Antiviral polypeptide modified based on Cecropin D and application of antiviral polypeptide in inhibiting porcine reproductive and respiratory syndrome virus infection

Abstract

The invention discloses an antiviral polypeptide modified on the basis of Cecropin D (CD) and application of the antiviral polypeptide to inhibition of infection of a porcine reproductive and respiratory syndrome virus (PRRSV), and particularly discloses an antiviral polypeptide modified on the basis of Cecropin D (CD) and application of the antiviral polypeptide modified on the basis of Cecropin D (CD). The amino acid sequence of the antiviral polypeptide is as shown in SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4. Based on the balance of hydrophilicity and hydrophobicity, a parent peptide CD is modified, and three derivatives of CD-2, CD-3 and CD-4 are designed and synthesized. Compared with the research of other known antiviral polypeptides, the polypeptide disclosed by the invention shows remarkable anti-PRRSV activity at a lower concentration (100mu g / mL), which indicates that the polypeptide designed and synthesized by the invention has higher safety and application potential in the development of anti-PRRSV drugs, and the proposal of the invention provides a new technical means for inhibiting PRRSV infection.

Description

Technical Field

[0001] The present invention relates to a novel antiviral polypeptide modified based on Cecropin D, and also relates to the application of the antiviral polypeptide in inhibiting porcine blue ear disease virus infection. The present invention belongs to the field of medical technology. Background Art

[0002] Viral infection of pigs is one of the difficulties in the development of the global pig industry. Widely spread pig pathogenic viruses mainly include pseudorabies virus (PRV), porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV), which have caused huge economic losses in the pig industry. Among them, PRRSV poses a huge threat to the global pig industry, so vaccination has become the main means of preventing and controlling the disease. However, although a variety of traditional blue ear disease vaccines have been approved and widely used in clinical practice, they still face many challenges. Although traditional inactivated vaccines have no biosafety risks, they have low neutralizing antibody levels, cannot induce cellular immunity, and require multiple vaccinations. In contrast, although live vaccines can provide immune protection, they are insufficient to protect heterologous strains, and there are problems such as virulence reversion, recombination of vaccine strains and wild strains, and lack of markers. In addition, although subunit vaccines and virus-like particle vaccines are non-pathogenic, their ability to protect susceptible animals is limited, and although nucleic acid vaccines have a simple production process, they have potential biosafety risks, such as the possibility of foreign DNA integrating into the host genome. At the same time, the high mutation rate and high recombination rate of PRRSV make existing vaccines easily ineffective, which further increases the difficulty of vaccine development.

[0003] Due to the huge challenges posed by PRRSV worldwide, more and more peptides with anti-PRRSV activity have been discovered and designed. Antimicrobial peptides are small molecule proteins that are widely found in animals, plants and microorganisms. They have potential antibacterial, antiviral, antifungal, antitumor and antiparasitic activities and can inhibit viral replication by interfering with multiple stages of the viral life cycle. Their diverse mechanisms of action mainly include destroying the viral envelope, preventing the virus from binding to host cells, and interacting with specific receptors of host cells.

[0004] Cecropin D (CD) belongs to the Cecropin family and was originally isolated from Cecropia pupae [HULTMARK D, ENGSTROMA, BENNICH H, et al. Insect immunity: isolation and structure of cecropinD and four minor antibacterial components from Cecropia pupae [J]. Eur J Biochem, 1982, 127 (1): 207-17]. It is a multifunctional antimicrobial peptide with multiple effects such as antiviral, antifungal, antitumor and immunomodulatory.

[0005] The invention first studies the antiviral effect of Cecropin D, and then further enhances the anti-PRRSV activity of Cecropin D by introducing different cationic amino acids, thereby providing a new technical means for inhibiting PRRSV infection. Summary of the invention

