Pea active peptide and application thereof in inhibiting porcine coronavirus

Abstract

The invention relates to the technical field of biology, and particularly discloses a pea active peptide and application thereof in inhibiting porcine coronavirus. The toxicity of pea active peptides EHYDSEAILFKK with different concentrations on Vero cells and ST cells is determined through a CCK-8 test, and it is proved that the pea peptide has no toxic effect on the cells; indirect immunofluorescence, virus copy number, immunoblotting and half tissue infection amount are used for determining that the pea active peptide EHYDSEAILFKK has an inhibition effect on porcine epidemic diarrhea virus in Vero cells and porcine transmissible gastroenteritis virus in ST cells; animal experiments show that piglets with 200 [mu] M of pea active peptide can significantly inhibit in-vivo replication of PEDV and TGEV. Therefore, the pea active peptide can obviously inhibit the replication of the porcine epidemic diarrhea virus and the porcine transmissible gastroenteritis virus in vivo and in vitro, and has a broad-spectrum effect of resisting the porcine coronavirus.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a pea active peptide and application thereof in inhibiting porcine coronavirus. BACKGROUND

[0002] Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) both belong to the family of Coronaviridae and the genus of Coronavirus, and their genomic compositions are also very similar. Both of them are linear single-stranded positive RNA with a full length of about 28 kb, and at least include seven open reading frames (ORFs) encoding ORF1, S, ORF3, E, M and N proteins. ORF1a and ORF1b encode two polyproteins, which account for about 2 / 3 of the full length of PEDV genome, and are cut into 16 non-structural proteins (nsp) such as nsp1-nsp16 by virus-encoded papain and 3C protease, and are involved in life activities such as replication, transcription and virus particle assembly of viral genome.

[0003] Porcine epidemic diarrhea (PED) is a highly contagious disease of pigs caused by porcine epidemic diarrhea virus (PEDV) infection, and the clinical manifestations are mainly vomiting, watery diarrhea and dehydration, and the mortality rate can reach 100%. It causes huge economic losses to the pig industry. In December 2010, a large-scale diarrhea of suckling piglets broke out in China, and the mortality rate can reach 50-100%, which seriously affected the pig breeding industry in China. Subsequent tests proved that the main pathogen of the piglet diarrhea was PEDV. Up to now, porcine epidemic diarrhea is still one of the important diseases that threaten the healthy development of the pig industry.

[0004] Transmissible gastroenteritis (TGE) is a highly contagious acute infectious disease of class B stipulated by the International Office of Epizootics, and its pathogen is transmissible gastroenteritis virus (TGEV). The main symptoms of the disease are dehydration, vomiting and severe diarrhea, and it often occurs in cold seasons in spring and winter. Pigs of different ages are susceptible, and the mortality rate of two-week-old piglets can be as high as 100%. Since it was discovered in Illinois, USA in 1933, the disease has been found in the United Kingdom, South America, Canada, and North Korea (Mullan et al 1994). In 1964, TGEV was first isolated in China, and since then, TGEV has existed in most parts of the country. TGEV often co-infects with porcine epidemic diarrhea virus (PEDV), which is the main cause of pig diarrhea and death in large-scale pig farms. PEDV and TGEV are very similar in clinical symptoms and are difficult to distinguish with the naked eye, which undoubtedly increases the difficulty of disease diagnosis.

[0005] Injection of vaccines is the main method to prevent PED and TGE. However, PEDV and TGEV constantly recombine and mutate to produce new recombinant viruses, and changes in viral antigen sites greatly affect the antigenicity of the virus, causing the mutated virus to escape the immune neutralization of the vaccine, often resulting in vaccine immunization failure. Therefore, the development of new broad-spectrum antiviral small molecule drugs is of great significance for the prevention and control of porcine coronavirus.

