CN113493496A

CN113493496A - Epitope polypeptide of 2019-nCoV new coronavirus S protein and application thereof

Info

Publication number: CN113493496A
Application number: CN202110297823.5A
Authority: CN
Inventors: 胡兰靛; 张宇超; 吴振川; 郭威
Original assignee: Anda Biological Drug Development Shenzhen Co ltd
Current assignee: Anda Biological Drug Development Shenzhen Co ltd
Priority date: 2020-03-20
Filing date: 2021-03-19
Publication date: 2021-10-12
Anticipated expiration: 2041-03-19
Also published as: CN113493496B

Abstract

The invention discloses epitope polypeptides of 2019-nCoV new coronavirus S protein and application thereof. The amino acid sequence of the epitope polypeptide is selected from one of the following: (1) an amino acid sequence shown as SEQ ID NO. 1; or an amino acid sequence which has more than 90 percent of homology with the amino acid sequence shown in SEQ ID NO.1 and has the same function; (2) an amino acid sequence shown as SEQ ID NO. 2; or an amino acid sequence which has more than 90 percent of homology with the amino acid sequence shown in SEQ ID NO.2 and has the same function; (3) an amino acid sequence shown as SEQ ID NO. 3; or an amino acid sequence which has more than 90 percent of homology with the amino acid sequence shown in SEQ ID NO.3 and has the same function. The high immunogenicity polypeptide screened by the invention can effectively induce an organism to generate immune response, simultaneously avoids the interference of irrelevant antigens and reduces the side reaction of the vaccine.

Description

Epitope polypeptide of 2019-nCoV new coronavirus S protein and application thereof

Technical Field

The invention belongs to the technical field of polypeptide vaccines, and particularly relates to an epitope polypeptide of 2019-nCoV new coronavirus S protein and application thereof.

Background

The current research and development aiming at the novel coronavirus (2019-nCoV) vaccine is basically the same as the strategy of developing the SARS-CoV vaccine in the last 20 years, and comprises (i) inactivated virus vaccine, (ii) virus vector vaccine and (iii) subunit vaccine. The polypeptide vaccine designed by the invention belongs to a novel subunit vaccine. Subunit vaccine is prepared by extracting special protein structure of bacteria and virus through chemical decomposition or controlled protein hydrolysis process and screening out immunological active segment. In the immune reaction process of pathogens and organisms, only a small amount of antigens have immunogenicity so as to stimulate the organisms to generate immune response, so that the subunit vaccine prepared by screening the antigens with immune activity can effectively play the role of the vaccine, simultaneously avoid the antibodies induced by irrelevant antigens and reduce the side reaction of the vaccine. At present, China is in a relatively laggard position in the aspect of development of subunit vaccines.

The novel coronavirus 2019-nCoV particle comprises 4 structural proteins, namely spike protein, membrane protein, envelope protein and nucleocapsid protein. Wherein the spike protein is encoded by the S gene and is a coat protein for enveloped viruses, also called S protein. The S protein is directed to the endoplasmic reticulum by its amino-terminal signal peptide and is highly N-glycosylated, and its trimer forms a unique spike structure on the surface of the virus. The S protein can be cleaved by furin (furin) of the host into polypeptide fragments S1 and S2, wherein S1 is the receptor binding domain and S2 serves as the stem of the spike molecule. The S protein mediates virus entry primarily through binding to host cell receptors and determines viral tissue or host tropism. Research shows that the S protein is a key component of a new coronavirus infected organism and is also a key target for developing a new coronary pneumonia vaccine and an antibody inhibitor.

Disclosure of Invention

The invention aims to provide a group of antigen epitope polypeptides of 2019-nCoV new coronavirus S protein and application thereof.

