CN113493496B

CN113493496B - Epitope polypeptide of 2019-nCoV novel coronavirus S protein and application thereof

Info

Publication number: CN113493496B
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: 2024-03-22
Anticipated expiration: 2041-03-19
Also published as: CN113493496A

Abstract

The invention discloses a group of epitope polypeptides of 2019-nCoV novel coronavirus S protein and application thereof. The amino acid sequence of the antigen 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 having more than 90% homology with the amino acid sequence shown in SEQ ID NO.1 and having the same function; (2) an amino acid sequence shown as SEQ ID NO. 2; or an amino acid sequence having more than 90% homology to the amino acid sequence shown in SEQ ID NO.2 and having the same function; (3) an amino acid sequence shown as SEQ ID NO. 3; or an amino acid sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO.3 and having the same function. The high immunogenicity polypeptide screened by the invention can effectively induce organisms to generate immune response, simultaneously avoid the interference of irrelevant antigens and reduce the side reaction of vaccines.

Description

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

Background

The current development of a vaccine against the novel coronavirus (2019-nCoV) is essentially the same as the strategy of developing SARS-CoV vaccine in the last 20 years, including (1) an inactivated viral vaccine, (2) a viral vector vaccine, and (3) a subunit vaccine. The polypeptide vaccine designed by the invention belongs to a novel subunit vaccine. Subunit vaccine refers to vaccine prepared by extracting specific protein structure of bacteria and virus by chemical decomposition or controlled proteolysis method, and screening out fragment with immunological activity. 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 subunit vaccines prepared by screening the antigens with the immune activity can effectively play a role of the vaccine, meanwhile, the antibodies induced by irrelevant antigens are avoided, and the side reaction of the vaccine is reduced. At present, china is still in a relatively laggard position in the aspect of subunit vaccine development.

The novel coronavirus 2019-nCoV particle comprises 4 structural proteins, respectively 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 packaging viruses, also known as the S protein. The S protein is directed onto the endoplasmic reticulum by means of its amino-terminal signal peptide and is highly N-glycosylated, and its trimers form a unique spike structure on the viral surface. The S protein is cleavable 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 viral invasion primarily through binding to host cell receptors and determines viral organization or host tropism. The research shows that the S protein is a key component of a new coronavirus infected organism and is also a key target point for developing new coronavirus vaccine and antibody inhibitor.

Disclosure of Invention

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

According to the invention, immune affinity analysis is carried out on patient MHC molecules and virus protein molecules, and antigen epitopes with high immunogenicity are predicted to be used as polypeptide vaccines for stimulating T cell immune response. Meanwhile, the antigen epitope with high affinity of B cell epitope is screened and used as a polypeptide vaccine for inducing B cell humoral immune response. The peptide segment bound by MHC molecule is 8-11 in length, and the molecule has a groove which is an antigen binding site, called peptide binding groove (peptide-binding groove or cleft), and the groove bottom has 6 pockets: pocket A-F, pocket A and Pocket F are responsible for fixing peptide fragments, pocket B-E are responsible for combining different peptide fragments, and the specificity is maintained. The characteristic of binding of the antigen binding groove of the MHC molecule to the antigen peptide often has two or more key amino acid (anchor residue) specificities combined with the polypeptide binding motif in the peptide binding groove of the MHC molecule. The invention uses a machine learning model, and uses the known combination condition of MHC molecules and antigens to train to obtain an affinity model, so as to predict the affinity of antigen peptides of the novel coronavirus and the MHC molecules. The B cell epitopes are predicted from protein sequences using a random forest algorithm that trains the epitope and non-epitope amino acids determined from the crystal structure, screening peptides scored above a set threshold 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 2019-nCoV novel coronavirus S protein to serve as a candidate antigen, and then the antigen epitope polypeptide with a vaccine effect is further obtained through the verification of a validity experiment of a polypeptide vaccine.

The first aspect of the present invention provides a group of epitope polypeptides of 2019-nCoV novel coronavirus S protein, wherein the amino acid sequence of the epitope polypeptides is selected from one of the following:

(1) An amino acid sequence as shown in SEQ ID NO. 1; or an amino acid sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO.1 and having the same function;

(2) An amino acid sequence as shown in SEQ ID NO. 2; or an amino acid sequence having more than 90% homology to the amino acid sequence shown in SEQ ID NO.2 and having the same function;

(3) An amino acid sequence as shown in SEQ ID NO. 3; or an amino acid sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO.3 and having the same function.