[0006] Peas (Pisum sativum L.) It has high economic value and is rich in dietary fiber, trypsin inhibitor, phenolic compounds and plant lectin, and can be used for food production, food processing and feed and other purposes. Pea protein can be obtained by protease hydrolysis and purification. Pea bioactive peptides have various biological functions, such as lowering blood lipids, antioxidant, anti-inflammatory and other biological functions, and are a class of natural bioactive substances with development potential. However, although pea bioactive peptides exhibit diverse physiological activities, their effects and related mechanisms in resisting porcine coronaviruses (including PEDV and TGEV) have not been reported, and they cannot provide new candidate substances or ideas for the prevention and control of porcine coronaviruses. In the face of the bottleneck of vaccine prevention and control caused by PEDV and TGEV variation, and the urgent need for new antiviral small molecule drugs, there is a lack of research on the effects of pea bioactive peptides on PEDV and TGEV in the prior art, and it is urgent to explore their application potential in inhibiting porcine coronaviruses to fill the technical gap in this field. SUMMARY

[0007] In view of the defects and problems existing in the control of porcine coronavirus at present, the application provides a pea active peptide and application thereof in inhibiting porcine coronavirus.

[0008] The application provides a pea active peptide, which has an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 1 is EHYDSEAILFKK; and the amino acid sequence of SEQ ID NO: 2 is EHYDSEAILF.

[0009] The application further provides application of the above-mentioned pea active peptide in inhibiting porcine coronavirus.

[0010] In the application, the non-toxic concentration of the pea active peptide in Vero cells is 12.5-200 μM.

[0011] In the application, the non-toxic concentration of the pea active peptide in ST cells is 12.5-200 μM.

[0012] In the application, the pea active peptide achieves the effect of inhibiting porcine coronavirus by inhibiting the replication of porcine coronavirus.

[0013] In the application, 200 μM of the pea active peptide can significantly inhibit the replication of porcine coronavirus in vivo.

[0014] The application further provides application of the above-mentioned pea active peptide in preparing a product for inhibiting porcine coronavirus.

[0015] Compared with the prior art, the application has the following beneficial effects: The application proves for the first time that the pea active peptide EHYDSEAILFKK can significantly inhibit the replication of porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus in vivo and in vitro at the cellular level and in a mammalian piglet, and has a broad-spectrum effect of resisting porcine coronavirus. The CCK-8 test is used to determine the toxicity of the pea active peptide with different concentrations on Vero cells and ST cells, and it is proved that the pea peptide has no toxic effect on the cells. The indirect immunofluorescence, virus copy number, immunoblotting and half quantity of tissue infection are used to determine the inhibiting effect of the pea active peptide EHYDSEAILFKK on porcine epidemic diarrhea virus in Vero cells and on porcine transmissible gastroenteritis virus in ST cells. And the pea active peptide EHYDSEAILFKK has no significant difference in the survival rate of Vero cells and ST cells at a concentration of 12.5-200 μM. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1The results of the cytotoxicity test of the pea bioactive peptide of the present invention on Vero cells are shown in the figure. Figures 1-7 show the cell viability of Vero cells and ST cells after treatment with EHYDSEAILFKK peptide at concentrations of 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively, which are the negative control and the cell viability of ST cells after treatment with EHYDSEAILFKK peptide at concentrations of 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM.

[0017] Figure 2 The results of the toxicity test of the pea bioactive peptides of the present invention on ST cells are shown in the figure. Figures 1-7 show the cell viability of Vero cells and ST cells after treatment with EHYDSEAILFKK peptides at concentrations of 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively, which are the negative control and the cell viability of ST cells after treatment with EHYDSEAILFKK peptides at concentrations of 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM.

[0018] Figure 3 The results of Western blot analysis of the inhibition of PEDV hubei-2016 replication by the pea bioactive peptide of this invention are shown in the figure. In the figure, NC, 1, and 2 are the negative control, the group incubated with pea bioactive peptide and then inoculated with PEDV hubei-2016 virus strain, and the group inoculated with PEDV hubei-2016 virus strain without pea bioactive peptide, respectively.