The invention carries out immune affinity analysis on MHC molecules and virus protein molecules of patients, predicts the antigen epitope with high immunogenicity, and is used as a polypeptide vaccine for stimulating T cell immune response. Meanwhile, the antigen epitope with high affinity of B cell epitope is screened to be used as a polypeptide vaccine for inducing the humoral immune response of the B cell. The length of a peptide segment combined by MHC molecules is 8-11, a groove is formed on the molecule and is a part for combining antigens, namely a peptide-binding groove or cleft, and 6 pockets are formed at the bottom of the groove: pocket A-F, Pocket A and Pocket F are responsible for fixing peptide segments, and Pocket B-E are responsible for combining different peptide segments and keeping specificity. The characteristic of the combination of the MHC molecule antigen binding groove and the antigen peptide is combined with the antigen peptide of the MHC binding complex, which often has two or more than two key amino acid (anchoring residue) specialties and a polypeptide binding motif in the MHC molecule peptide binding groove, and the two have certain specificity. The invention utilizes a machine learning model, trains by using the known combination condition of MHC molecules and antigens to obtain an affinity model, thereby predicting the affinity of the antigen peptide of the novel coronavirus and the MHC molecules. Predicting B cell antigenic determinants according to protein sequences by using a random forest algorithm, training epitope and non-epitope amino acids determined according to a crystal structure by using the algorithm, and screening peptide fragments with scores higher than a set threshold value to serve as potential BCR recognition vaccine molecules.

According to the invention, through a biological information algorithm, the recognition mode of a virus antigen and a host immune system is simulated, a polypeptide sequence with high immunogenicity is screened and predicted from the 2019-nCoV new coronavirus S protein to serve as a candidate antigen, and then the antigen epitope polypeptide with the vaccine effect is obtained through further validity experimental verification of a polypeptide vaccine.

The invention provides an epitope polypeptide of 2019-nCoV new coronavirus S protein, wherein the amino acid sequence of the epitope polypeptide is selected from one of the following:

(1) an amino acid sequence shown as SEQ ID NO. 1; or an amino acid sequence which has more than 90 percent of homology with the amino acid sequence shown in SEQ ID NO.1 and has the same function;

(2) an amino acid sequence shown as SEQ ID NO. 2; or an amino acid sequence which has more than 90 percent of homology with the amino acid sequence shown in SEQ ID NO.2 and has the same function;

(3) an amino acid sequence shown as SEQ ID NO. 3; or an amino acid sequence which has more than 90 percent of homology with the amino acid sequence shown in SEQ ID NO.3 and has the same function.

The second aspect of the invention provides a polynucleotide sequence encoding the epitope polypeptide of the 2019-nCoV neo-coronavirus S protein.

The third aspect of the invention provides the application of one or more of the antigen epitope polypeptides in the preparation of human coronavirus vaccines. Preferably, the human coronavirus is 2019-nCoV neocoronavirus.

The fourth aspect of the invention provides the application of one or more of the antigen epitope polypeptides in preparing medicines for resisting human coronavirus infection. Preferably, the human coronavirus is 2019-nCoV neocoronavirus. The mechanism of the epitope polypeptide as an anti-2019-nCoV new coronavirus drug is as follows: as blocking peptide for blocking virus infection, the S protein of the virus can be prevented from being combined with an ACE2 receptor, so that the infection of 2019-nCoV new corona virus can be resisted.

In a fifth aspect, the invention provides an antibody that specifically binds to the epitope polypeptide.

The sixth aspect of the invention provides a detection kit, which comprises any one of the epitope polypeptides, or the antibody, or the combination of the epitope polypeptide and the antibody.

The detection kit provided by the invention is used for detecting whether human coronavirus or anti-human coronavirus antibody exists in a sample in vitro. Preferably, the human coronavirus is 2019-nCoV neocoronavirus.

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

the invention screens and designs the polypeptide vaccine based on the known virus sequence characteristics and a computer algorithm, the sequence of the polypeptide molecule is clear, and the production and the preparation are convenient. Overcoming the technical problem of separating specific virus components by using complicated experimental means.

The vaccine of the present invention is designed in consideration of the affinity between Major Histocompatibility Complex (MHC) and polypeptide molecules, and thus can be efficiently recognized by T cells, thereby inducing cellular immune responses in the body. Compared with the conventional subunit vaccine or inactivated virus vaccine, the polypeptide vaccine can only activate humoral immune response, and the immune efficacy of the polypeptide vaccine is stronger.

3, the high-immunogenicity polypeptide obtained by artificial screening can effectively induce an organism to generate immune response, simultaneously avoids the interference of irrelevant antigens and reduces the side reaction of the vaccine. On the other hand, the polypeptide vaccine has clear components and no harm to human bodies, and the safety is guaranteed.

