a host cell expressing the S protein is bound to a cell expressing ACE2, the R815 site of the S protein is cleaved again by transmembrane serine protease (TMPRSS2), exposing the Fusion Peptide (FP) which is inserted into the target cell membrane. The C-terminal of FP has two domains of HR1 and HR2, HR1 and HR2 form alpha-helix to draw the distance between virus envelope and cell membrane, to initiate cell fusion, form syncytia and finally death. The new corona infects the lungs of dead patients and the presence of multiple such syncytia is found.

As mentioned above, human angiotensin converting enzyme 2(ACE2) is the main receptor for SARS-CoV-2 and plays a key role in virus invasion. ACE2 expression levels varied in different organs. In normal tissues, ACE2 is mainly highly expressed in organs such as kidney, heart, vascular endothelium, small intestine and the like, and is low in tissues such as spleen, brain, muscle, pituitary and skin, and the expression difference of ACE2 among different genders in different organs is not significant. In most tumor tissues, ACE2 expression levels were generally higher.

At present, no report shows that the novel coronavirus S protein can be used for treating tumors.

Disclosure of Invention

Object of the Invention

The invention aims to provide a new idea for treating tumors, and particularly provides application of S protein of a novel coronavirus (particularly SARS-CoV-2 virus) or related products thereof in preparing a medicament for treating tumors.

Solution scheme

In order to achieve the purpose, the invention provides the following technical scheme:

in a first aspect, the present invention provides a use of the S protein of the novel coronavirus or a functionally related variant thereof, a nucleotide encoding the S protein of the novel coronavirus or a functionally related variant thereof, an expression construct comprising the nucleotide, an expression vector comprising the expression construct, or a transformed host cell comprising the nucleotide, the expression construct or the expression vector, in the preparation of a medicament for preventing and/or treating a tumor, wherein the tumor is a tumor associated with high expression of angiotensin converting enzyme 2.

In a preferred embodiment of the above use, the novel coronavirus is SARS-CoV-2 virus.

Preferably, the functionally related variant of the novel coronavirus S protein is a variant thereof capable of binding to ACE2, preferably with high affinity.

Preferably, the S protein of the new coronavirus has an amino acid sequence shown as SEQ ID NO. 1.

Preferably, the nucleotide encoding the S protein of the novel coronavirus has a sequence shown as SEQ ID NO. 2.

In a particular embodiment, the S protein of the novel coronaviruses or functionally related variants thereof is used for the prevention and/or treatment of tumors by interacting with angiotensin converting enzyme 2 on the surface of tumor cells, preferably forming syncytia.

Preferably, the S protein of the novel coronaviruses or functionally related variants thereof is fused to a tumor-specific binding peptide or antibody to specifically target tumors.

Preferably, the medicament for preventing and/or treating tumors further comprises other antitumor agents.

Preferably, the tumor is selected from the group consisting of: liver cancer, cervical cancer, renal papillary cell carcinoma, colon adenocarcinoma, pancreatic cancer, rectal adenocarcinoma, stomach adenocarcinoma, and lung adenocarcinoma, and other ACE 2-highly expressing tumors.

In a second aspect, the present invention provides a method for inhibiting or treating a tumor, wherein the tumor is a tumor associated with high expression of angiotensin-converting enzyme 2, the method comprising: administering to a subject in need thereof an effective amount of an S protein of a novel coronavirus, or a functionally related variant thereof, a fusion protein of said S protein of a novel coronavirus, or a functionally related variant thereof, a nucleotide encoding said S protein of a novel coronavirus, or a functionally related variant thereof, or said fusion protein, an expression construct comprising said nucleotide, an expression vector comprising said expression construct, or a transformed host cell comprising said nucleotide, expression construct or expression vector.

In a preferred embodiment of the above method, the novel coronavirus is SARS-CoV-2 virus.

Preferably, the functionally related variant of the novel coronavirus S protein is a variant thereof capable of binding to ACE2, preferably with high affinity.

Preferably, the S protein of the new coronavirus has an amino acid sequence shown as SEQ ID NO.1, and the nucleotide encoding the S protein of the new coronavirus has a sequence shown as SEQ ID NO. 2.

In a particular embodiment, the S protein of the novel coronaviruses or functionally related variants thereof is used for the prevention and/or treatment of tumors by interacting with angiotensin converting enzyme 2 on the surface of tumor cells, preferably forming syncytia.

Preferably, the S protein of the novel coronaviruses or a functionally related variant thereof is fused to a tumor-specific binding peptide or antibody to specifically target tumors.

Preferably, the method further comprises administering an additional anti-neoplastic agent, either simultaneously or sequentially.

Preferably, the tumor is selected from the group consisting of: liver cancer, cervical cancer, renal papillary cell carcinoma, colon adenocarcinoma, pancreatic cancer, rectal adenocarcinoma, stomach adenocarcinoma, and lung adenocarcinoma, and other ACE 2-highly expressing tumors.

Advantageous effects

The inventors of the present application discovered, by chance, in long-term scientific research practice, a phenomenon in which tumor cell death is induced by S protein-mediated syncytia formation of new coronaviruses (particularly SARS-CoV-2 virus); based on the phenomenon, the invention provides the application of S protein of a novel coronavirus (particularly SARS-CoV-2 virus) or related products thereof in preparing a medicament for treating tumors.

Through a large number of scientific researches and experiments, the inventor finds that the S protein of SARS-CoV-2 virus can obviously inhibit the activity of tumor cells (such as mouse colon cancer cell MC-38) in vitro by interacting with ACE2 on the surface of the tumor cells (such as forming syncytia), and can obviously inhibit the formation of tumor bodies and the growth of tumors in vivo (such as in a mouse), so that the S protein has a good clinical application prospect in the aspect of tumor treatment. The invention provides a brand-new treatment strategy for treating tumors, which is completely different from the existing treatment method.