In a second aspect, the invention provides a polynucleotide sequence encoding an epitope polypeptide of the 2019-nCoV novel coronavirus S protein.

The third aspect of the invention provides the use of one or more of said epitope polypeptides in the preparation of a human coronavirus vaccine. Preferably, the human coronavirus is a 2019-nCoV novel coronavirus.

The fourth aspect of the invention provides the use of one or more of said epitope polypeptides in the preparation of a medicament for the treatment of human coronavirus infection. Preferably, the human coronavirus is a 2019-nCoV novel coronavirus. The antigen epitope polypeptide is used as a mechanism of a novel anti-2019-nCoV coronavirus drug: as a blocking peptide blocking viral infection, the S protein of the virus can be prevented from binding to ACE2 receptor, thus resisting infection by 2019-nCoV new coronavirus.

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

In a sixth aspect, the invention provides a test kit comprising any one of said epitope polypeptides, or said antibodies, or a combination of epitope polypeptides and antibodies.

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

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

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

2, the vaccine design of the present invention considers the affinity of the Major Histocompatibility Complex (MHC) to polypeptide molecules and is therefore able to be efficiently recognized by T cells, thereby inducing a cellular immune response in the body. Compared with the conventional subunit vaccine or inactivated virus vaccine, the polypeptide vaccine can only activate humoral immune response and has stronger immune efficacy.

3, the high immunogenicity polypeptide obtained by manual screening can effectively induce organisms to generate immune response, meanwhile, the interference of irrelevant antigens is avoided, and the side reaction of vaccines is reduced. On the other hand, the polypeptide vaccine has clear components, has no harm to human body and ensures the safety.

4, the polypeptide vaccine provided by the invention can effectively generate stronger neutralization reaction with IgG and IgM antibodies in serum of patients in the convalescence of new coronaries. In serum of convalescence patients, the specific antibody of healthier people has the function of resisting the new coronavirus, so the polypeptide vaccine of the invention has the capability of inducing neutralizing antibodies resisting the new coronavirus in human bodies, thereby improving the immunity of organisms to the new coronavirus. In addition, the polypeptide vaccine of the invention can competitively bind to ACE2, which indicates that the polypeptide vaccine of the invention can prevent viruses from binding to host cells and has a defensive effect on new coronavirus infection.

Drawings

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

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

FIG. 2B is a graph showing the comparison of IgG antibody responses at different recovery phases after the same patient is cured in the detection of IgG antibody of SEQ ID NO. 1.

FIG. 2C is a graph showing the comparison of IgG antibody responses of each patient sample to the healthy group, grouped according to the different phases of recovery in the IgG antibody assay of SEQ ID NO. 1.

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

FIG. 3B is a graph showing the comparison of IgM antibody responses at different recovery phases after the same patient is cured in the IgM antibody detection of SEQ ID NO. 1.

FIG. 3C is a graph showing the IgM antibody response of each patient sample compared to the healthy group, grouped at different stages of recovery in the IgM antibody assay of SEQ ID NO. 1.

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

FIG. 4B is a graph showing the comparison of IgG antibody responses at different recovery phases after the same patient is cured in the detection of IgG antibody of SEQ ID NO. 2.

FIG. 4C is a graph showing the comparison of IgG antibody responses of each patient sample to the healthy group, grouped according to the different phases of recovery in the IgG antibody assay of SEQ ID NO. 2.

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

FIG. 5B is a graph showing the comparison of IgM antibody responses at different recovery phases after healing of the same patient in IgM antibody detection of SEQ ID NO. 2.

FIG. 5C is a graph showing the IgM antibody response of each patient sample compared to the healthy group, grouped at different stages of recovery in the IgM antibody assay of SEQ ID NO. 2.

FIG. 6A is a graph comparing the maximum intensity of response in an IgG antibody response test with that of a healthy group for each patient sample in the IgG antibody detection of SEQ ID NO. 3.

FIG. 6B is a graph showing the comparison of IgG antibody responses at different recovery phases after the same patient is cured in the detection of IgG antibody of SEQ ID NO. 3.

FIG. 6C is a graph showing the comparison of IgG antibody responses of each patient sample to the healthy group, grouped according to the different phases of recovery in the IgG antibody assay of SEQ ID NO. 3.

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

FIG. 7B is a graph showing the comparison of IgM antibody responses at different recovery phases after the same patient is cured in IgM antibody detection of SEQ ID NO. 3.

FIG. 7C is a graph showing the IgM antibody response of each patient sample compared to the healthy group, grouped at different stages of recovery in the IgM antibody assay of SEQ ID NO. 3.

