Polypeptide for inhibiting replication of porcine epidemic diarrhea virus and application thereof

Abstract

The invention relates to a bioengineering technology, and aims to provide a polypeptide for inhibiting replication of a porcine epidemic diarrhea virus and application of the polypeptide. The polypeptide is a transmembrane polypeptide targeting a PEDV replication stage, and 58th arginine in a nucleocapsid N protein sequence of PEDV is used as a target; the polypeptide is named as R58A polypeptide, and the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1. According to the present invention, the key site affecting virus replication in the N-terminal region of the PEDV N protein is adopted as the new target, such that the inhibition effect in the PEDV infection model verification experiment of IPEC-J2, Vero and ST cells is significant, and the new target can be further adopted as the active component to prepare the porcine epidemic diarrhea resistance drug.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a polypeptide for inhibiting the replication of porcine epidemic diarrhea virus and an application thereof. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is an acute, highly contagious enteric disease pathogen that causes fatal watery diarrhea in newborn piglets. It has become one of the most important pathogens threatening the pig industry because it poses a huge public health threat and economic burden to the pig industry. Therefore, clarifying the infection regulation, immune escape and pathogenic mechanism of the virus is of great significance for the prevention and treatment of the disease.

[0003] PEDV belongs to the genus Alphacoronavirus of the family Coronaviridae. It is a single-stranded positive-sense RNA virus wrapped by an envelope membrane. Its genome is approximately 28 kb in length and encodes four structural proteins, namely spike (S) protein, envelope (E) protein, membrane (M) protein, nucleocapsid (N) protein, one accessory protein (ORF3), and 16 non-structural proteins (Nsp 1-16).

[0004] In terms of PEDV prevention and control, as an RNA virus, it is very easy to mutate, undergoing complex and rapid evolution and recombination, so that the vaccinated pigs still show a high mortality rate from 2010 to the present. Currently, more than 98% of the prevalent strains belong to the G2 group, and there is no sufficiently safe and effective vaccine for the G2 group PEDV. Once an epidemic occurs in a pig farm, the importance of therapeutic drugs gradually becomes apparent. Clinically, interferon administration and passive immunization injection of PEDV-specific yolk antibodies are usually used as treatment methods. Interferon is a general drug for the treatment of viral diseases, lacks specificity, and has limited therapeutic effects. Yolk antibodies have problems such as limited protection period and uneven quality. Neither of them directly targets the PEDV virus, making it difficult to obtain a good inhibitory effect on the PEDV virus in practical applications.

[0005] Therefore, it is necessary to adjust new ideas to suppress the spread of PEDV virus and propose new solutions. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a polypeptide for inhibiting the replication of porcine epidemic diarrhea virus and its application.

[0007] To solve the technical problem, the solution of the present invention is:

[0008] Provided is a polypeptide for inhibiting the replication of porcine epidemic diarrhea virus. The polypeptide is a membrane-penetrating polypeptide targeting the replication stage of PEDV, and uses the 58th arginine in the nucleocapsid N protein sequence of PEDV as a target. The polypeptide is named R58A polypeptide, and its amino acid sequence is shown in SEQ ID NO:1.

[0009] As a preferred embodiment of the present invention, the N-terminus of the polypeptide is modified with a cell-penetrating peptide, and the cell-penetrating peptide is CCPs, and its sequence is shown in SEQ ID NO:2.

[0010] The present invention further provides a method for inhibiting PEDV virus replication using the aforementioned polypeptide, which comprises pre-incubating IPEC-J2, Vero or ST cells with the polypeptide, adding the same amount of polypeptide as in the pre-incubation again after infection with PEDV, lysing the cells and harvesting samples, and detecting the expression of PEDV-N protein in the cells.

[0011] As a preferred embodiment of the present invention, when performing polypeptide pre-incubation, the concentration of the polypeptide used is 1 to 10 μg / mL.

[0012] The present invention also provides a pharmaceutical composition for inhibiting the replication of porcine epidemic diarrhea virus, wherein the pharmaceutical composition comprises the aforementioned polypeptide and a pharmaceutically acceptable carrier or adjuvant.

[0013] The present invention further provides the use of the polypeptide composition in preparing a vaccine or medicine for preventing and / or treating porcine epidemic diarrhea virus.

[0014] Description of the invention principle:

[0015] In view of the fact that the actual application effect of the existing research results on the prevention of PEDV in the industry is not ideal, the present invention proposes a new solution, which is to interrupt the epidemic process of PEDV by designing a polypeptide for inhibiting the replication ability of PEDV.