Drawings

One or more embodiments are illustrated by the corresponding figures in the drawings, which are not meant to be limiting. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

FIG. 1A shows the amino acid sequences of the fusion genes S-eGFP, S-S1/S2mut-eGFP and hACE2-mCherry constructed by the plasmid construction method in example 1 of the present invention; FIG. 1B shows the fusion of 293T cells with cells expressing hACE2 on the cell surface after being mixed uniformly under the condition of expressing SARS-CoV-2S protein by the method of fluorescence microscope observation in example 3 of the present invention; from left to right, fusion conditions of cells expressing wild type S protein and S1/S2(682/683/685) locus mutant type S protein and cells expressing hACE2 after being mixed uniformly are shown; FIG. 1C shows the calculation of fusion efficiency of each group of cells by the method of nuclear counting in example 3 of the present invention; FIG. 1D shows the detection of the expression level of the wild-type S protein, S1/S2(682/683/685) site-mutated S protein in cells by immunoblotting in example 3 of the present invention.

FIG. 2A is a diagram showing the results of observing the formation of tumors by subcutaneous injection after mixing MC-38 cells expressing wild-type S protein and MC-38 cells expressing hACE2, by the method of mouse subcutaneous tumor formation in example 4 of the present invention; FIG. 2B shows the length and width of tumor body measured by vernier caliper measurement in example 4 of the present invention, according to the formula V ═ 0.5X length X width ² Calculating the volume of tumor bodies of each group; FIG. 2C shows the detection of the expression levels of MC-38 cells stably expressing wild-type S protein, S1/S2(682/683/685) site-mutated S protein, and hACE2, respectively, by immunoblotting as described in example 2 of the present invention.

FIG. 3 shows the inhibitory effect of VSV-S expressing wild-type S protein on the growth of a tumor expressing hACE2, as determined by construction of VSV pseudovirus, and the length and width of the tumor body as measured by vernier caliper measurement in example 5 of the present invention, according to the formula V of 0.5X length X width ² The volume of tumor bodies of each group was calculated.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention. Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, and the like that are well known to those skilled in the art are not described in detail in order to not unnecessarily obscure the present invention.

In the following examples, the biomaterials and experimental methods used were as follows:

biological material

MC-38 cells, 293T cells (from ATCC cell bank), C57 mice (from Sibefu)

Experimental method for cell fusion

Respectively adding 4 mu g of expression vectors of wild type S-eGFP fusion gene, 4 mu g of hACE2-mCherry fusion gene and 4 mu g of S-eGFP fusion gene with mutant site of S1/S2(682/683/685) into 400 mu L of transfection Buffer, uniformly mixing and standing for 5min, adding 8 mu L of transfection reagent (Jetprime from PolyPlus), uniformly mixing and standing for 15min, adding the solution into a cell culture medium, expressing the fusion protein for 24h, and then taking 293T cells expressing the wild type S-eGFP fusion protein, 293T cells expressing the hACE 2-mChery fusion protein, 293T cells expressing the S-eGFP fusion protein with mutant site of S1/S2(682/683/685), and 293T cells expressing the S-eGFP fusion protein with mutant site of hACE 2-mChery fusion protein and expressing the hACE 2-mChery fusion proteinThe 293T cells of the protein are respectively and uniformly mixed according to the cell amount of 1:1 and then planted in a six-hole plate, and the total cell amount is 2 multiplied by 10 ⁵ After 4h, nuclei were stained with Hoechst33342 (Cell Signaling Technology) dye for 15min and observed for fusion by fluorescence microscopy.

Experimental method for detecting S protein and ACE2 expression amount by immunolblotting

Centrifuging cells, adding 100 μ L PBS for resuspension, adding 100 μ L4% SDS cell lysate, boiling for 10min, collecting supernatant at 12000rpm/10min, loading with 8% prefabricated gel, loading 10 μ L, 80V running out concentrated gel, adjusting voltage to 120V, stopping electrophoresis when the solution runs to the lower end of bromophenol blue distance of 1cm, transferring membrane at 18V for 70min, sealing PVDF membrane in rapid sealing solution (Biosharp) for 5min, cutting membrane, adding anti-Spike (antibody: 5% skimmed milk powder ═ 1: 1000, available from GeneTex corporation), anti- α -Tubulin (antibody: 5% skimmed milk powder ═ 1:5000, available from sigma corporation), anti-ACE2 (antibody: 5% skimmed milk powder ═ 1: 1000, available from Abcam corporation), incubating at 37 deg.C for 2h, washing rabbit ST three times, adding secondary antibody (antibody: 5% skimmed milk powder ═ 1: 1000, 7000% skimmed milk powder) respectively, and incubating at 7000 bridge gold bridge corporation, A mouse secondary antibody (antibody: 5% nonfat milk powder: 1:7000, available from Canon Goldia) was incubated at 37 ℃ for 45min, washed three times with TBST, blotted with PVDF membrane, homogenized with a developer (available from PerKinElmer), and developed with a developing apparatus (available from Tanon).

Experimental method for subcutaneous tumor formation of mice

Respectively taking 1 × 10 ⁶ MC-38 cells stably expressing wild-type S-eGFP fusion protein and 1X 10 ⁶ 1X 10 MC-38 cells stably expressing hACE2-mCherry ⁶ MC-38 cells stably expressing wild-type S-eGFP fusion protein and 1X 10 ⁶ 1X 10 MC-38 cells ⁶ MC-38 cells stably expressing hACE2-mCherry and 1 × 10 cells ⁶ MC-38 cells and 2X 10 ⁶ Mixing MC-38 cells, inoculating to C57 mouse, taking out tumor after 21d, measuring the length and width of tumor according to formula V of 0.5 × length × width ² The volume of tumor bodies of each group was calculated.