4, the polypeptide vaccine provided by the invention can effectively generate stronger neutralization reaction with IgG and IgM antibodies in serum of a patient in the convalescent period of new coronary pneumonia. In the serum of a patient in the convalescent period, the specific antibody of a healthy person has the effect of resisting the new coronavirus, so that the polypeptide vaccine has the capability of inducing a neutralizing antibody resisting the new coronavirus in a human body, and the immunity of the organism to the new coronavirus is improved. In addition, the polypeptide vaccine of the invention can competitively bind with ACE2, which shows that the polypeptide vaccine of the invention can prevent the combination of virus and host cell and has defense effect on new coronavirus infection.

Drawings

FIG. 1 is a flow chart of the present invention for screening epitope polypeptides.

FIG. 2A is a graph comparing the maximum intensity of the IgG antibody response test in the IgG antibody response test for each patient sample in the IgG antibody assay of SEQ ID No.1 with that of the healthy group.

FIG. 2B is a graph comparing IgG antibody responses at different convalescent stages after the same patient is cured in the IgG antibody assay of SEQ ID No. 1.

FIG. 2C is a graph showing the comparison of IgG antibody responses of individual patient samples with those of healthy groups, which were grouped according to different stages of the convalescent period in the IgG antibody assay of SEQ ID NO. 1.

FIG. 3A is a graph comparing the maximal response intensity in the IgM antibody response test for each patient sample in the IgM antibody detection of SEQ ID NO.1 with that of the healthy group.

FIG. 3B is a graph comparing IgM antibody responses at different convalescent stages after the same patient is cured in IgM antibody assay of SEQ ID NO. 1.

FIG. 3C is a graph showing the IgM antibody responses of each patient sample in the IgM antibody assay of SEQ ID NO.1, grouped according to different stages of the convalescent period, compared with that of the healthy group.

FIG. 4A is a graph comparing the maximum intensity of the IgG antibody response test in the IgG antibody response test for each patient sample in the IgG antibody assay of SEQ ID No.2 with that of the healthy group.

FIG. 4B is a graph comparing IgG antibody responses at different convalescent stages of the same patient after healing in the IgG antibody assay of SEQ ID No. 2.

FIG. 4C is a graph showing the comparison of IgG antibody responses of individual patient samples with those of healthy groups, grouped according to different stages of convalescence in the IgG antibody assay of SEQ ID NO. 2.

FIG. 5A is a graph comparing the maximal response intensity in the IgM antibody response test for each patient sample in the IgM antibody detection of SEQ ID NO.2 with that of the healthy group.

FIG. 5B is a graph showing a comparison of IgM antibody responses in different convalescent phases after the same patient is cured in the IgM antibody assay of SEQ ID NO. 2.

FIG. 5C is a graph showing the IgM antibody responses of each patient sample in the IgM antibody assay of SEQ ID NO.2, grouped according to different stages of the convalescent period, compared with that of the healthy group.

FIG. 6A is a graph comparing the maximal response intensity in an IgG antibody response assay for each patient sample in the IgG antibody assay of SEQ ID No.3 with that of a healthy group.

FIG. 6B is a graph comparing IgG antibody responses at different convalescent stages of the same patient after healing in the IgG antibody assay of SEQ ID No. 3.

FIG. 6C is a graph showing the comparison of IgG antibody responses of individual patient samples with those of healthy groups, grouped according to different stages of convalescence in the IgG antibody assay of SEQ ID NO. 3.

FIG. 7A is a graph comparing the maximal response intensity in the IgM antibody response test for each patient sample in the IgM antibody detection of SEQ ID NO.3 with that of the healthy group.

FIG. 7B is a graph comparing IgM antibody responses at different convalescent stages after the same patient is cured in IgM antibody assay of SEQ ID NO. 3.

FIG. 7C is a graph showing the IgM antibody responses of each patient sample in the IgM antibody assay of SEQ ID NO.3, grouped according to different stages of the convalescent period, compared with that of the healthy group.

FIG. 8 is an electrophoretogram of the epitope polypeptide of the present invention competitively inhibiting the binding of RBD region of S protein to human ACE2 protein.

Detailed Description

The technical solution of the present invention will be described in detail with reference to examples. The reagents and biomaterials used below were all commercial products unless otherwise specified.