[0016] During the research, the applicant noticed that the nucleocapsid (N) protein in the PEDV structural protein is highly conserved and forms a complex with the genomic RNA, affecting the synthesis, packaging and assembly of viral RNA particles. The PEDV N protein also has the effects of prolonging the cellular DNA synthesis phase (S phase), inducing endoplasmic reticulum stress and inhibiting the production of type III IFN. The N-terminal domain (NTD) of the N protein of PEDV mediates RNA binding and is a key domain that affects the function of the N protein. In addition, the arginine contained in the N protein is a basic amino acid that participates in a variety of biological processes and can provide key nutrients required during viral infection.

[0017] Based on the above research results on the properties of N protein and arginine, the applicant's research team clarified the infection regulation mechanism of PEDV and innovatively proposed to use arginine at a specific target in the N protein of PEDV as the object of modification, thus providing new ideas for solutions. In the existing research results, there is no clear record of using the properties of nucleocapsid (N) protein and arginine to interrupt the PEDV replication process and thus achieve the purpose of suppressing the spread of the virus.

[0018] The applicant conducted in-depth research and complex screening on the arginine in the N-terminal domain (NTD) of the PEDV N protein, and finally confirmed that the arginine at position 58 (N-R58) of the nucleocapsid protein determines the replication ability of PEDV, and further proposed the following polypeptide design scheme on this basis:

[0019] (1) Based on the N-terminal sequence characteristics of the PEDV N protein, 20 amino acids including the site are intercepted with arginine as the center. Since the purity, reactivity and difficulty of synthesis of the polypeptide are related to the number of amino acids, about 30 amino acids are generally appropriate. Therefore, except for the cell-penetrating peptide containing 11 amino acids, the length of the target polypeptide proposed in this application is 20 amino acids.

[0020] (2) After determining the amino acid sequence of the polypeptide, the central arginine was mutated to alanine, and a transmembrane peptide sequence was inserted at the N-terminus to assist the polypeptide in entering the cell, and finally a polypeptide (named R58A) was synthesized. The technical purpose of mutating the central arginine to alanine is to disable the function of this site.

[0021] Through verification in the natural infection model of PEDV, the results showed that the R58A polypeptide obtained in the present application can significantly inhibit the replication of PEDV.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention has found the key site in the N-terminal region of the PEDV N protein that affects viral replication through in-depth research. Taking advantage of the highly conserved characteristics of this site in different coronaviruses, it is proposed to use it as a new target for broad-spectrum anti-coronavirus drugs and attenuated coronavirus vaccines.

[0024] 2. The present invention further provides a membrane-penetrating polypeptide targeting the replication stage of PEDV based on a new target. The polypeptide has a significant inhibitory effect in the PEDV infection model verification experiment of IPEC-J2, Vero, and ST cells, and can be further used as an active ingredient for the preparation of anti-porcine epidemic diarrhea drugs.

[0025] 3. The present invention clarifies that the conserved arginine at the N-terminus of the nucleocapsid protein of PEDV is a key site that affects PEDV infection. On this basis, the design of a membrane-penetrating polypeptide targeting this site can exert an antiviral function. The proposal of the present invention further enriches the research strategy on the regulation and pathogenic mechanism of PEDV infection and provides new ideas for the development of new prevention and treatment methods.

[0026] 4. This invention can also provide new inspiration for researchers to gain a deeper understanding of the regulation of viral infection and find new clues for prevention and control in other research work similar to coronaviruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a structural description of the R58A polypeptide of the present invention.

[0028] Figure 2 This is the identification result of the R58A polypeptide in the present invention.

[0029] Figure 3 This is the cytotoxicity analysis of different concentrations of R58A peptide on IPEC-J2 cells.

[0030] Figure 4 Analysis of the effect of R58A polypeptide on PEDV replication.

[0031] Figure 5 To analyze the role of R58A polypeptide during PEDV infection in other susceptible cells. DETAILED DESCRIPTION

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention. The present invention is not limited to the following embodiments, and the test methods not described in detail in the embodiments are all conventional methods in the art.

[0033] Example 1 R58A polypeptide inhibits PEDV replication

[0034] (1) Synthesis of peptides targeting PEDV N protein R58

[0035] The PEDV N protein sequence (GenBank: MK409659.1) was downloaded from the NCBI database. The arginine at position 58 (N-R58) of the nucleocapsid protein can affect the replication ability of PEDV. The amino acids before and after the arginine were designed according to the description of the principle of the invention, and then the arginine at this site was mutated to alanine, and cell-penetrating peptides (CPPs) were added to finally form the R58A polypeptide. The specific sequence of the polypeptide is shown in Table 1, and the underlined abbreviation A in the sequence is alanine mutated from arginine.

[0036] The synthesis of the peptide product was commissioned to Shanghai Hongpeptide Biotechnology Co., Ltd. The molecular weight of the peptide was 4133.72 and the purity was 92.13%. Figure 2 .