[0019] Figure 4 The results of Western blot analysis of the inhibition of TGEV TS replication by pea bioactive peptides of the present invention are shown. In the figure, NC, 1, and 2 are the negative control, the TGEV TS virus strain group after incubation with pea bioactive peptides, and the TGEV TS strain group without pea bioactive peptides, respectively.

[0020] Figure 5 The results of indirect immunofluorescence detection of PEDV hubei-2016 by pea bioactive peptides of the present invention are shown. In the figure, N represents the green fluorescence of PEDV N protein expression, DAPI staining is the cell nucleus of Vero cells, and MERGE is the table showing the fusion of green fluorescent protein and DAPI. 1 and 2 are the groups incubated with pea bioactive peptides and then inoculated with PEDV hubei-2016 strain, and the groups inoculated with PEDV hubei-2016 strain without pea bioactive peptides, respectively.

[0021] Figure 6 The results of indirect immunofluorescence detection of TGEV TS by pea bioactive peptides of the present invention are shown in the figure. N in the figure represents the green fluorescence of TGEV N protein expression, DAPI staining is the cell nucleus of Vero cells, and MERGE is the table showing the fusion of green fluorescent protein and DAPI. 1 and 2 are the TGEV TS strain group after incubation with pea bioactive peptides and the TGEV TS strain group without pea bioactive peptides, respectively.

[0022] Figure 7The results of the detection of the titer of pea bioactive peptide against PEDV hubei-2016 in this invention are shown in the figure. NC, 1, and 2 are the negative control, the group incubated with pea bioactive peptide EHYDSEAILFKK and then inoculated with PEDV hubei-2016 strain, and the group inoculated with PEDV hubei-2016 strain without pea bioactive peptide EHYDSEAILFKK.

[0023] Figure 8 The results of the detection of TGEV TS titer by pea bioactive peptides of the present invention are shown in the figure. NC, 1, and 2 are the negative control, the group incubated with pea bioactive peptide EHYDSEAILFKK and then inoculated with TGEV TS virus strain, and the group inoculated with TGEV TS strain without pea bioactive peptide EHYDSEAILFKK, respectively.

[0024] Figure 9 The figure shows the viral copy number of PEDV hubei-2016 against the pea bioactive peptide of this invention; NC, 1, and 2 in the figure represent the negative control, the group incubated with pea bioactive peptide EHYDSEAILFKK and then inoculated with PEDV hubei-2016 strain, and the group inoculated with PEDV hubei-2016 strain without pea bioactive peptide EHYDSEAILFKK.

[0025] Figure 10 The figure shows the viral copy number of TGEV TS by the pea active peptide of the present invention; NC, 1, and 2 in the figure are the negative control, the TGEV TS strain group after incubation with pea active peptide EHYDSEAILFKK, and the TGEV TS strain group without pea active peptide EHYDSEAILFKK.

[0026] Figure 11 This is a time-process analysis diagram of the pea bioactive peptides on PEDV hubei-2016 according to the present invention.

[0027] Figure 12 The figures show the adsorption, invasion, and replication results of pea bioactive peptides on PEDV hubei-2016 in this invention. In the figures, 1, 2, 3, 4, 5, and 6 represent the peptide co-incubated with PEDV during adsorption, the peptide not incubated during adsorption, the peptide co-incubated with PEDV during invasion, the peptide not incubated during invasion, the peptide co-incubated with PEDV during replication, and the peptide not incubated during replication, respectively.

[0028] Figure 13 This is a grayscale analysis diagram of the adsorption, invasion, and replication of PEDV hubei-2016 by the pea bioactive peptides of this invention; in the diagram, 1, 2, 3, 4, 5, and 6 represent the peptide co-incubated with TGEV during adsorption, the peptide not incubated during adsorption, the peptide co-incubated with TGEV during invasion, the peptide not incubated during invasion, the peptide co-incubated with TGEV during replication, and the peptide not incubated during replication, respectively.