[0006] The object of the present invention is to provide an antiviral polypeptide based on Cecropin D modification and its application in inhibiting porcine blue ear disease virus infection.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The present invention designs and synthesizes Cecropin D and its three derivatives CD-2 to CD-4 based on the balance between hydrophilicity and hydrophobicity. Among them, more positively charged lysine is introduced into CD-2 to enhance the interaction between the polypeptide and the membrane, and tryptophan with strong hydrophobicity and positive charge is introduced into CD-3; and in CD-4, valine (V) is replaced by phenylalanine (F) to promote the stability of the secondary structure. The results of HPLC and CD spectra show that the designed polypeptides have high purity and all form significant α-helical structures. Especially in the environment of simulating cell membrane, the polypeptides show significant structural changes, which verifies that they can form stable secondary structures. Studies have shown that alpha helix can be inserted into the viral envelope, causing membrane destruction, interfering with its structural integrity and thus inhibiting viral infection [HUANGY, HE L, LI G, et al. Role of helicity of alpha-helical antimicrobial peptides to improve specificity [J]. Protein Cell, 2014, 5 (8): 631-42. HUAN Y, KONG Q, MOU H, et al. Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields [J]. Front Microbiol, 2020, 11 (582779.] G W. Structures of human host defense cathelicidin LL-37 and its smallest antimicrobial peptide KR-12 in lipid micelles [J]. The Journal of biological Chemistry, 2008,] Based on these structural characteristics, it is speculated that it should have good antiviral activity, so we tested its antiviral activity through a variety of experimental methods. The results showed that the polypeptides of the present invention exhibited significant anti-PRRSV activity at a lower concentration (100 μg / mL). These polypeptides inhibit the replication of PRRSV by blocking the key step of PRRSV invasion of cells.

[0009] Therefore, based on the above research, the present invention proposes an antiviral polypeptide modified based on Cecropin D, the amino acid sequence of the antiviral polypeptide is shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0010] Furthermore, the present invention also proposes the use of the antiviral polypeptide in the preparation of a drug for inhibiting porcine reproductive and respiratory syndrome virus.

[0011] Among them, preferably, the porcine reproductive and respiratory syndrome virus is highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV).

[0012] Preferably, the antiviral polypeptide inhibits the replication of PRRSV by blocking the key step of PRRSV invading cells.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] Based on the balance between hydrophilicity and hydrophobicity, the present invention designed and synthesized three derivatives by modifying the parent peptide Cecropin D. Compared with the research on other known antiviral peptides, the polypeptide of the present invention exhibited significant antiviral activity at lower concentrations. For example, Liu et al. demonstrated that the polypeptide Cecropin D exhibited effective antiviral activity against PRRSV infection and replication at a concentration of 300 μg / mL in vitro. [ XL,CG,YH,et al.Inhibition of porcinereproductive andrespiratory syndrome virus by Cecropin D in vitro[J].Infection,genetics and evolution:journal of molecular epidemiology andevolutionary genetics in infectious diseases,2015, ] , while the polypeptide designed by the present invention exhibited excellent anti-PRRSV activity at a concentration of 100 μg / mL. This indicates that the polypeptide designed and synthesized by the present invention has high safety and application potential in the development of anti-PRRSV drugs, and the present invention provides a new technical means for inhibiting PRRSV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A -D is the HPLC chromatogram and mass spectrum of peptides CD and CD-2 to CD-4;

[0016] Figure 1E is the CD value of the peptide and CD-2 to CD-4 in aqueous solution;

[0017] Figure 1F is the CD value of the peptide and CD-2 to CD-4 in a simulated cell membrane environment;

[0018] Figure 2 The results of the toxicity test of peptide CD and CD-2 to CD-4 on MARC-145 cells;

[0019] Among them, ad are the toxicity results of peptide CD and CD-2 to CD-4 on MARC-145 cells;

[0020] Figure 3 The results of the direct antiviral activity test of peptide CD and CD-2 to CD-4;

[0021] Among them, A is the relative expression level of PRRSV mRNA; B is the Western blot result of PRRSV-N protein expression; C is the bar graph of Western blot result of PRRSV-N protein expression;

[0022] Figure 4 The results of the antiviral activity test of peptides CD and CD-2 to CD-4 in the pretreatment stage;

[0023] Among them, A is the relative expression level of PRRSV mRNA; B is the Western blot result of PRRSV-N protein expression; C is the bar graph of Western blot result of PRRSV-N protein expression; D is the result of indirect immunofluorescence detection.