[0037] Table 1 Synthetic peptide sequences

[0038] Sequence number Peptide name sequence SEQ ID NO:1 R58A YGRKKRRQRRRQIGYWNEQIAWRMRRGERIE SEQ ID NO:2 CCPs YGRKKRRQRRR

[0039] (2) R58A peptide has no toxicity to IPEC-J2 cells

[0040] R58A peptide was used to treat normal porcine intestinal epithelial cells (IPEC-J2 cells) at different concentrations (1, 5, 10, 20 and 50 μg / mL), and Mock wells and Blank wells were set up for incubation for 24 hours. 10 μL CCK-8 dye was added to 100 μL culture medium, and the OD450 value was measured with a microplate reader after incubation for 1 hour to calculate the cell viability. The results showed that there was no significant difference after R58A peptide treatment compared with the Mock well, indicating that it was non-toxic to IPEC-J2 cells ( Figure 3 ).

[0041] (3) PEDV N-R58A peptide can act as an antiviral effector to inhibit PEDV replication

[0042] IPEC-J2 cells were pre-incubated with different concentrations of R58A peptide (1, 5, 10, 20 and 50 μg / mL) for 1 hour, and then infected with PEDV. After 4 hours of adsorption, 4 μg / mL trypsin was added for maintenance, and the same amount of peptide as in pre-incubation was added. RNA and protein of the samples were harvested at 24 hours of infection. After RNA extraction, probe detection found that the R58A mutant peptide significantly reduced the transcription level of the PEDV N gene, and the inhibitory effect of 1-10 μg / mL was the best ( Figure 4 ), but had no significant effect on the transcription level of PEDV ORF1 gene. Western blot was used to detect the expression levels of PEDV S1 protein and PEDV N protein. The results showed that the R58A mutant polypeptide significantly reduced the expression of PEDV N and PEDV S1 proteins ( Figure 4 ).

[0043] Example 2 Application of PEDV N-R58A polypeptide in other susceptible cells

[0044] According to the operation in Example 1, African green monkey kidney cells (Vero cells) and pig testicular cells (ST cells) were pre-incubated with 1, 5, and 10 μg / mL R58A polypeptide for 1 hour, and PEDV infected the cells with MOI = 1 for 4 hours. Then, 4 μg / mL trypsin and the same amount of polypeptide as in the pre-incubation were added to the culture medium, and the cells were lysed at 24 hours after infection to detect the expression of PEDV-N protein in the cells. The results showed that the PEDV-N protein level in the R58A mutant polypeptide group in Vero cells gradually decreased in a dose-dependent manner ( Figure 5 In ST cells, as the concentration of PEDV N-R58A polypeptide increased, the expression of PEDV N protein was inhibited in a dose-dependent manner ( Figure 5 ).

[0045] Analysis and Conclusion:

[0046] Based on the results of Examples 1 and 2, it can be seen that the R58A polypeptide provided by the present invention can interrupt the PEDV replication process, and can therefore be further used as an inhibitor targeting the PEDV virus replication stage, thereby achieving the suppression of the PEDV virus epidemic. At the same time, based on the non-toxic effects of the polypeptide on susceptible cells such as IPEC-J2, Vero and ST during application, its application value as an active component of an antiviral drug is further verified.

[0047] Based on the results of multiple examples verified by the applicant, it is confirmed that the polypeptide has the best inhibitory effect on PEDV-N protein expression when used at a concentration of 1 to 10 μg / mL.

[0048] The protection scope of the present invention is not limited to the above-mentioned specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments rather than to limit the protection scope of the present invention. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the technical personnel in the field of the present invention's mastery of the prior art and the records of the present invention, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to implement the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A polypeptide for inhibiting the replication of porcine epidemic diarrhea virus, characterized in that: The polypeptide is a membrane-penetrating polypeptide targeting the replication stage of PEDV, and takes the 58th arginine in the nucleocapsid N protein sequence of PEDV as a target; the polypeptide is named R58A polypeptide, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that The N-terminus of the polypeptide is modified with a cell-penetrating peptide; the cell-penetrating peptide is CCPs, and its sequence is shown in SEQ ID NO:

2.

3. A method for inhibiting PEDV virus replication using the polypeptide according to claim 1, characterized in that: IPEC-J2, Vero or ST cells are pre-incubated with the polypeptide, and after infection with PEDV, the same amount of polypeptide as that in the pre-incubation is added again, the cells are lysed and samples are harvested to detect the expression of PEDV-N protein in the cells.

4. The method according to claim 3, characterized in that When performing polypeptide pre-incubation, the concentration of the polypeptide used is 1 to 10 μg / mL.

5. A pharmaceutical composition for inhibiting the replication of porcine epidemic diarrhea virus, characterized in that: The pharmaceutical composition comprises the polypeptide according to claim 1, and a pharmaceutically acceptable carrier or adjuvant.

6. Use of the polypeptide composition according to claim 5 in the preparation of a vaccine or a medicine for preventing and / or treating porcine epidemic diarrhea virus.

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