Example 1 design screening of epitope Polypeptides

Referring to fig. 1, a flow chart for designing and screening epitope polypeptides of the present invention is shown. And training by using a machine learning model and using the known combination condition of the MHC molecules and the antigens to obtain an affinity model, thereby predicting the affinity of the antigen peptides of the novel coronavirus and the MHC molecules. Predicting B cell antigenic determinants according to protein sequences by using a random forest algorithm, training epitope and non-epitope amino acids determined according to a crystal structure by using the algorithm, and screening peptide fragments with scores higher than a set threshold value to serve as potential BCR recognition vaccine molecules. For a polypeptide sequence screened by a computer model, a polypeptide fragment is obtained by a solid phase synthesis method, and then the effectiveness of the polypeptide as a vaccine is evaluated by a neutralizing antibody test and a cytokine stimulation test to obtain the final epitope polypeptide.

Aiming at the sequence characteristics and spatial conformation of the 2019-nCoV new coronavirus S protein, 3 antigen epitope polypeptide sequences are designed and screened by utilizing the algorithm, and the sequence information is as follows:

SEQ ID NO.1：GCVIAWNSNNLDSKVGG

SEQ ID NO.2：LKPFERDISTEIYQAGSTPCNGVEGFN

SEQ ID NO.3：PLQSYGFQPTNGVGYQPY

EXAMPLE 2 Synthesis of epitope Polypeptides

3 antigen epitope polypeptide sequences (SEQ ID NO.1-SEQ ID NO.3) designed and screened are synthesized by a solid phase synthesis method. The specific process for solid phase synthesis of the polypeptide is as follows:

chloromethyl polystyrene resin as insoluble solid phase carrier, first of all an amino acid with one amino group protected by a blocking group is covalently linked to the solid phase carrier. Under the action of trifluoroacetic acid, the amino protecting group is removed, so that the first amino acid is attached to the solid phase carrier. The carboxyl group of the second amino acid, whose amino group is blocked, is then activated by N, N' -Dicyclohexylcarbodiimide (DCC), and the DCC-activated second amino acid reacts with the amino group of the first amino acid, which has been grafted onto the solid support, to form a peptide bond, thus forming a dipeptide with a protecting group on the solid support. The above peptide bond formation reaction is repeated to grow the peptide chain from the C-terminus to the N-terminus until the desired peptide chain length is reached. Finally, the protecting group is removed, and the ester bond between the peptide chain and the solid phase carrier is hydrolyzed by HF, so that the synthesized peptide is obtained.

Example 3 evaluation of the effectiveness of epitope-polypeptide vaccines

The experimental method comprises the following steps: and marking the synthesized epitope polypeptides with a C-terminal biotin group, and printing the epitope polypeptides on the cut pieces by using a biotin-streptavidin chemical method to prepare polypeptide chips. The prepared polypeptide chip is used for detecting the neutralization reaction degree of IgG and IgM antibodies and epitope polypeptides in serum.

Serum samples of 19 patients in recovery period of new coronary pneumonia and 10 healthy persons were collected from the hospital. Convalescent patients were diagnosed with new coronary pneumonia before receiving treatment, and therefore had been exposed to new coronary virus, and antibodies against the new coronary virus remained in the serum after the cure. By detecting the intensity of antibody responses to each epitope polypeptide in these samples and comparing them with those of healthy persons, the epitope polypeptides prepared in the present invention can be evaluated for their ability to induce neutralizing antibodies in humans.

1. IgG antibody detection result of SEQ ID NO.1

In the test of the epitope polypeptide shown in SEQ ID NO.1 reacting with IgG antibody, all (100%) of 19 patient samples showed the strongest antibody reaction higher than that of the healthy group. Referring to fig. 2A, a graph of the maximum response intensity in an IgG antibody response test for each patient sample compared to the healthy group is shown, where gray is the healthy group, red is the patient with an antibody response intensity greater than that of the healthy group, i.e., the positive group, and blue is the patient with an antibody response intensity less than that of the healthy group, i.e., the negative group. As can be seen from fig. 2A: the antibody response intensity was greater in 19 patients than in the healthy group. Referring to fig. 2B, a graph comparing IgG antibody responses at different recovery stages after cure for the same patient is shown with several stages above healthy labeled red and one stage below healthy labeled blue. As can be seen from fig. 2B: in 13 cases (68.4%) of the patients the antibody response was higher in all phases of the convalescent period (1-7 months) than in the healthy group, while in the remaining 6 cases a certain phase lower than in the healthy group appeared. See fig. 2C for a comparison of IgG antibody responses of individual patient samples to healthy groups, grouped by different stages of recovery. As can be seen from fig. 2C: there were 16 (88.9%) serum samples with higher antibody responses than the healthy group in the first month, 14 (77.8%) serum samples with higher antibody responses than the healthy group in the 4 th month, and 13 (78.6%) serum samples with higher antibody responses than the healthy group in the 7 th month.