Experimental method for oncolytic effect of oncolytic virus on hACE2 expressing tumor

Take 2X 10 ⁶ C57 mice were injected subcutaneously into MC-38 cells stably expressing hACE2-mCherry, and 10 cells were injected after 15d tumor formation ⁸ pfuVSV-S, VSV-S inactivated by ultraviolet for 30min and physiological saline of the same volume, removing tumor body at 21d, measuring the length and width of the tumor body, and determining the formula V as 0.5 x length x width ² The volume of tumor bodies of each group was calculated.

Examples

Example 1S-eGFP fusion proteins, S-eGFP fusion proteins with mutations at S1/S2(682/683/685) and preparation of hACE2-mCherry fusion protein

The experimental method comprises the following steps: amplifying SARS-CoV-2-spike fragment by PCR method, seamlessly connecting the amplified fragment with the eGFP-N2 carrier after enzyme digestion of BamHI to form S-eGFP fusion gene; designing a primer to mutate 682/683/685 th arginine in the S sequence into alanine, amplifying a fragment containing a mutation site by PCR, and seamlessly connecting the amplified fragment with an eGFP-N2 carrier after enzyme digestion of BamHI to form an S-eGFP fusion gene with a mutation at the S1/S2(682/683/685) site; the hACE2 fragment was amplified by PCR and seamlessly ligated to the pmCherry-N1 vector to form the hACE2-mCherry fusion gene. The recombinant plasmid is transformed into DH5 alpha competent bacteria, plates are coated at 37 ℃ overnight, monoclonal bacteria are selected and shaken for sequencing, and then the plasmid is extracted.

The experimental results are as follows: as shown in FIG. 1A, the amino acid sequences (shown in SEQ ID NO:3,4 and 5) of the wild-type S-eGFP fusion gene, the S1/S2(682/683/685) site mutated S-eGFP fusion gene and the hACE2-mCherry fusion gene after sequencing are the same as those of the template, and the plasmid construction is successful.

Example 2S-eGFP fusion proteins, S1/S2(682/683/685) site mutated S-eGFP fusion proteins and stable expression of hACE2-mCherry fusion protein in MC-38 cells

The experimental method comprises the following steps: respectively amplifying the S-eGFP fusion gene, the S-eGFP fusion gene with the S1/S2(682/683/685) site mutation and the hACE 2-mChery fusion gene in the example 1 by using a PCR method, and seamlessly connecting the amplified fragments with the PCDH-CMV-MCS-EF1-Puro after enzyme digestion of BamHI to form PCDH-S-eGFP, PCDH-S1/S2 mut-eGFP and PCDH-hACE 2-mChery vectors; 8 mu g of PCDH-S-eGFP or PCDH-S-S1/S2 mut-eGFP or PCDH-hACE 2-mChery is co-transfected with 4 mu g of PsPAX2 and 3 mu g of PMD2.G plasmid into 293T cells, culture supernatant is collected after 48h, centrifugation is carried out at 3000g/10min, lentivirus is obtained after filtration by a 0.45 mu m filter, 4ml of lentivirus is added into MC-38 with 60 percent of confluence, culture medium is supplemented to 10ml, liquid is changed after 24h, the culture medium is changed into DMEM with 4 mu g/ml of puromycin after 48h, single clone is selected for amplification culture, and the expression of the fusion protein is verified by an Immunobot method.

The experimental results are as follows: as shown in fig. 2C, the level of expression of hACE2 was significantly higher in cells expressing hACE2 than in control cells into which hACE2 was not introduced; furthermore, the wild-type S-eGFP fusion protein has the same expression level as the S-eGFP fusion protein with the mutation at the S1/S2(682/683/685) site, which indicates that S, S-S1/S2mut and hACE2 realize stable expression in MC-38 cells.

Example 3 fusion of cells expressing wild-type S protein with cells expressing hACE2

The experimental method comprises the following steps:

1. the expression vectors of 4. mu.g of wild-type S-eGFP fusion gene, 4. mu.g of hACE2-mCherry fusion gene and 4. mu.g of S-eGFP fusion gene with mutant S1/S2(682/683/685) sites were added to 400. mu.L of transfection Buffer, mixing, standing for 5min, adding 8 μ L transfection reagent (JetPRIME, purchased from Polyplus), mixing, standing for 15min, adding the solution into cell culture medium, after 24h of fusion protein expression, 293T cells expressing wild type S-eGFP fusion protein, 293T cells expressing hACE2-mCherry fusion protein, 293T cells expressing S-eGFP fusion protein with S1/S2(682/683/685) site mutation and 293T cells expressing hACE 2-mChery fusion protein are uniformly mixed according to the cell amount of 1:1 and planted in a six-well plate, and the total cell amount is 2 multiplied by 10. ⁵ 4h later, the cell nucleus was stained with Hoechst33342 dye for 15min, and the fusion was observed and counted by a fluorescence microscope (the results are shown in FIGS. 1B and 1C). furthermore, in order to exclude the influence of the expression level of S protein on the experimental results,the inventors also examined the expression level of the wild-type S protein, S1/S2(682/683/685) site-mutated S protein, in cells by immunoblotting (the results are shown in FIG. 1D).