[0024] Figure 5 The results of the antiviral activity test of peptide CD and CD-2 to CD4 in the adsorption stage;

[0025] Among them, A is the relative expression level of PRRSV mRNA; B is the Western blot result of PRRSV-N protein expression; C is the bar graph of Western blot result of PRRSV-N protein expression;

[0026] Figure 6 The results are the antiviral activity test results of peptides CD and CD-2 to CD-4 at the invasion stage;

[0027] Among them, A is the relative expression level of PRRSV mRNA; B is the Western blot result of PRRSV-N protein expression; C is the bar graph of Western blot result of PRRSV-N protein expression; D is the result of indirect immunofluorescence detection. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0029] Example 1 Synthesis of antiviral polypeptides and their application in preventing PRRSV infection

[0030] 1 Materials and methods

[0031] 1.1 Cells and viruses

[0032] African green monkey kidney epithelial cells (MeatAnimal Research Center-145, MARC-145) were preserved by our laboratory.

[0033] Strain: High pathogenicity porcine reproductive and respiratory syndrome virus (HP-PRRSV) is preserved in this laboratory.

[0034] 1.2 Synthesis and purification of antiviral peptides

[0035] Based on the structural characteristics of Cecropin D (CD) and the characteristics of PRRSV with envelope structure, CD was modified with cationization, and three derivatives from CD-2 to CD-4 were designed and synthesized. Subsequently, the synthesis of these antiviral peptides was completed using solid phase synthesis technology, and they were purified and analyzed for purity by high performance liquid chromatography, and antiviral peptides with a purity of up to 95% were obtained.

[0036] 1.3 Structural analysis

[0037] The secondary structures of these antiviral peptides were characterized by circular dichroism. We used a circular polarization spectrometer produced by JASCO to dissolve these antiviral peptides in water and 50% trifluoroethanol, and measured the CD spectra in the range of 190nm to 260nm.

[0038] 1.4 Cytotoxicity assay

[0039] To evaluate the toxicity of CD and CD-2 to CD-4 to MARC-145 cells, a cell viability assay (MTS method) was used. MARC-145 cells (density 1×10 6cell / mL) were inoculated into a 96-well culture plate and cultured for 24 hours until the cell confluence reached 80% to 90%. After the peptide CD and CD-2 to CD-4 were diluted proportionally starting from 100 μg / mL, the supernatant was discarded, 100 μL of the peptide solution was added to each well, and the cells were cultured in a cell culture incubator for 24 hours. 10 μL of MTS solution was added to each well, and the culture was continued for 1 to 2 hours. The absorbance at a wavelength of 490 nm was measured with an ELISA instrument to calculate the percentage of relative cell survival rate.

[0040] 1.5 Anti-PRRSV virus experiment

[0041] 1.5.1Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

[0042] MARC-145 cells were cultured at a rate of 1×10 6 The cells were inoculated into 6-well plates at a density of 10 cells / mL, and the cells were treated with peptides CD and CD-2 to CD-4 and highly pathogenic porcine blue ear disease virus (HP-PRRSV). After 18 hours, RNA was extracted from the cells, reverse transcribed, and cDNA was obtained and detected by real-time quantitative PCR. The reaction procedures and primers are shown in Table 1. Each sample was repeated three times, and the data were finally analyzed.

[0043] Table 1 RT-qPCR primers

[0044]

[0045] 1.5.2 Western blotting (WB)

[0046] MARC-145 cells were inoculated in a 6-well plate and treated when the cell confluence reached 70%. After 18 hours, the culture medium was discarded, and the cells were treated with 5× loading buffer and denatured at 100°C for 10 minutes. The samples were added to the wells of the protein gel and electrophoresed at a voltage of 80V. The proteins were then transferred to the NC membrane by electrotransfer. After the transfer, it was blocked with 5% skim milk for 2 hours. Next, the PRRSV-Nprotein antibody (primary antibody) was prepared at a dilution ratio of 1:1000 and incubated at 4°C overnight. The membrane was washed 5 times for 3 minutes each time, and HRP-labeled anti-rabbit IgG secondary antibody was added at a dilution ratio of 1:2000. After incubation at room temperature for 1 hour, it was washed 5 times. Finally, the luminescent solution was prepared and the membrane was placed in a chemiluminescent imager for detection.