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

Detailed Description

The following describes the technical scheme of the present invention in detail by referring to examples. The reagents and biological materials used hereinafter are commercial products unless otherwise specified.

Example 1 design selection of epitope Polypeptides

Referring to FIG. 1, a flow chart for screening epitope polypeptides is designed for the present invention. The affinity model is obtained by training a machine learning model by using the known combination condition of the MHC molecules and the antigens, so that the affinity of the antigen peptide of the novel coronavirus and the MHC molecules is predicted. The B cell epitopes are predicted from protein sequences using a random forest algorithm that trains the epitope and non-epitope amino acids determined from the crystal structure, screening peptides scored above a set threshold as potential BCR recognition vaccine molecules. And (3) for the polypeptide sequences screened by the computer model, obtaining polypeptide fragments by utilizing a solid-phase synthesis method, and then evaluating the effectiveness of the polypeptides as vaccines by a neutralizing antibody test and a cytokine stimulation test to obtain the final epitope polypeptides.

3 antigen epitope polypeptide sequences are designed and screened by utilizing the algorithm aiming at the sequence characteristics and the spatial conformation of the 2019-nCoV novel coronavirus S protein, 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 adopting a solid phase synthesis method. The specific process of solid phase synthesis of polypeptide is as follows:

chloromethyl polystyrene resin is used as insoluble solid phase carrier, and one amino acid with amino group protected by blocking group is first connected to the solid phase carrier. Under the action of trifluoroacetic acid, the protecting group of the amino group is removed, so that the first amino acid is attached to the solid support. The carboxyl group of the second amino acid with blocked amino group is then activated by N, N' -Dicyclohexylcarbodiimide (DCC), and the second amino acid with activated carboxyl group is reacted with the amino group of the first amino acid attached to 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 removing the protecting group, and hydrolyzing the ester bond between the peptide chain and the solid phase carrier by using HF to obtain the synthesized peptide.

Example 3 evaluation of the effectiveness of epitope polypeptide vaccine

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

Serum samples from 19 patients with new coronaries at recovery from pneumonia and serum samples from 10 healthy persons were collected from hospitals. Patients in convalescence are diagnosed with new coronavirus before receiving treatment, so they have been exposed to new coronavirus, and antibodies against new coronavirus remain in serum after cure. By detecting the intensity of the antibody response against each epitope polypeptide in these samples and comparing with healthy humans, the ability of the epitope polypeptides prepared in the present invention to induce neutralizing antibodies in humans can be assessed.

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

In the test of the epitope polypeptide shown in SEQ ID NO.1 and IgG antibody reaction, the strongest antibody reaction of all samples (100%) of 19 patient samples is higher than that of the healthy group. Referring to fig. 2A, a graph comparing the maximum intensity of response of each patient sample in an IgG antibody response test with that of a healthy group is shown, wherein gray is the healthy group, red is the positive group of patients having an intensity of antibody response greater than that of the healthy group, and blue is the negative group of patients having an intensity of antibody response lower than that of the healthy group. As can be seen from fig. 2A: the intensity of antibody response was greater in 19 patients than in the healthy group. Referring to fig. 2B, a comparison of IgG antibody responses at different recovery phases after healing of the same patient is shown, with several phases being red for higher than healthy groups and blue for lower than healthy groups. As can be seen from fig. 2B: 13 (68.4%) patients had higher antibody responses than the healthy group at all phases of the recovery period (1-7 months), while the remaining 6 patients had a lower phase than the healthy group. Referring to fig. 2C, a graph comparing IgG antibody responses of each patient sample to the healthy group was obtained for the grouping according to the different phases of recovery. As can be seen from fig. 2C: the antibody response was higher in 16 (88.9%) serum samples in the first month than in the healthy group, in 14 (77.8%) serum samples in the 4 th month than in the healthy group, and in 13 (78.6%) serum samples in the 7 th month than in the healthy group.