2. 293T cells transiently transformed with wild type S-eGFP fusion protein, 293T cell precipitates transiently transformed with S1/S2(682/683/685) site-mutated S-eGFP fusion protein were resuspended in 100. mu.L of PBS, 100. mu.L of 4% SDS cell lysate was added, the mixture was boiled for 10min, supernatant was collected at 12000rpm/10min, the supernatant was applied to 8% pre-made gel in an amount of 10. mu.L, 80V was discharged from the concentrated gel and the voltage was adjusted to 120V, electrophoresis was stopped when the solution was discharged to the lower end of the bromophenol blue distance of 1cm, PVDF membrane was applied to a rapid blocking solution (Biosharp) for 5min after 70min, anti-Spike (antibody: 5% nonfat milk powder: 1: 1000, available from GeneTex) was incubated for 1 min, anti- α -Tubulin (antibody: 5% nonfat milk powder: 5000, available from TBsigap) was incubated for 2h, and then antibody: ST 5% at antibody: 7000% from TBSIGMA (antibody: 1: 7000), purchased from sequoia ponticum), a murine secondary antibody (antibody: 5% skim milk powder 1:7000 from sequoia) for 45min, TBST three times, followed by blotting of PVDF membrane, application of developer (from PerKinElmer) and development with a developer (from Tanon).

The experimental results are as follows:

1) as shown in FIG. 1B, after the 293T cell transiently transformed with the wild-type S-eGFP fusion protein is mixed with the 293T cell transiently transformed into hACE2-mCherry, the cell is obviously fused, and after the 293T cell transiently transformed into the S1/S2(682/683/685) site mutation S-eGFP fusion protein is mixed with the 293T cell transiently transformed into hACE2-mCherry, the cell fusion phenomenon is not obvious;

2) as shown in fig. 1C, the fusion efficiency of 293T cells transiently transformed with wild-type S-eGFP fusion protein after being mixed with 293T cells transiently transformed with hACE2 was significantly higher than that of 293T cells of S-eGFP fusion protein mutated at S1/S2(682/683/685) site after being mixed with 293T cells of transient hACE2-mCherry, P < 0.05; p < 0.01; p < 0.001;

3) as shown in FIG. 1D, the transient wild-type S-eGFP fusion protein was expressed at the same level as the S-eGFP fusion protein with the mutation at the S1/S2(682/683/685) site; the results indicate that the above-mentioned difference in fusion efficiency is not caused by the difference in the expression amount of S protein.

Example 4 reduction of tumorigenicity ability of cells expressing wild-type S protein when mixed with cells expressing hACE2

The experimental method comprises the following steps:

1. will be 1 × 10 ⁶ MC-38 cells stably expressing wild-type S-eGFP fusion protein and 1X 10 ⁶ 1X 10 MC-38 cells stably expressing hACE2-mCherry ⁶ MC-38 cells stably expressing hACE2-mCherry and 1 × 10 cells ⁶ 1X 10 MC-38 cells ⁶ MC-38 cells stably expressing wild-type S-eGFP fusion protein and 1X 10 ⁶ MC-38 cells, 2X 10 ⁶ Mixing MC-38 cells, inoculating to C57 mouse, taking out tumor after 21d, measuring the length and width of tumor according to formula V of 0.5 × length × width ² The volume of tumor bodies of each group was calculated. The cells were prepared as described in example 2 above, except for the MC-38 cells.

2. MC-38 cells stably expressing wild type S-eGFP fusion protein, MC-38 cells stably expressing hACE2-mCherry, MC-38 cells stably expressing S1/S2(682/683/685) site mutated S-eGFP fusion protein and control cell precipitates are respectively added with 100 mu L PBS for heavy suspension, 100 mu L4% SDS cell lysate is added, after boiling for 10min, supernatant is taken at 12000rpm/10min, 8% pre-gel is used for sample loading, the sample loading amount is 10 mu L, 80V is run out of concentrated gel, the voltage is adjusted to 120V, the electrophoresis is stopped when the concentrated gel runs to the lower end of bromophenol blue distance of 1cm, PVDF membrane is placed in rapid blocking liquid (purchased from Biosharp company) after 70min of 18V membrane transfer, the membrane is cut after 5min of blocking, anti-Spike (antibody: 5% defatted milk powder: 1: 1000, purchased from GeneTex company) and anti-Tubulin (antibody: 5% defatted milk powder: 5000: 1: 5000), purchased from sigma), anti-ACE2 (antibody: 5% of skimmed milk powder as 1: 1000, purchased from Abcam), incubated at 37 ℃ for 2h, washed three times with TBST, and added with a rabbit secondary antibody (antibody: 5% skim milk powder 1:7000, available from sequoia ponticum), murine secondary antibody (antibody: 5% skim milk powder 1:7000 from sequoia jinqiao), 45min at 37 ℃, washed three times with TBST, blotted dry with developer (from PerKinElmer) and developed with a developer (from Tanon).

The experimental results are shown in fig. 2A, 2B and 2C, respectively. Fig. 2A shows: the MC-38 cell expressing the wild S protein and the MC-38 cell expressing hACE2 are mixed uniformly and then do not form tumor under the skin of the mouse, and the rest groups form tumor bodies; FIG. 2B shows: MC-38 cells expressing wild type S protein and MC-38 cells expressing hACE2 are mixed uniformly and injected subcutaneously, no tumor is formed, and the other groups form tumor bodies, so that the obvious difference is that P is less than 0.05; p < 0.01; p < 0.001; the results show that: the wild S protein can obviously inhibit the formation of tumor bodies in mice. Fig. 2C shows: the MC-38 cells stably expressing the wild type S-eGFP fusion protein and the MC-38 cells stably expressing the S-eGFP fusion protein with mutation at the S1/S2(682/683/685) site have consistent S protein expression amount which is obviously higher than that of a control cell, the MC-38 cells stably expressing hACE2 and ACE2 expression amount which is obviously higher than that of the control cell show that: the construction of the stable cell line is successful.