[0029] Figure 14 The results of the release of PEDV hubei-2016 by the pea bioactive peptides of this invention are shown.

[0030] Figure 15 This is an experimental analysis diagram showing the time process of pea bioactive peptides on TGEV TS according to the present invention.

[0031] Figure 16 This is a grayscale analysis diagram of the adsorption, invasion, and replication of TGEV TS by the pea bioactive peptides of the present invention; in the diagram, 1, 2, 3, 4, 5, and 6 represent the peptide co-incubated with TGEV during adsorption, the peptide not incubated during adsorption, the peptide co-incubated with TGEV during invasion, the peptide not incubated during invasion, the peptide co-incubated with TGEV during replication, and the peptide not incubated during replication, respectively.

[0032] Figure 17 This is a graph showing the effect of pea bioactive peptides of the present invention on TGEV TS release.

[0033] Figure 18 The results of this invention's pea bioactive peptide inhibiting PEDV hubei-2016 replication in piglets are shown in the figure. NC, 2, and 3 represent the negative control group, the group treated with bioactive peptide and challenged with PEDV hubei / 2016, and the group directly challenged with PEDV hubei / 2016, respectively.

[0034] Figure 19 The results of the pea active peptide inhibiting TGEVTS replication in piglets are shown in the figure; NC, 2, and 3 are the negative control group, the TGEVTS challenge group after active peptide treatment, and the TGEVTS direct challenge group, respectively. Detailed Implementation

[0035] This invention provides a pea bioactive peptide and its application in inhibiting porcine coronavirus. The invention is further illustrated below with examples and accompanying drawings. However, this invention is not limited to these specific embodiments; those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. In this invention, each set of data is set to 3 parallel groups. ImageJ software was used to analyze the grayscale values ​​of the Western blot results, and the values ​​are expressed as mean ± standard deviation (mean ± sd). One-way ANOVA was used to compare the differences among multiple groups. GraphPad Prism 9.0 was used for analysis and plotting; a p-value < 0.05 was considered statistically significant.

[0036] The materials, main reagents, and main instruments and equipment used in this invention are as follows: (1) Cells, viruses and bioactive peptides: Vero cells, PEDV CH-hubei-2016 strain (GenBank accession number: KY928065), were preserved by the Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences; The TGEV TS strain (GenBank accession number: DQ201447) was donated by Researcher Lan Xi of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The pea bioactive peptide EHYDSEAILFKK was synthesized by Jier Biochemical (Shanghai) Co., Ltd., with a purity of over 95%. The addition of KK after LF increases its water solubility. It was diluted to 1 mM with ultrapure water, filtered through a 0.22 μm filter, and stored at -20℃ for later use. (2) Main reagents: Fetal bovine serum, a product of Gibco. Dulbecco's modification of Eagle's medium, Dulbecco (DMEM) high glucose culture medium, trypsin, protein quality standards, products of Beijing Solarbio Biotechnology Co., Ltd. Cell Counting Kit-8 (CCK-8) reagent kit and ECL ultrasensitive luminescent solution are products of Shanghai Beyotime Biotechnology Co., Ltd. The N anti-PEDV monoclonal antibody was preserved by the Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences. The N anti-TGEV monoclonal antibody was donated by Professor Wei Zhanyong of Henan Agricultural University. Rabbit anti-β-actin antibody, a product of Cell Signaling Technology (CST), USA; HRP-labeled goat anti-rabbit IgG and goat anti-mouse IgG, products of Wuhan Sanying Biotechnology Co., Ltd. RNA extraction kit, reverse transcription kit, and real-time PCR enzyme are products of Takara Bio Engineering Co., Ltd.