[0047] 1.5.3 Indirect immunofluorescence assay (IFA)

[0048] MARC-145 cells were plated at 1 × 10 5 The cells were inoculated at a density of 100 μL / mL and treated when they grew to 70% confluence. After 18 hours, the medium was discarded and the cells were washed 3 times with PBS for 3 minutes each time. The cells were then fixed with 4% paraformaldehyde for 15 minutes and washed again with PBS. The cells were then treated with 0.1% Triton X-100 for 15 minutes and washed 3 times with PBS. The cells were blocked with 5% BSA, incubated at 37°C for 1 hour and the blocking solution was discarded. The primary antibody (dilution ratio 1:100) was added, 500 μL per well, incubated at 4°C overnight, and the cells were washed with PBS. The secondary antibody (dilution ratio 1:500) was then added, 500 μL per well, incubated at 37°C for 1 hour under light-proof conditions, the solution was discarded, and the cells were washed 5 times with PBS. DAPI was added to each well, incubated at room temperature for 10 minutes and discarded, then washed 3 times with PBS, and finally observed under a fluorescence microscope.

[0049] 2 Results Analysis

[0050] 2.1 Design and synthesis of antiviral peptides

[0051] Based on the structural characteristics of CD and the characteristics of PRRSV with an envelope structure, CD was cationic modified and three derivatives, CD-2, CD-3 and CD-4, were designed and synthesized. The antiviral peptide sequences are shown in Table 2.

[0052] Table 2 Peptide sequences and their physicochemical properties

[0053]

[0054] a: Molecular weight (MW) of peptides were measured by ESI-MS.

[0055] b: Retention time of peptides were determined by RP-HPLC.

[0056] 2.2 Peptide purity and structure analysis

[0057] The antiviral peptides CD and CD-2 to CD-4 were synthesized by solid phase synthesis technology, and purified and analyzed by high performance liquid chromatography and mass spectrometry. Figure 1A -D, the results show that the purity of all peptides reached more than 95%. Subsequently, the structure of the synthesized peptides was analyzed using circular dichroism spectroscopy. Figure 1EAs shown in Figure 2, the results indicate that the four polypeptides exhibit a random coil structure in aqueous solution. Figure 1F As shown in the figure, in the environment simulating the cell membrane, the structures of the four polypeptides changed significantly, and two typical negative peaks appeared at wavelengths of 208nm and 222nm, indicating the formation of significant α-helical structures. In order to simulate the behavior of proteins in the cell membrane, we selected 50% trifluoroethanol (2,2,2-Trifluoroethanol, TFE) with hydrophobicity as the simulation environment, and found that the circular dichroism curves of the four polypeptides changed significantly. This shows that the TFE environment can induce significant changes in the polypeptide structure and promote the formation of more stable α-helical structures in the cell membrane environment.

[0058] 2.3 Cytotoxicity

[0059] The toxicity of peptides CD and CD-2 to CD-4 to MARC-145 cells was evaluated by MTS assay. Figure 2 As shown in ad, the results show that within the effective concentration range, polypeptide CD and CD-2 to CD-4 exhibit low toxicity to MARC-145 cells. When the final concentration of the polypeptide reaches 100 μg / mL, the toxicity of the four polypeptides to cells is still low, and the cell viability remains above 90%. This result shows that even at higher concentrations, polypeptide CD and CD-2 to CD-4 have little effect on cells and do not significantly inhibit cell proliferation and growth. Based on this result, 100 μg / mL was determined as the highest test concentration in the antiviral activity evaluation experiment to ensure that the antiviral effects of polypeptides CD and CD-2 to CD-4 are evaluated at the maximum effective concentration while maintaining good cell viability.

[0060] 2.4 Evaluation of anti-PRRSV activity

[0061] 2.4.1 Peptides have a direct inactivation effect on PRRSV

[0062] In order to study whether the peptide CD and CD-2 to CD-4 have a direct viral inactivation effect on PRRSV, HP-PRRSV was incubated with CD, CD-2, CD-3 and CD-4 at a concentration of 100 μg / mL at 37°C for 2 hours. Then it was applied to the cells, and the antiviral activity of the peptide was detected by viral titer. RT-qPCR and Western blot were used to quantitatively analyze the viral RNA level and PRRSV-N protein expression. The results are shown in Figure 3 As shown in the results, the four peptides did not show significant virus inactivation effects. RT-qPCR data showed that the viral RNA level in the treatment group did not decrease significantly compared with the control group ( Figure 3 A). In addition, Western blot analysis results ( Figure 3 B, C) show that there is no significant difference in the expression level of PRRSV-N protein between the polypeptide-treated group and the control group. This indicates that under this condition, the polypeptide does not have a significant direct antiviral effect on PRRSV.