Example 5 oncolytic viruses expressing wild-type S protein significantly reduced the size of hACE2 expressing tumors

The experimental method comprises the following steps:

1. amplifying an S fragment of SARS-CoV-2 by using a PCR method, seamlessly connecting the amplified fragment with a pVSVAG-GFP vector to obtain a pVSVAG-GFP-S plasmid, co-transfecting the pVSVAG-GFP-S, PBS-N, PBS-P, PBS-G and PBS-L to BHK21 cells according to the ratio of 5:3:5:8:1 (mu G), collecting supernatant to 293T-ACE2 cells transfected with pCAGGS-G after 72h, collecting supernatant for two blind passages after 72h, centrifuging at 3000G/10min, and filtering by using a 0.45 mu m filter to obtain VSV-S.

2. Take 2X 10 ⁶ C57 mice were subcutaneously injected with MC-38 cells stably expressing hACE2-mCherry prepared in example 2, and 10 cells were injected after 15d tumor formation ⁸ Pfu VSV-S, VSV-S inactivated by ultraviolet for 30min and physiological saline of the same volume, removing tumor body at 21d, measuring the length and width of the tumor body, and determining the formula V as 0.5 x length x width ² Calculating the volume of tumor bodies of each group;

the results of the experiment are shown in FIG. 3, which shows: the volume of the tumor body of the oncolytic virus expressing VSV-S is obviously smaller than that of a control group which is not injected and a VSV-S control group which is inactivated by ultraviolet for 30min, wherein P is less than 0.05; p < 0.01; p < 0.001.

These results show that: wild-type S protein significantly reduced tumor growth in mice.

According to the above examples, wild-type S protein can significantly inhibit the viability of tumor cells in vitro, significantly inhibit tumor formation in vivo, and significantly inhibit tumor growth in vivo by forming a syncytium with ACE2, and therefore, has an obvious effect of inhibiting or regressing ACE of tumors expressing ACE2 on the cell surface, and is expected to become a new tumor treatment strategy.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Shunhu biomedical technology, Inc. of Beijing

Application of novel coronavirus S protein in preparation of drugs for treating tumors

<130> 1048-210174F

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 1295

<212> PRT

<213> SARS-CoV-2

<220>

<221> DOMAIN

<222> (1)..(1295)

<223> amino acid sequence of SARS-CoV-2 virus S protein

<400> 1

Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly

1 5 10 15

Met Leu Val Ala Ser Val Leu Ala Gln Cys Val Asn Leu Thr Thr Arg

20 25 30

Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr

35 40 45

Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp

50 55 60

Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His

65 70 75 80

Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro

85 90 95

Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile

100 105 110

Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu

115 120 125

Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe

130 135 140

Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn

145 150 155 160

Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn

165 170 175

Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly

180 185 190

Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile

195 200 205

Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val

210 215 220

Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu

225 230 235 240

Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His

245 250 255

Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly

260 265 270

Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu

275 280 285

Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu

290 295 300

Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu

305 310 315 320

Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser

325 330 335

Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val

340 345 350

Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg

355 360 365

Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser

370 375 380

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

385 390 395 400

Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp

405 410 415

Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr

420 425 430

Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn

435 440 445

Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr

450 455 460

Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser

465 470 475 480

Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly

485 490 495

Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn

500 505 510

Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu

515 520 525

Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu

530 535 540

Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr

545 550 555 560

Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe

565 570 575

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

580 585 590

Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val

595 600 605

Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln

610 615 620

Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu

625 630 635 640

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

645 650 655

Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu

660 665 670

Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln

675 680 685

Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala Ser Gln Ser Ile Ile

690 695 700

Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn

705 710 715 720

Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu

725 730 735

Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr

740 745 750

Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly

755 760 765

Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu

770 775 780

Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr

785 790 795 800

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

805 810 815

Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu

820 825 830

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

835 840 845

Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln

850 855 860

Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met

865 870 875 880

Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly

885 890 895

Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln

900 905 910

Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr

915 920 925

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

930 935 940

Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln

945 950 955 960

Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln

965 970 975

Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu

980 985 990

Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile

995 1000 1005

Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu

1010 1015 1020

Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr

1025 1030 1035

Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

1040 1045 1050

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro

1055 1060 1065

His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu

1070 1075 1080

Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala

1085 1090 1095

His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp

1100 1105 1110

Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr

1115 1120 1125

Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile

1130 1135 1140

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

1145 1150 1155

Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro

1160 1165 1170

Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val

1175 1180 1185

Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn

1190 1195 1200

Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu

1205 1210 1215

Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala

1220 1225 1230

Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met

1235 1240 1245

Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser

1250 1255 1260

Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly

1265 1270 1275

Val Lys Leu His Tyr Thr Gly Gly Thr Glu Thr Ser Gln Val Ala

1280 1285 1290

Pro Ala

1295

<210> 2

<211> 3888

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(3888)