2. IgM antibody detection result of SEQ ID NO.1

In the test of the reactivity of the epitope polypeptide shown in SEQ ID NO.1 with IgM antibodies, 17 (89.5%) of 19 patient samples responded more strongly than those in the healthy group. Referring to fig. 3A, a graph of the maximal response intensity in the IgM antibody response test for each patient sample compared to the healthy group is shown with gray for the healthy group, red for patients with antibody response intensity greater than the healthy group, i.e., the positive group, and blue for patients with antibody response intensity less than the healthy group, i.e., the negative group. As can be seen from fig. 3A: the antibody response intensity was greater in 17 patients than in the healthy group, and lower in 2 patients than in the healthy group. Referring to fig. 3B, a graph comparing IgM antibody responses at different convalescent stages after cure for the same patient is shown with several phases above healthy labeled red and one phase below healthy labeled blue. As can be seen from fig. 3B: in 10 cases (52.6%) of the patients the antibody response was higher in all phases of the convalescent period (1-7 months) than in the healthy group, while in the remaining 9 cases a certain phase lower than in the healthy group appeared. See fig. 3C, which is a graph comparing IgM antibody responses of individual patient samples to healthy groups, grouped by different stages of the convalescent phase. As can be seen from fig. 3C: there were 13 (72.2%) serum samples with higher antibody responses than the healthy group in the first month, 12 (66.6%) serum samples with higher antibody responses than the healthy group in the 4 th month, and 12 (80%) serum samples with higher antibody responses than the healthy group in the 7 th month.

3. IgG antibody detection result of SEQ ID NO.2

The epitope polypeptide shown in SEQ ID NO.2 reacted with IgG antibodies in the assay, 7 (36.8%) of the 19 patient samples had the strongest antibody responses higher than those in the healthy group. Referring to fig. 4A, a graph of the maximum response intensity in an IgG antibody response test for each patient sample compared to the healthy group is shown, where gray is the healthy group, red is the patient with an antibody response intensity greater than that of the healthy group, i.e., the positive group, and blue is the patient with an antibody response intensity less than that of the healthy group, i.e., the negative group. As can be seen from fig. 4A: the antibody response intensity was greater in 7 patients than in the healthy group, and lower in 12 patients than in the healthy group. Referring to fig. 4B, a graph comparing IgG antibody responses at different recovery stages after cure for the same patient is shown with several stages above healthy labeled red and one stage below healthy labeled blue. As can be seen from fig. 4B: there were 0 (0%) patients who had higher antibody responses than the healthy group during all phases of the convalescent period (1-7 months), and the remaining 19 patients had a certain phase lower than the healthy group. See fig. 4C, which is a graph comparing IgG antibody responses of individual patient samples to healthy groups, grouped by different stages of recovery. As can be seen from fig. 4C: there were 5 (27.8%) serum samples with higher antibody responses than the healthy group in the first month, 2 (11.1%) serum samples with higher antibody responses than the healthy group in the 4 th month, and 3 (20%) serum samples with higher antibody responses than the healthy group in the 7 th month.