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

In the test of the reaction of the epitope polypeptide shown in SEQ ID No.1 with IgM antibodies, 17 (89.5%) patients in 19 patient samples had the strongest antibody reaction than in the healthy group. Referring to fig. 3A, a graph comparing the maximum intensity of response of each patient sample in IgM antibody response test with that of the healthy group is shown, wherein gray is the healthy group, red is the positive group of patients with higher intensity of antibody response than the healthy group, and blue is the negative group of patients with lower intensity of antibody response than the healthy group. As can be seen from fig. 3A: the antibody response intensity was greater for 17 patients than for the healthy group, and for 2 patients than for the healthy group. Referring to fig. 3B, a comparison of IgM antibody responses at different recovery phases after healing of the same patient is shown, with several phases being red for higher than healthy groups and blue for lower than healthy groups. As can be seen from fig. 3B: there were 10 (52.6%) patients with higher antibody responses than the healthy group at all phases of the recovery period (1-7 months), and the remaining 9 patients showed a lower phase than the healthy group. Referring to fig. 3C, a graph comparing IgM antibody responses of each patient sample to healthy groups, grouped according to different phases of recovery. As can be seen from fig. 3C: the antibody response was higher for 13 (72.2%) serum samples in the first month than for the healthy group, for 12 (66.6%) serum samples in the 4 th month than for the healthy group, and for 12 (80%) serum samples in the 7 th month than for the healthy group.

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

In the test of the epitope polypeptide shown in SEQ ID NO.2 and IgG antibody reaction, 7 (36.8%) patients in 19 patient samples have the strongest antibody reaction higher than that of the healthy group. Referring to fig. 4A, a graph comparing the maximum intensity of response of each patient sample in an IgG antibody response test with that of the healthy group is shown, wherein gray is the healthy group, red is the positive group of patients having an intensity of antibody response greater than that of the healthy group, and blue is the negative group of patients having an intensity of antibody response lower than that of the healthy group. As can be seen from fig. 4A: the antibody response intensity was greater for 7 patients than for the healthy group, and for 12 patients than for the healthy group. Referring to fig. 4B, a comparison of IgG antibody responses at different recovery phases after healing of the same patient is shown, with several phases being red for higher than healthy groups and blue for lower than healthy groups. As can be seen from fig. 4B: there were 0 (0%) patients with higher antibody responses than the healthy group at all phases of the recovery period (1-7 months), and the remaining 19 patients showed a phase lower than the healthy group. Referring to fig. 4C, a graph comparing IgG antibody responses of each patient sample to the healthy group was obtained for the grouping according to the different phases of recovery. As can be seen from fig. 4C: the antibody response was higher for 5 (27.8%) serum samples in the first month than for the healthy group, 2 (11.1%) serum samples in the 4 th month than for the healthy group, and 3 (20%) serum samples in the 7 th month than for the healthy group.

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

In the test of the reaction of the epitope polypeptide shown in SEQ ID No.2 with IgM antibodies, the strongest antibody reaction of 15 (78.9%) patients in 19 patient samples was higher than that of the healthy group. Referring to fig. 5A, a graph comparing the maximum intensity of response of each patient sample in IgM antibody response test with that of the healthy group is shown, wherein gray is the healthy group, red is the positive group of patients with higher intensity of antibody response than the healthy group, and blue is the negative group of patients with lower intensity of antibody response than the healthy group. As can be seen from fig. 5A: the antibody response intensity was greater in 15 patients than in the healthy group, and the antibody response intensity was lower in 4 patients than in the healthy group. Referring to fig. 5B, a comparison of IgM antibody responses at different recovery phases after healing of the same patient is shown, with several phases being red for higher than healthy groups and blue for lower than healthy groups. As can be seen from fig. 5B: there were 7 (36.8%) patients with higher antibody responses than the healthy group at all phases of the recovery period (1-7 months), and the remaining 12 patients showed a lower phase than the healthy group. Referring to fig. 5C, a graph comparing IgM antibody responses of each patient sample to healthy groups, grouped according to different phases of recovery. As can be seen from fig. 5C: the different phases were compared in healthy groups, and 11 (61.1%) serum samples were higher in antibody response than in healthy groups in the first month, 8 (44.4%) serum samples were higher in antibody response than in healthy groups in the 4 th month, and 9 (60%) serum samples were higher in antibody response than in healthy groups in the 7 th month.

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

In the test of the reaction of the epitope polypeptide shown in SEQ ID NO.3 with IgG antibodies, the strongest antibody reaction of 15 (78.9%) patients in 19 patient samples was higher than that of the healthy group. Referring to fig. 6A, a graph comparing the maximum intensity of response of each patient sample in an IgG antibody response test with that of the healthy group is shown, wherein gray is the healthy group, red is the positive group of patients having an intensity of antibody response greater than that of the healthy group, and blue is the negative group of patients having an intensity of antibody response lower than that of the healthy group. As can be seen from fig. 6A: the antibody response intensity was greater in 15 patients than in the healthy group, and the antibody response intensity was lower in 4 patients than in the healthy group. Referring to fig. 6B, a comparison of IgG antibody responses at different recovery phases after healing of the same patient, with several phases higher than healthy groups marked red and with some phases lower than healthy groups marked blue. As can be seen from fig. 6B: there were 7 (36.8%) patients with higher antibody responses than the healthy group at all phases of the recovery period (1-7 months), and the remaining 12 patients showed a lower phase than the healthy group. Referring to fig. 6C, a graph comparing IgG antibody responses of each patient sample to healthy groups was obtained for the different stages of recovery. As can be seen from fig. 6C: the antibody response was higher for 12 (66.7%) serum samples in the first month than for the healthy group, 11 (61.1%) serum samples in the 4 th month than for the healthy group, and 7 (46.7%) serum samples in the 7 th month than for the healthy group.