[0037] (3) Major instruments and equipment: Visible spectrophotometer, Shanghai Opler Instruments Co., Ltd.; Electrophoresis apparatus, Beijing Liuyi Biotechnology Co., Ltd.; Transfer apparatus, Bio-RAD; Gene-geniu fully automated gel imaging system, Syngene, UK; Clean bench, Suzhou Antai Co., Ltd.; Carbon dioxide incubator, Thermo, USA; Fluorescence microscope, Zeiss, Germany; qPCR instrument, ABI, USA.

[0038] Example 1: Detection of the toxicity of pea bioactive peptide EHYDSEAILFKK to Vero and ST cells. Vero and ST cells in good growth condition were seeded into 96-well plates and cultured at 37°C with 5% CO2. When the cells reached approximately 80% confluence, they were inoculated with the pea bioactive peptide EHYDSEAILFKK. The bioactive peptide was diluted to 12.5–200 μM in DMEM medium, with three biological replicates for each concentration. The cells were cultured at 37°C with 5% CO2 for 24 h, then the medium was discarded. After inoculating Vero and ST cells with 12.5–200 μM EHYDSEAILFKK for 24 h, DMEM medium containing 10% CCK-8 solution was added, and the cells were cultured at 37°C for 1 h. The absorbance was then read at 450 nm using a microplate reader. Cell viability was calculated according to the instructions. The cell viability results are shown below. Figure 1 and Figure 2 .

[0039] Depend on Figure 1 and Figure 2 It was found that 200 μM had no significant toxic effect on Vero cells and ST cells (P>0.05), so 200 μM was selected for subsequent experiments.

[0040] Example 2: Western blot detection Cells were plated in 24-well cell culture plates. When cell confluence reached 80%, 200 μM of pea bioactive peptide EHYDSEAILFKK was added. After 24 hours of culture, cells were inoculated with 0.1 MOI PEDV hubei-2016 and 0.1 MOI TGEV TS strains, respectively. Cells were harvested after 18 hours and lysed on ice for 30 minutes using RIPA lysis buffer. Loading buffer was added, and the cells were boiled in water for 10 minutes. Proteins were then separated using SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 50 g / L skim milk powder at 37 °C for 1 hour. After washing three times with TBST, the corresponding antibodies were added, and the membranes were incubated overnight at 4 °C. After washing three times with TBST, the membranes were incubated with HRP-labeled goat anti-mouse and goat anti-rabbit IgG antibodies for 2 hours. After washing with TBST, the membranes were detected using ECL ultrasensitive luminescence buffer. The results are shown below. Figure 3 and Figure 4 As shown.

[0041] Depend on Figure 3 It can be seen that the N protein band in group 1 was significantly lower than that in group 2, and the difference was statistically significant, proving that incubation with the active peptide EHYDSEAILFKK can significantly inhibit the Hubei-2016 strain. Figure 4 It can be seen that the N protein band in group 1 was significantly lower than that in group 2, and the difference was statistically significant, proving that incubation with the active peptide EHYDSEAILFKK can significantly inhibit the expression of N protein in the TGEV TS strain. Combined with...Figure 3 and Figure 4 The study demonstrated that the pea bioactive peptide EHYDSEAILFKK can inhibit the replication of PEDV hubei-2016 strain and TGEV TS strain.

[0042] Example 3: Indirect immunofluorescence detection Pea bioactive peptide EHYDSEAILFKK was incubated in 24-well plates, and cells were seeded and cultured for 24 h. The cells were then inoculated with 0.1 MOI PEDV hubei-2016 strain and 0.1 MOI TGEV TS strain, respectively. After 18 h, the cells were washed with PBST, fixed with 4% paraformaldehyde at room temperature for 30 min, blocked with 50 g / L bovine serum albumin (BSA) at 37 ℃ for 1 h, incubated overnight at 4 ℃ with anti-N protein monoclonal antibody, and incubated for 1 h at 37 ℃ in the dark with FITC-labeled goat anti-mouse secondary antibody. After staining with 4,6-diamidino-2-phenylin-dole-dihydrochloride (DAPI), the cells were observed and photographed under a fluorescence microscope. The results are shown below. Figure 5 and Figure 6 As shown.

