Polypeptide for enhancing porcine epidemic diarrhea virus replication and application thereof

Abstract

The invention relates to a bioengineering technology, and aims to provide a polypeptide for enhancing porcine epidemic diarrhea virus replication and application thereof. 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 R58-WT polypeptide, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1. According to the invention, a key site influencing virus replication in an N-terminal region of PEDV N protein is used as a new target spot, the polypeptide has a very significant promotion effect in PEDV infection model verification experiments of IPEC-J2, Vero and ST cells, and the problems of weak low-generation virulence, poor cell adaptability, difficulty in passage and the like in the process of rescuing PEDV low virulent strains can be perfectly solved.

Description

Technical Field

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

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious intestinal disease that causes vomiting, dehydration, and fatal watery diarrhea in piglets. Porcine epidemic diarrhea virus (PEDV), the pathogen of PED, belongs to the genus Alphacoronavirus of the family Coronaviridae. It is a single-stranded positive-sense RNA virus wrapped in an envelope, with a diameter of about 90-190 nm. The envelope has petal-like filaments with large spacing, regular arrangement, and a crown shape. Its genome is about 28 kb in length, including a non-coding region (5'-UTR) with a cap at the 5' end, a non-coding region (3'-UTR) with a poly(A) tail at the 3' end, and 7 open reading frames (ORFs). ORFs encode 4 structural proteins, namely spike (S) protein, envelope (E) protein, membrane (M) protein, nucleocapsid (N) protein, 1 auxiliary protein (ORF3), and 16 non-structural proteins (Nsp 1-16).

[0003] PEDV N protein has been reported to participate in viral replication and transcription, encapsulate the viral RNA genome, and is dynamically associated with the viral RNA replication site, playing a role in protecting the viral genome and mediating the transfer of the genome to the budding site. In addition, PEDV N can inhibit the expression of IFN-β and mediate the immune escape of the virus in two ways: destroying the RIG-I / MDA5 cascade reaction; blocking the interaction between TBK1 and IRF3. Research on PEDV N protein helps to reveal the laws of viral infection and transmission, and is of great significance for the development of new epidemic prevention measures.

[0004] In the research process on PEDV, since a large amount of complicated testing and verification work is required on the effect of virus infection under different conditions, providing sufficient amount of virus for experiment is a prerequisite.

[0005] The isolation of PEDV has always been the "gold standard" for clinical diagnosis of PED. Researchers usually isolate PEDV by mixing the feces and diseased small intestine samples of diseased pigs with phosphate buffer, then freeze-thaw centrifugation, and taking the supernatant to infect Vero cells in vitro. After continuous passage and expansion, the clinical strain of PEDV is purified. This method is limited by PEDV's weak resistance to the environment, poor cell adaptability, trypsin dependence, and weak cell-to-cell transmission ability.

[0006] PEDV live attenuated vaccine has been widely studied in vaccine development due to its high immunogenicity. In the preparation of attenuated vaccines, researchers currently use two main methods. One is to isolate the virus and continuously propagate it in vitro to make it show higher virus titer, lower pathogenicity and higher immunogenicity. For example, the live attenuated vaccine developed from the 28th generation cell culture of PEDV CV777 strain can provide more than 80% protection for immunized piglets; the second is based on the study of PEDV virulence factors and immune-related epitopes, using PEDV's efficient reverse genetic technology to artificially modify and screen the viral genome to obtain safer and more effective PEDV attenuated vaccine candidate strains.

[0007] However, in the process of rescuing PEDV attenuated strains, the above current practices or research results all face the problems of weak virulence of low-generation viruses, poor cell adaptability, and difficulty in propagation, which cannot meet the needs of large-scale experiments for PEDV viruses in practical applications. Therefore, it is necessary to propose new solutions. Summary of the invention

[0008] 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.

[0009] To solve the technical problem, the solution of the present invention is:

[0010] Provided is a polypeptide for enhancing the replication of porcine epidemic diarrhea virus, which 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 R58-WT polypeptide, and its amino acid sequence is shown in SEQ ID NO:1.