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

In the test of the reaction of the epitope polypeptide shown in SEQ ID No.3 with IgM antibodies, 17 (89.5%) patients in 19 patient samples had the strongest antibody reaction than in the healthy group. Referring to fig. 7A, a graph comparing the maximum intensity of response of each patient sample in IgM antibody response test with that of the healthy group is shown, wherein gray is the healthy group, red is the positive group of patients with higher intensity of antibody response than the healthy group, and blue is the negative group of patients with lower intensity of antibody response than the healthy group. As can be seen from fig. 7A: the antibody response intensity was greater for 17 patients than for the healthy group, and for 2 patients than for the healthy group. Referring to fig. 7B, a comparison of IgM antibody responses at different recovery phases after healing of the same patient is shown, with several phases being red for higher than healthy groups and blue for lower than healthy groups. As can be seen from fig. 7B: 11 (57.9%) patients had higher antibody responses than the healthy group at all phases of the recovery period (1-7 months), while the remaining 8 patients had a lower phase than the healthy group. Referring to fig. 7C, a graph comparing IgM antibody responses of each patient sample to healthy groups, grouped according to different phases of recovery. As can be seen from fig. 7C: the different phases were compared to the healthy group, and the antibody response was higher in 14 (77.8%) serum samples in the first month than in the healthy group, in 12 (66.7%) serum samples in the 4 th month than in the healthy group, and in 12 (80%) serum samples in the 7 th month than in the healthy group.

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

ACE2 is also known as ACEH and is known as angiotensin converting enzyme 2. The protein coded by the gene belongs to the family of angiotensin converting enzymes of dipeptidyl carboxyl dipeptidase and has quite large homology with human angiotensin converting enzyme 1. This secreted protein catalyzes the cleavage of angiotensin I into angiotensin 1-9 and angiotensin II into the vasodilator angiotensin 1-7.ACE2 has a strong affinity for Ang II type 1 and type 2 receptors, regulating blood pressure, humoral balance, inflammation, cell proliferation, hypertrophy and fibrosis. At the same time, the organ and cell specific expression of the gene suggests that it may play a role in regulating cardiovascular and renal functions and fertility. ACE2 is considered as an important functional receptor for coronaviruses such as SARS. In the structure of SARS spike glycoprotein (S) protein-bound ACE2, the catalytically active site of ACE2 is not blocked by the SARS S protein. ACE2 therefore functions as a SARS receptor irrespective of its peptidase activity. 2019-nCoV, like SARS-CoV, infects human airway epithelial cells by invasion through mediation of the S-protein with the human cell surface ACE2 receptor, but 1 of the 3 Receptor Binding Domains (RBDs) in the S protein protrudes spirally upward to allow the S protein to bind more readily to the host receptor ACE 2.

Therefore, the detection of the competitive binding effect of the epitope polypeptide and ACE2 can prove that the epitope polypeptide prepared by the invention has the capability of preventing the combination of the novel coronavirus and host cells and has the defending effect on the infection of the novel coronavirus.

The test procedure is as follows:

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

(2) Eluting with Lysis buffer for 3 times;

(3) Preincubating with a polypeptide;

(4) Adding cell lysate overnight;

(5) Eluting the lysate for 3 times;

(6) 2 Xbuffer was added and left at 100℃for 5 minutes.

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

The foregoing 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 scope of the technical solution of the present invention, and any changes and modifications are within the scope of the present invention.

Sequence listing

<110> Anda biopharmaceutical development (Shenzhen Co., ltd.)

<120> 2019-nCoV novel 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. A polypeptide of 2019-nCoV new coronavirus S protein, characterized in that: the amino acid sequence of the polypeptide is selected from the amino acid sequences shown in SEQ ID NO. 3.

2. A polynucleotide, characterized in that: encoding the polypeptide of claim 1.

3. A test kit, characterized in that: comprising the polypeptide of claim 1.