<223> nucleotide sequence encoding S protein of SARS-CoV-2 virus

<400> 2

atgcccatgg ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct 60

tccgtgctag cccagtgcgt gaacctgacc acaaggaccc agctgccccc tgcctatacc 120

aattccttca cacggggcgt gtactatccc gacaaggtgt ttagaagctc cgtgctgcac 180

tctacacagg atctgtttct gcctttcttt agcaacgtga cctggttcca cgccatccac 240

gtgagcggca ccaatggcac aaagcggttc gacaatccag tgctgccctt taacgatggc 300

gtgtacttcg cctctaccga gaagagcaac atcatcagag gctggatctt tggcaccaca 360

ctggactcca agacacagtc tctgctgatc gtgaacaatg ccaccaacgt cgtgatcaag 420

gtgtgcgagt tccagttttg taatgatcct ttcctgggcg tgtactatca caagaacaat 480

aagagctgga tggagtccga gtttcgcgtg tattctagcg ccaacaattg cacatttgag 540

tacgtgtccc agccattcct gatggacctg gagggcaagc agggcaattt caagaacctg 600

agggagttcg tgtttaagaa tatcgatggc tacttcaaga tctactctaa gcacacccca 660

atcaacctgg tgcgcgacct gccacagggc ttcagcgccc tggagccact ggtggatctg 720

cccatcggca tcaacatcac ccggtttcag acactgctgg ccctgcacag aagctacctg 780

acacctggcg actcctctag cggatggacc gcaggagctg ccgcctacta tgtgggctat 840

ctgcagccaa ggaccttcct gctgaagtac aacgagaatg gcaccatcac agacgcagtg 900

gattgcgcac tggaccccct gagcgagacc aagtgtacac tgaagtcctt taccgtggag 960

aagggcatct atcagacatc caatttcagg gtgcagccca ccgagtctat cgtgcgcttt 1020

cccaatatca caaacctgtg cccttttggc gaggtgttca acgcaaccag gttcgcaagc 1080

gtgtacgcat ggaataggaa gcggatcagc aactgcgtgg ccgactatag cgtgctgtac 1140

aactccgcct ctttcagcac ctttaagtgc tatggcgtgt cccccacaaa gctgaatgac 1200

ctgtgcttta ccaacgtgta cgccgattct ttcgtgatca ggggcgacga ggtgcgccag 1260

atcgcaccag gacagacagg caagatcgca gactacaatt ataagctgcc tgacgatttc 1320

accggctgcg tgatcgcctg gaacagcaac aatctggatt ccaaagtggg cggcaactac 1380

aattatctgt accggctgtt tagaaagtct aatctgaagc cattcgagag ggacatctct 1440

acagagatct accaggcagg cagcacccca tgcaatggag tggagggctt taactgttat 1500

ttccctctgc agagctacgg cttccagcca acaaacggcg tgggctatca gccctaccgc 1560

gtggtggtgc tgagctttga gctgctgcac gcacctgcaa cagtgtgcgg accaaagaag 1620

tccaccaatc tggtgaagaa caagtgcgtg aacttcaact tcaacggact gaccggcaca 1680

ggcgtgctga ccgagtccaa caagaagttc ctgccctttc agcagttcgg cagggacatc 1740

gcagatacca cagacgccgt gcgcgaccct cagaccctgg agatcctgga catcacacca 1800

tgctctttcg gcggcgtgag cgtgatcaca cctggcacca atacaagcaa ccaggtggcc 1860

gtgctgtatc aggacgtgaa ttgtaccgag gtgcccgtgg caatccacgc agatcagctg 1920

acccctacat ggcgggtgta cagcaccggc tccaacgtgt tccagacaag agccggatgc 1980

ctgatcggag cagagcacgt gaacaattcc tatgagtgcg acatccctat cggcgccggc 2040

atctgtgcct cttaccagac ccagacaaac tctccaagga gagcccggag cgtggcatcc 2100

cagtctatca tcgcctatac aatgagcctg ggcgccgaga acagcgtggc ctactctaac 2160

aatagcatcg ccatccctac caacttcaca atctccgtga ccacagagat cctgccagtg 2220

tccatgacca agacatctgt ggactgcaca atgtatatct gtggcgattc taccgagtgc 2280

agcaacctgc tgctgcagta cggcagcttt tgtacccagc tgaatagagc cctgacaggc 2340

atcgccgtgg agcaggacaa gaacacacag gaggtgttcg cccaggtgaa gcagatctac 2400

aagaccccac ccatcaagga ctttggcggc ttcaattttt cccagatcct gcccgatcct 2460

tccaagcctt ctaagcggag ctttatcgag gacctgctgt tcaacaaggt gaccctggcc 2520

gatgccggct tcatcaagca gtatggcgat tgcctgggcg acatcgccgc cagagacctg 2580

atctgtgccc agaagtttaa tggcctgacc gtgctgcctc cactgctgac agatgagatg 2640

atcgcacagt acacaagcgc cctgctggca ggcaccatca catccggatg gaccttcggc 2700

gcaggagccg ccctgcagat ccccttcgct atgcagatgg cctatcggtt caacggcatc 2760

ggcgtgaccc agaatgtgct gtacgagaac cagaagctga tcgccaatca gtttaactcc 2820

gccatcggca agatccagga cagcctgtcc tctacagcct ccgccctggg caagctgcag 2880

gatgtggtga atcagaacgc ccaggccctg aataccctgg tgaagcagct gagctccaac 2940

ttcggcgcca tctctagcgt gctgaatgac atcctgagcc ggctggacaa ggtggaggca 3000

gaggtgcaga tcgaccggct gatcacaggc agactgcagt ctctgcagac ttacgtgaca 3060

cagcagctga tcagggcagc agagatcagg gcaagcgcca atctggcagc aaccaagatg 3120

tccgagtgcg tgctgggcca gtctaagaga gtggactttt gtggcaaggg ctatcacctg 3180

atgtccttcc cacagtctgc ccctcacgga gtggtgtttc tgcacgtgac ctacgtgcca 3240

gcccaggaga agaacttcac cacagcacca gcaatctgcc acgatggcaa ggcacacttt 3300

cccagggagg gcgtgttcgt gagcaacggc acccactggt ttgtgacaca gcgcaatttc 3360

tacgagcctc agatcatcac cacagacaat acattcgtgt ccggcaactg tgacgtggtc 3420

atcggcatcg tgaacaatac cgtgtatgat cctctgcagc cagagctgga cagctttaag 3480

gaggagctgg ataagtactt caagaatcac acctccccag acgtggatct gggcgacatc 3540

agcggcatca atgcctccgt ggtgaacatc cagaaggaga tcgacaggct gaacgaggtg 3600

gccaagaatc tgaacgagag cctgatcgat ctgcaggagc tgggcaagta tgagcagtac 3660

atcaagtggc cctggtatat ctggctgggc ttcatcgccg gcctgatcgc tatcgtgatg 3720

gtgaccatca tgctgtgctg tatgacatcc tgctgttctt gcctgaaggg ctgctgtagc 3780

tgtggctcct gctgtaagtt tgatgaggac gattccgagc cagtgctgaa gggcgtgaag 3840

ctgcactaca ccggcggcac cgagacatct caggtggccc ccgcctaa 3888

<210> 3

<211> 1541

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(1541)