[0011] 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.

[0012] The present invention further provides a method for promoting the expression of PEDV-N protein using the aforementioned polypeptide, which comprises pre-incubating IPEC-J2, Vero or ST cells with the polypeptide, adding the same amount of polypeptide as that 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.

[0013] As a preferred embodiment of the present invention, when performing polypeptide pre-incubation, the concentration of the polypeptide used is 10 μg / mL.

[0014] The present invention also provides the use of the above polypeptide in preparing a reagent for promoting the replication of porcine epidemic diarrhea virus.

[0015] Description of the invention principle:

[0016] In view of the fact that the existing work in the industry on the isolation, propagation and preparation of attenuated vaccine candidate strains of PEDV virus is not ideal, the present invention proposes a new solution, which proposes to enhance the virus replication efficiency by designing a polypeptide for enhancing the replication ability of PEDV.

[0017] 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.

[0018] 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 the arginine at a specific target position in the N protein of PEDV as the regulation center of the PEDV replication process, providing new ideas for solutions. In the existing research results, there is no clear record of using the characteristics of nucleocapsid (N) protein and arginine to regulate the PEDV replication process.

[0019] To this end, 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:

[0020] (1) Based on the N-terminal sequence characteristics of PEDV N protein, arginine is centered and some amino acids are retained before and after it. Since the purity, reactivity and preparation difficulty of the polypeptide are limited by the length of the polypeptide, about 30 amino acids are generally appropriate. Therefore, in this application, except for the penetrating peptide (11 amino acids), the length of the target polypeptide is designed to be 20 amino acids.

[0021] (2) After the amino acid sequence of the polypeptide is determined, a transmembrane peptide sequence is inserted at the N-terminus to assist the polypeptide in entering the cell, and finally a polypeptide (named R58-WT) is synthesized.

[0022] Through verification in the natural infection model of PEDV, the results showed that the R58-WT polypeptide obtained in the present application can significantly enhance the replication of PEDV.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 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, and uses the highly conserved characteristics of this site in different coronaviruses to propose it as a new target for adjusting the viral replication process.

[0025] 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 promoting effect in the PEDV infection model verification experiment of IPEC-J2, Vero, and ST cells, and can perfectly solve the problems of weak virulence of low-generation viruses, poor cell adaptability, and difficulty in propagation in the process of rescuing PEDV weak strains.

[0026] 3. The present invention clarifies the important regulatory function of the conserved arginine at the N-terminus of the nucleocapsid protein of PEDV in the replication process of PEDV. On this basis, the design of a membrane-penetrating peptide targeting this site can promote viral replication. The proposal of the present invention further enriches the application of membrane-penetrating peptides in enhancing the replication ability of viruses and improving the rescue efficiency, and further plays a role in the preparation of candidate strains of viral vaccines.

[0027] 4. This invention also provides 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

[0028] Figure 1 This is a description of the R58-WT polypeptide structure in the present invention.

[0029] Figure 2 This is a description of the properties of the R58-WT polypeptide in the present invention.

[0030] Figure 3 Analysis of the effects of different concentrations of R58-WT polypeptide on IPEC-J2 cell viability.

[0031] Figure 4 Analysis of the effect of R58-WT polypeptide on PEDV replication.

[0032] Figure 5 To analyze the effect of R58-WT polypeptide during PEDV infection in other susceptible cells. DETAILED DESCRIPTION

[0033] 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.

[0034] Example 1 R58-WT polypeptide promotes PEDV replication

[0035] 1) Synthesis of peptides containing N-terminal arginine of PEDV N protein

[0036] 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. According to the description of the principle of the invention, the amino acids before and after the arginine are designed: the arginine site is taken as the center, 20 amino acids including the site are intercepted, and cell-penetrating peptides (CPPs) are added to finally form the R58-WT polypeptide. The specific sequence of the polypeptide is shown in Table 1, and the underlined abbreviation R in the sequence is the arginine as the target.