4. IgM antibody detection result of SEQ ID NO.2

In the test of the reactivity of the epitope polypeptide shown in SEQ ID NO.2 with IgM antibodies, 15 (78.9%) of the 19 patient samples responded with the strongest antibodies higher than those of the healthy group. Referring to fig. 5A, a graph of the maximal response intensity in the IgM antibody response test for each patient sample compared to the healthy group is shown with gray for the healthy group, red for patients with antibody response intensity greater than the healthy group, i.e., the positive group, and blue for patients with antibody response intensity less than the healthy group, i.e., the negative group. As can be seen from fig. 5A: the antibody response intensity was greater in 15 patients than in the healthy group, and lower in 4 patients than in the healthy group. Referring to fig. 5B, a graph comparing IgM antibody responses at different convalescent stages after cure for the same patient is shown with several phases above healthy labeled red and one phase below healthy labeled blue. As can be seen from fig. 5B: in 7 (36.8%) patients the antibody response was higher in all phases of the convalescent period (1-7 months) than in the healthy group, and in the remaining 12 cases a certain phase lower than in the healthy group appeared. See fig. 5C, which is a graph comparing IgM antibody responses of individual patient samples to healthy groups, grouped by different stages of the convalescent phase. As can be seen from fig. 5C: the comparison between the different stages in the healthy group resulted in a higher antibody response in 11 (61.1%) serum samples in the first month, 8 (44.4%) serum samples in the 4 th month and 9 (60%) serum samples in the 7 th month.

5. IgG antibody detection result of SEQ ID NO.3

The epitope polypeptide shown in SEQ ID NO.3 reacted with IgG antibodies in the assay, 15 (78.9%) of the 19 patient samples reacted more strongly than in the healthy group. Referring to fig. 6A, a graph of the maximum response intensity in an IgG antibody response test for each patient sample compared to the healthy group is shown, where gray is the healthy group, red is the patient with an antibody response intensity greater than that of the healthy group, i.e., the positive group, and blue is the patient with an antibody response intensity less than that of the healthy group, i.e., the negative group. As can be seen from fig. 6A: the antibody response intensity was greater in 15 patients than in the healthy group, and lower in 4 patients than in the healthy group. Referring to fig. 6B, a graph comparing IgG antibody responses at different recovery stages after cure for the same patient is shown with several stages above healthy labeled red and one stage below healthy labeled blue. As can be seen from fig. 6B: in 7 (36.8%) patients the antibody response was higher in all phases of the convalescent period (1-7 months) than in the healthy group, and in the remaining 12 cases a certain phase lower than in the healthy group appeared. See fig. 6C, which is a graph comparing IgG antibody responses of individual patient samples to healthy groups, grouped by different stages of recovery. As can be seen from fig. 6C: there were 12 (66.7%) serum samples with higher antibody responses than the healthy group in the first month, 11 (61.1%) serum samples with higher antibody responses than the healthy group in the 4 th month, and 7 (46.7%) serum samples with higher antibody responses than the healthy group in the 7 th month.

6. IgM antibody detection result of SEQ ID NO.3

The epitope polypeptide shown in SEQ ID NO.3 reacted with IgM antibodies in the test, 17 (89.5%) of 19 patients reacted with the strongest antibodies higher than those in the healthy group. Referring to fig. 7A, a graph of the maximal response intensity in the IgM antibody response test for each patient sample compared to the healthy group is shown with gray for the healthy group, red for patients with antibody response intensity greater than the healthy group, i.e., the positive group, and blue for patients with antibody response intensity less than the healthy group, i.e., the negative group. As can be seen from fig. 7A: the antibody response intensity was greater in 17 patients than in the healthy group, and lower in 2 patients than in the healthy group. Referring to fig. 7B, a graph comparing IgM antibody responses at different convalescent stages after cure for the same patient, with several stages above healthy labeled red and one stage below healthy labeled blue. As can be seen from fig. 7B: in 11 cases (57.9%) the patients had a higher antibody response than the healthy group during all phases of the convalescent period (1-7 months), while in the remaining 8 cases a certain phase was found which was lower than the healthy group. See fig. 7C, which is a graph comparing IgM antibody responses of individual patient samples to healthy groups, grouped by different stages of the convalescent phase. As can be seen from fig. 7C: the comparison between the different phases in the healthy group resulted in 14 (77.8%) serum samples with higher antibody responses in the first month, 12 (66.7%) serum samples with higher antibody responses in the 4 th month and 12 (80%) serum samples with higher antibody responses in the 7 th month.