<223> amino acid sequence of fusion protein S-eGFP

<400> 3

Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly

1 5 10 15

Met Leu Val Ala Ser Val Leu Ala Gln Cys Val Asn Leu Thr Thr Arg

20 25 30

Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr

35 40 45

Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp

50 55 60

Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His

65 70 75 80

Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro

85 90 95

Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile

100 105 110

Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu

115 120 125

Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe

130 135 140

Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn

145 150 155 160

Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn

165 170 175

Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly

180 185 190

Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile

195 200 205

Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val

210 215 220

Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu

225 230 235 240

Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His

245 250 255

Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly

260 265 270

Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu

275 280 285

Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu

290 295 300

Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu

305 310 315 320

Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser

325 330 335

Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val

340 345 350

Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg

355 360 365

Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser

370 375 380

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

385 390 395 400

Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp

405 410 415

Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr

420 425 430

Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn

435 440 445

Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr

450 455 460

Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser

465 470 475 480

Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly

485 490 495

Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn

500 505 510

Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu

515 520 525

Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu

530 535 540

Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr

545 550 555 560

Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe

565 570 575

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

580 585 590

Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val

595 600 605

Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln

610 615 620

Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu

625 630 635 640

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

645 650 655

Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu

660 665 670

Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln

675 680 685

Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala Ser Gln Ser Ile Ile

690 695 700

Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn

705 710 715 720

Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu

725 730 735

Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr

740 745 750

Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly

755 760 765

Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu

770 775 780

Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr

785 790 795 800

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

805 810 815

Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu

820 825 830

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

835 840 845

Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln

850 855 860

Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met

865 870 875 880

Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly

885 890 895

Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln

900 905 910

Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr

915 920 925

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

930 935 940

Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln

945 950 955 960

Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln

965 970 975

Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu

980 985 990

Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile

995 1000 1005

Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu

1010 1015 1020

Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr

1025 1030 1035

Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

1040 1045 1050

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro

1055 1060 1065

His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu

1070 1075 1080

Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala

1085 1090 1095

His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp

1100 1105 1110

Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr

1115 1120 1125

Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile

1130 1135 1140

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

1145 1150 1155

Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro

1160 1165 1170

Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val

1175 1180 1185

Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn

1190 1195 1200

Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu

1205 1210 1215

Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala

1220 1225 1230

Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met

1235 1240 1245

Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser

1250 1255 1260

Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly

1265 1270 1275

Val Lys Leu His Tyr Thr Gly Gly Thr Glu Thr Ser Gln Val Ala

1280 1285 1290

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

1295 1300 1305

Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly

1310 1315 1320

Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly

1325 1330 1335

Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr

1340 1345 1350

Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr

1355 1360 1365

Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln

1370 1375 1380

His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

1385 1390 1395

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala

1400 1405 1410

Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu

1415 1420 1425

Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys

1430 1435 1440

Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp

1445 1450 1455

Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn

1460 1465 1470

Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn

1475 1480 1485

Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr

1490 1495 1500

Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg

1505 1510 1515

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr

1520 1525 1530

Leu Gly Met Asp Glu Leu Tyr Lys

1535 1540

<210> 4

<211> 1541

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(1541)

<223> amino acid sequence of fusion protein S1/S2(682, 683, 685) -mut-eGFP

<400> 4

Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly

1 5 10 15

Met Leu Val Ala Ser Val Leu Ala Gln Cys Val Asn Leu Thr Thr Arg

20 25 30

Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr

35 40 45

Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp

50 55 60

Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His

65 70 75 80

Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro

85 90 95

Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile

100 105 110

Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu

115 120 125

Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe

130 135 140

Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn

145 150 155 160

Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn

165 170 175

Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly

180 185 190

Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile

195 200 205

Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val

210 215 220

Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu

225 230 235 240

Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His

245 250 255

Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly

260 265 270

Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu

275 280 285

Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu

290 295 300

Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu

305 310 315 320

Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser

325 330 335

Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val

340 345 350

Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg

355 360 365

Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser

370 375 380

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

385 390 395 400

Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp

405 410 415

Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr

420 425 430

Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn

435 440 445

Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr

450 455 460

Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser

465 470 475 480

Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly

485 490 495

Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn

500 505 510

Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu

515 520 525

Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu

530 535 540

Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr

545 550 555 560

Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe

565 570 575

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

580 585 590

Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val

595 600 605

Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln

610 615 620

Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu

625 630 635 640

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

645 650 655

Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu

660 665 670

Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln

675 680 685

Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala Ser Gln Ser Ile Ile

690 695 700

Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn

705 710 715 720

Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu

725 730 735

Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr

740 745 750

Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly

755 760 765

Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu

770 775 780

Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr

785 790 795 800

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

805 810 815

Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu

820 825 830

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

835 840 845

Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln

850 855 860

Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met

865 870 875 880

Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly

885 890 895

Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln

900 905 910

Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr

915 920 925

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

930 935 940

Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln

945 950 955 960

Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln

965 970 975

Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu

980 985 990

Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile

995 1000 1005

Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu

1010 1015 1020

Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr

1025 1030 1035

Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

1040 1045 1050

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro

1055 1060 1065

His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu

1070 1075 1080

Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala

1085 1090 1095

His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp

1100 1105 1110

Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr

1115 1120 1125

Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile

1130 1135 1140

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

1145 1150 1155

Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro

1160 1165 1170

Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val

1175 1180 1185

Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn

1190 1195 1200

Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu

1205 1210 1215

Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala

1220 1225 1230

Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met

1235 1240 1245

Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser

1250 1255 1260

Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly

1265 1270 1275

Val Lys Leu His Tyr Thr Gly Gly Thr Glu Thr Ser Gln Val Ala

1280 1285 1290

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

1295 1300 1305

Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly

1310 1315 1320

Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly

1325 1330 1335

Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr

1340 1345 1350

Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr

1355 1360 1365

Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln

1370 1375 1380

His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

1385 1390 1395

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala

1400 1405 1410

Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu

1415 1420 1425

Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys

1430 1435 1440

Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp

1445 1450 1455

Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn

1460 1465 1470

Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn

1475 1480 1485

Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr

1490 1495 1500

Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg

1505 1510 1515

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr

1520 1525 1530

Leu Gly Met Asp Glu Leu Tyr Lys

1535 1540

<210> 5

<211> 1064

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(1064)