[0037] The synthesis of R58-WT peptide product was entrusted to Shanghai Hongpeptide Biotechnology Co., Ltd. The molecular weight of the peptide was 4218.82 and the purity was 93.99%. Figure 2 .

[0038] Table 1 Synthetic peptide sequences

[0039] Sequence number Peptide name sequence SEQ ID NO:1 R58-WT YGRKKRRQRRRQIGYWNEQIRWRMRRGERIE SEQ ID NO:2 CCPs YGRKKRRQRRR

[0040] 2) R58-WT peptide is not toxic to IPEC-J2 cells

[0041] The porcine intestinal epithelial cell line (IPEC-J2 cells) was treated with R58-WT peptide at concentrations of 1, 5, 10, 20 and 50 μg / mL, respectively, and the Mock and Blank wells were used as controls. Cell viability was measured after 24 hours. CCK-8 dye was added to the wells, incubated for 1 hour, and the absorbance was measured at 450nm to calculate the cell viability. The results showed that the cell viability values ​​of R58-WT at different concentrations were similar to those of the Mock group, indicating that the peptide was non-toxic to IPEC-J2 cells ( Figure 3 ).

[0042] 3) PEDV N-R58-WT peptide can act as a viral enhancer to promote PEDV replication

[0043] IPEC-J2 cells were pre-incubated with R58-WT polypeptide at different concentrations (1, 5, 10, 20 and 50 μg / mL) for 1 h, and then PEDV (MOI = 1) infected cells. After 4 h, trypsin (4 μg / mL) was added and the same amount of polypeptide as in the pre-incubation was added. After 24 h, the cells were lysed and the cell RNA and protein were harvested. The transcription levels of PEDV ORF1 and PEDV N genes were detected by probe method, and the protein levels of PEDV N and PEDV S1 were detected by Western blot.

[0044] The results showed that PEDV N-R58-WT peptide significantly promoted the expression of PEDV N in a dose-dependent manner between 1 and 10 μg / mL ( Figure 4 ). The transcription level of PEDV ORF1 reached a peak when the peptide dosage was 5 μg / mL, and decreased significantly when it was greater than 10 μg / mL. The protein level of PEDV S1 was highest when the peptide dosage was 10 μg / mL ( Figure 4 ).

[0045] Example 2 Application of PEDV N-R58-WT polypeptide in other susceptible cells

[0046] The R58-WT polypeptide was added to the African green monkey kidney cell line (Vero cell) and the pig testicular cell line (ST cell) at the appropriate concentration in Example 1, pre-incubated for 1 hour, and supplemented with polypeptide and trypsin (4 μg / mL) after infection with PEDV for 4 hours. Cell protein was harvested after 24 hours, and the expression level of PEDV-N protein was detected by Western blot.

[0047] The results showed that in Vero cells, the level of PEDV N protein increased in a dose-dependent manner after treatment with wild-type peptide (WT). Figure 5 ), while in ST cells, different concentrations of wild-type peptide (WT) could promote the expression of PEDV N protein ( Figure 5 ). The above results are the same as those in Example 1, confirming the promoting effect of PEDV N-R58-WT on viral replication.

[0048] Analysis and Conclusion:

[0049] Based on the results of Examples 1 and 2, it can be seen that the R58-WT polypeptide provided by the present invention can significantly promote the PEDV replication process, and can therefore be further used as a promoter targeting the PEDV virus replication stage, thereby increasing the rescue of weak strains for virus isolation and passage. 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 a pharmaceutically active component of a generally applicable value-added promoter is further verified.

[0050] Based on the results of multiple examples verified by the applicant, it is confirmed that when the polypeptide is used at a concentration of 10 μg / mL, the effect of promoting the replication process of the PEDV virus is best.

[0051] 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 enhancing the replication of porcine epidemic diarrhea virus, characterized in that 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 R58-WT 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 promoting the expression of PEDV-N protein 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 peptide pre-incubation, the peptide concentration used was 10 μg / mL.

5. Use of the polypeptide according to claim 1 in the preparation of an agent for promoting the replication of porcine epidemic diarrhea virus.

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