[0043] from Figure 5 The results show that the green fluorescence of the N protein in group 1 is significantly weaker than that in group 2, proving that incubation with the active peptide EHYDSEAILFKK can significantly inhibit the Hubei-2016 strain. Figure 6 It can be seen that the green fluorescence of the N protein in group 1 is significantly weaker than that in group 2, proving that incubation with the active peptide EHYDSEAILFKK can significantly inhibit the replication of the TGEV TS strain.

[0044] Example 4: Half-maximum tissue culture infection dose (TCID) 50 ) detection Healthy cells were seeded into 96-well plates, washed with PBS, and then inoculated with 0.1 MOI PEDVhubei-2016 and 0.1 MOI TGEV TS strains, respectively. Samples from different treatment groups were serially diluted 10-fold (100 μL virus solution + 900 μL DMEM) and added to the culture plates, with 8 replicates per group. After incubation at 37 ℃ for 1 h, the virus solution was discarded, and DMEM containing 2% trypsin was added for further incubation for 5–7 days. Disease lesions were observed daily until the number of diseased wells no longer increased. The half-maximal infectious dose (TCID50) was calculated using the REED-MUENCH method. 50 The result is as follows Figure 7 and Figure 8 As shown.

[0045] Depend on Figure 7 The results show that TCID in group 1 50 The titer was significantly lower than that in group 2, and the difference was statistically significant, demonstrating that incubation with the active peptide EHYDSEAILFKK significantly reduced the titer of the Hubei-2016 strain. Figure 8 The results show that TCID in group 1 50 The titer was significantly lower than that of Group 2, and the difference was statistically significant, demonstrating that incubation of the active peptide EHYDSEAILFKK can significantly inhibit the titer of the TGEV TS strain.

[0046] Example 5: Virus copy number detection After incubating cells with EHYDSEAILFKK, 0.1 MOI PEDV hubei-2016 strain and 0.1 MOITGEV TS strain were inoculated, respectively. Cells were harvested after 18 h, and RNA was extracted using the Trizol method. 1 μg of RNA was reverse transcribed into cDNA, and the viral copy number was determined using a laboratory-established fluorescent probe method. The results are as follows: Figure 9 and Figure 10 As shown.

[0047] Depend on Figure 9 It can be seen that the viral copy number in group 1 was significantly lower than that in group 2, and the difference was statistically significant, proving that incubation with the active peptide EHYDSEAILFKK can significantly reduce the replication of the Hubei-2016 strain. Figure 10 The results showed that the viral copy number in group 1 was significantly lower than that in group 2, and the difference was significant, proving that the incubation of the active peptide EHYDSEAILFKK can significantly inhibit the replication of the TGEV TS strain.

[0048] Example 6: Time Process Analysis Experiment The life cycle of PEDVs and TGEVs includes stages such as adsorption, invasion, replication, and release. For example... Figure 11 and 15 As shown, pea bioactive peptide EHYDSEAILFKK was added at different stages, and the bioactivity of the peptide was detected. The virus was also inoculated with 0.1 MOI PEDV hubei-2016 strain and 0.1 MOI TGEV TS strain. The effects of pea bioactive peptide EHYDSEAILFKK on different stages of the PEDV and TGEV lifecycle were determined through time-process analysis. The results are shown below. Figures 12-14 and Figures 16-17 As shown.

[0049] Combination Figures 12-14It can be seen that the pea bioactive peptide EHYDSEAILFKK can significantly promote the adsorption (P<0.05) and invasion (P<0.05) of PEDV hubei-2016 strain into Vero cells, but inhibit viral replication in cells (P<0.05), while having no significant effect on viral release.

[0050] Combination Figure 16 and 17 It can be seen that the pea bioactive peptide EHYDSEAILFKK can significantly promote the adsorption of TGEV TS strain (P<0.05), but inhibit viral replication in cells (P<0.05), and has no significant effect on viral release and invasion into cells.