<223> amino acid sequence of fusion protein hACE2-mCherry

<400> 5

Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala

1 5 10 15

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

20 25 30

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

35 40 45

Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu

225 230 235 240

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

245 250 255

Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly

260 265 270

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

275 280 285

Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala

290 295 300

Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr

485 490 495

Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe

580 585 590

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

595 600 605

Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu

610 615 620

Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met

625 630 635 640

Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu

645 650 655

Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val

660 665 670

Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro

675 680 685

Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile

690 695 700

Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn

705 710 715 720

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

725 730 735

Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val

740 745 750

Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg

755 760 765

Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile

770 775 780

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

785 790 795 800

Val Gln Thr Ser Phe Gly Ser Gly Ser Gly Ser Pro Ile Leu Gln Ser

805 810 815

Thr Val Pro Arg Ala Arg Asp Pro Pro Val Ala Thr Met Val Ser Lys

820 825 830

Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe Lys

835 840 845

Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly

850 855 860

Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys

865 870 875 880

Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro

885 890 895

Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala Asp Ile

900 905 910

Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg

915 920 925

Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser

930 935 940

Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr

945 950 955 960

Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp

965 970 975

Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Gly

980 985 990

Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly His Tyr Asp Ala

995 1000 1005

Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro

1010 1015 1020

Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser His Asn

1025 1030 1035

Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg

1040 1045 1050

His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys

1055 1060

Claims (10)

1. Use of the S protein of a novel coronavirus or a functionally related variant thereof, a nucleotide encoding the S protein of a novel coronavirus or a functionally related variant thereof, an expression construct comprising the nucleotide, an expression vector comprising the expression construct or a transformed host cell comprising the nucleotide, the expression construct or the expression vector for the preparation of a medicament for the prevention and/or treatment of a tumor, wherein the tumor is a tumor associated with high expression of angiotensin converting enzyme 2(ACE 2).

2. The use of claim 1, wherein the new coronavirus is a SARS-CoV-2 virus;

preferably, the functionally related variant of the novel coronavirus S protein is a variant thereof capable of binding, preferably with high affinity, to ACE 2;

preferably, the S protein of the new coronavirus has an amino acid sequence shown as SEQ ID NO. 1;

preferably, the nucleotide encoding the S protein of the novel coronavirus has a sequence shown as SEQ ID NO. 2.

3. Use according to claim 1 or 2, wherein the S protein of the neocoronaviruse or a functionally related variant thereof is used for the prevention and/or treatment of tumors by interacting with angiotensin converting enzyme 2 on the surface of tumor cells, preferably forming syncytia.

4. The use according to any one of claims 1 to 3, wherein the S protein of the neocoronaviruse or a functionally related variant thereof is fused to a tumor-specific binding peptide or antibody;

preferably, the medicament also comprises other antineoplastic agents.

5. The use of any one of claims 1-4, wherein the tumor is selected from the group consisting of: liver cancer, cervical cancer, renal papillary cell carcinoma, colon adenocarcinoma, pancreatic cancer, rectal adenocarcinoma, stomach adenocarcinoma, and lung adenocarcinoma, and other ACE 2-highly expressing tumors.

6. A method for preventing or treating tumors, wherein the tumors are tumors related to high expression of angiotensin-converting enzyme 2, and the method comprises the following steps: administering to a subject in need thereof a prophylactically or therapeutically effective amount of a fusion protein of the S protein of a novel coronavirus, or a functionally related variant thereof, with a tumor-specific targeting peptide, a nucleotide encoding the S protein of said novel coronavirus, or a functionally related variant thereof, or said fusion protein, an expression construct comprising the nucleotide, an expression vector comprising the expression construct, or a transformed host cell comprising the nucleotide, the expression construct or the expression vector.

7. The method of claim 6, wherein the novel coronavirus is SARS-CoV-2 virus;

preferably, the functionally related variant of the novel coronavirus S protein is a variant thereof capable of binding, preferably with high affinity, to ACE 2;

preferably, the S protein of the new coronavirus has an amino acid sequence shown as SEQ ID NO. 1;

preferably, the nucleotide encoding the S protein of the novel coronavirus has a sequence shown as SEQ ID NO. 2.

8. The method according to claim 6 or 7, wherein the S protein of the neocoronaviruse or a functionally related variant thereof is used for the prevention or treatment of tumors by interacting with angiotensin-converting enzyme 2 on the surface of tumor cells, preferably forming syncytia.

9. The method according to any one of claims 6 to 8, wherein the S protein of the neocoronavirus, or a functionally related variant thereof, is fused to a tumor-specific binding peptide or antibody;

preferably, the method further comprises administering an additional anti-neoplastic agent, either simultaneously or sequentially.

10. The method of any one of claims 6-9, wherein the tumor is selected from the group consisting of: liver cancer, cervical cancer, renal papillary cell carcinoma, colon adenocarcinoma, pancreatic cancer, rectal adenocarcinoma, stomach adenocarcinoma, and lung adenocarcinoma, and other tumors highly expressing ACE 2.

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