[0063] 2.4.2 Peptide inhibits PRRSV infection of cells

[0064] In order to evaluate whether the polypeptide CD and CD-2 to CD-4 designed in the present invention can protect MARC-145 cells from PRRSV infection, the preventive effect of polypeptide CD and CD-2 to CD-4 on MARC-145 was studied. The cells were treated with CD, CD-2, CD-3 and CD-4 at a concentration of 100 μg / mL for 2 hours, and then the supernatant was removed and the cells were washed twice with serum-free DMEM. Subsequently, the cells were infected with HP-PRRSV (MOI=1) for 2 hours, the cells were washed, and the samples were prepared after continuing to culture for 18 hours to detect the virus content in the cells. The RT-qPCR results are shown in Figure 4 As shown in A, the results showed that the viral RNA levels in the CD and CD-2 treatment groups were significantly decreased compared with the control group, with obvious differences. The results of Western blot analysis are shown in Figure 4 As shown in B and C, the results show that the expression levels of PRRSV-N protein were significantly different between the CD and CD-2 treatment groups and the control group, while there were no significant differences in the other treatment groups. Figure 4 D. The above results show that the mother peptide CD and the structure-optimized peptide CD-2 have a certain protective effect on cells, and PRRSV is effectively inhibited.

[0065] 2.4.3 Peptide inhibition of PRRSV adsorption to cells

[0066] MARC-145 cells were precooled at 4°C for 30 min, and then CD (100 μg / mL), CD-2 (100 μg / mL), CD-3 (25 μg / mL), and CD-4 (25 μg / mL) were added to DMEM and infected with HP-PRRSV (MOI = 1) for 2 h at 4°C. The supernatant was discarded, and the cells were washed twice with serum-free DMEM to prepare samples. The viral content in the cells was detected by Western blot and RT-qPCR. The results are shown in Figure 5 As shown, the results showed that the mother peptide CD exhibited good anti-disease activity in the virus adsorption stage, but the results of the other treatment groups showed that the viral RNA levels were not significantly different from those of the control group, and Western blot results showed that the expression of PRRSV-N protein was not significantly inhibited.

[0067] 2.4.4 Peptides inhibit PRRSV invasion of cells

[0068] MARC-145 cells were pre-cooled at 4°C and then infected with HP-PRRSV (MOI = 1) at 4°C for 2 hours. The supernatant was discarded after infection and the cells were washed twice with serum-free DMEM to remove unbound virus particles. Subsequently, CD, CD-2, CD-3 and CD-4 peptides were added at a concentration of 100 μg / mL and incubated for 2 hours at 37°C. Samples were collected for antiviral activity testing. The results are shown in Figure 2. Figure 6 As shown in the results of this experiment, the four peptides significantly inhibited the replication of HP-PRRSV through Western blot and RT-qPCR analysis. The RT-qPCR results showed that ( Figure 6 A), the viral RNA levels of all peptide-treated groups were significantly lower than those of the control group, and the differences were significant, indicating that the peptides had a significant inhibitory effect on viral invasion. The inhibition rate of the mother peptide CD on HP-PRRSV was 50.42%. After structural optimization, the inhibition rate was effectively improved. The inhibition rates of CD-2, CD-3 and CD-4 on HP-PRRSV were 72.06%, 72.12% and 71.92%, respectively. At the same time, Western blot results further supported this conclusion ( Figure 6 B, C), the four peptides all significantly reduced the expression of PRRSV-N protein, indicating that the virus was also inhibited at the protein level. Figure 6 D. The above results indicate that these peptides inhibit the replication of PRRSV by blocking the key steps of PRRSV invasion of cells.

Claims

1. An antiviral polypeptide based on Cecropin D (CD), characterized in that: The amino acid sequence of the antiviral polypeptide is shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.

4.

2. Use of the antiviral polypeptide according to claim 1 in the preparation of a drug for inhibiting porcine reproductive and respiratory syndrome virus (PRRSV).

3. The use according to claim 2, characterized in that The porcine reproductive and respiratory syndrome virus is a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV).

4. The use according to claim 2, characterized in that The antiviral polypeptide inhibits the replication of PRRSV by blocking the key step of PRRSV invading cells.

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