[0051] Experimental example: Animal experiment Thirty suckling piglets were randomly divided into six groups of five each: a negative control group, an active peptide treatment group, and a challenge group. Since both PEDV and TGEV can infect suckling piglets, it was possible for a single piglet to be infected with both. To avoid affecting the reliability of the experimental data, negative control groups were set up for both groups infected with PEDV and TGEV. Different experiments were conducted in separate animal laboratory rooms. Specifically, Group 1 was the negative control group (NC) and was fed normally. Group 2 involved each piglet being orally administered the active peptide EHYDSEAILFKK at a dose of 10 mg / kg for 2 consecutive days, followed by inoculation with the PEDV hubei-2016 strain at a dose of 104 TCID50 / pig. Group 3 was fed normally, and 2 days later, it was inoculated with the PEDVhubei-2016 strain at the same time as Group 2, at a dose of 104 TCID50 per piglet. Group 4 was the negative control group (NC) and was fed normally. Group 5 involved each piglet being orally administered the active peptide EHYDSEAILFKK at a dose of 10 mg / kg for 2 consecutive days, followed by inoculation with the TGEV TS strain at a dose of 104 TCID50 / pig. Group 6 was fed normally, and 2 days later, it was inoculated with TGEV TS strain at the same time as Group 4, at a dose of 104 TCID50 per piglet.

[0052] All groups were fed continuously for 15 days. The disease status of the experimental animals was observed and recorded daily. Blood samples were collected on days 5, 10, and 15 after challenge. RNA was extracted using the Takara MiniBEST viral RNA / DNA Extraction Kit from Takara Bio Inc., and the RNA concentration was measured. 1 μg of RNA was used for reverse transcription using the Takara Bio PrimeScript RT Master Mix kit. The reverse transcription system was as follows: 4 μL 5×MLV buffer, 1 μL dNTP (10mM), 2 μL random primer, 2 μL MLV, 1 μL RNase inhibitor, 1 mg RNA, and RNase-free water to make up to 20 μL. After offline mixing, the mixture was incubated at 42℃ for 10 min and 95℃ for 2 min.

[0053] Viral copy number was determined using a fluorescent probe method. The reaction system was as follows: 10 μL Ex Taq (probe qPCR), 0.4 μL each of forward and reverse primers, 0.2 μL probe, 1 μL cDNA, and double-distilled water to a final volume of 20 μL. The reaction program was: 95 ℃ for 20 s, 95 ℃ for 3 s, 60 ℃ for 30 s, for a total of 40 cycles. Refer to R... 2 Determine data availability. Verify the preventive effect of pea bioactive peptides against PEDV and TGEV infections using qRT-PCR; results are as follows: Figure 18 and 19 As shown.

[0054] Depend on Figure 18 and 19 It can be seen that the pea bioactive peptide EHYDSEAILFKK can inhibit the replication of PEDV hubei-2016 and TGEV TS in piglets.

[0055] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pea bioactive peptide, characterized in that: It has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; the amino acid sequence of SEQ ID NO:1 is EHYDSEAILFKK; the amino acid sequence of SEQ ID NO:2 is EHYDSEAILF.

2. The application of the pea bioactive peptide as described in claim 1 in inhibiting porcine coronavirus.

3. The application as described in claim 2, characterized in that: The non-toxic concentration of the pea bioactive peptide in Vero cells is 12.5-200 μM.

4. The application as described in claim 2, characterized in that: The non-toxic concentration of the pea bioactive peptide in ST cells is 12.5-200 μM.

5. The application as described in claim 2, characterized in that: The pea bioactive peptides inhibit the replication of porcine coronavirus, thereby suppressing its replication.

6. The application according to claim 2, characterized in that: 200 μM of pea bioactive peptides can significantly inhibit the replication of porcine coronavirus in vivo.

7. The use of the pea bioactive peptide as described in claim 1 in the preparation of products that inhibit porcine coronavirus.

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