7. Binding experiments of RBD region of competitive inhibition S protein to human ACE2 protein

ACE2 is also known as achh and is known as angiotensin converting enzyme 2. The protein coded by the gene belongs to an angiotensin converting enzyme family of dipeptidyl carboxyl dipeptidase and has considerable homology with human angiotensin converting enzyme 1. This secreted protein catalyzes the cleavage of angiotensin I to angiotensin 1-9 and angiotensin II to the vasodilator angiotensin 1-7. ACE2 has strong affinity for Ang type II type 1 and type 2 receptors, and regulates blood pressure, fluid balance, inflammation, cell proliferation, hypertrophy, and fibrosis. Meanwhile, the specific expression of organs and cells of the gene suggests that the gene may play a role in regulating cardiovascular and renal functions and fertility. ACE2 is considered to be an important functional receptor for coronaviruses such as SARS. In the structure of ACE2 bound by the SARS spike glycoprotein (S) protein, the catalytically active site of ACE2 was not blocked by the SARS S protein. Thus, ACE2 functions as the SARS receptor, independent of its peptidase activity. 2019-nCoV like SARS-CoV infects human airway epithelial cells by invasion mediated by the S-protein and human cell surface ACE2 receptor, but 1 of the 3 receptor junction Regions (RBDs) in the S protein protrudes in an upward spiral to allow the S protein to bind more readily to host receptor ACE 2.

Therefore, the detection of the competitive binding effect of the epitope polypeptide and ACE2 shows that the epitope polypeptide prepared by the invention has the capability of preventing the binding of the new coronavirus and host cells and has a defense effect on the infection of the new coronavirus.

The test procedure is as follows:

(1)1ug RBD-HIS + HIS-resin was ligated at 4 ℃ for 2-4 hours;

(2) eluting 3 times by using lysine buffer;

(3) pre-co-incubation with a polypeptide;

(4) adding cell lysate overnight;

(5) the lysate was eluted 3 times;

(6) add 2 Xbuffer and let stand at 100 ℃ for 5 minutes.

The reaction system after adding the buffer was subjected to electrophoresis, and the results of electrophoresis are shown in FIG. 8. ACE2 is a receptor for S protein invasion cells, the RBD region of S protein is combined with ACE2 protein (interaction), the two proteins form a large complex and run slowly on an electrophoretogram, after polypeptide is added, the two proteins are combined with ACE2 protein competitively with RBD, so that the combination of ACE2 and RBD is blocked, a large complex cannot be formed, and the band on the electrophoretogram is weakened. Therefore, the weaker the band on the electrophoretogram, the more obvious the inhibition effect of the epitope polypeptide on the binding of ACE2 and RBD is. As can be seen from fig. 8: the epitope polypeptides shown in SEQ ID NO.2 and SEQ ID NO.3 have obvious inhibiting effect on the combination of ACE2 and RBD. SEQ ID NO.1 shows a certain inhibitory effect when 250nM is used.

The above description is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the spirit of the invention, and any changes and modifications made are within the scope of the invention.

Sequence listing

<110> Anda biopharmaceutical development (Shenzhen) Limited

<120> 2019-nCoV new coronavirus S protein epitope polypeptide and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

Gly

<210> 2

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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

1 5 10 15

Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn

20 25

<210> 3

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

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

1 5 10 15

Pro Tyr

Claims

1. An epitope polypeptide of 2019-nCoV neo-coronavirus S protein, which is characterized in that: the amino acid sequence of the epitope polypeptide is selected from one of the following:

2. A polynucleotide sequence characterized by: encoding the epitope polypeptide of claim 1.

3. Use of one or more of the epitope polypeptides of claim 1 for the preparation of a human coronavirus vaccine.

4. Use of one or more of the epitope polypeptides of claim 3 for the preparation of a human coronavirus vaccine, wherein: the human coronavirus is 2019-nCoV new coronavirus.

5. Use of one or more of the epitope polypeptides of claim 1 for the preparation of a medicament against human coronavirus infection.

6. Use of one or more of the epitope polypeptides of claim 5 for the preparation of a medicament against human coronavirus infection, wherein: the human coronavirus is 2019-nCoV new coronavirus.

7. An antibody, characterized by: the antibody specifically binds to the epitope polypeptide of claim 1.

8. A detection kit, characterized in that: comprising any one of the epitope polypeptides of claim 1, or the antibody of claim 7, or a combination of an epitope polypeptide and an antibody.

9. The test kit of claim 8 for use in vitro testing of a sample for the presence of human coronavirus or anti-human coronavirus antibodies.

10. The test kit of claim 9 for use in vitro testing of a sample for the presence of human coronavirus or anti-human coronavirus antibody, wherein: the human coronavirus is 2019-nCoV new coronavirus.