CN103623391A - Application of antimicrobial peptide Protegrin-1 for preventing and controlling porcine reproductive and respiratory syndrome - Google Patents

Abstract

The invention belongs to the technical field of medicines, and in particular discloses application of an antimicrobial peptide Protegrin-1 for preventing and controlling porcine reproductive and respiratory syndrome. According to the invention, an antimicrobial peptide Protegrin-1 is chemically synthesized according to an amino acid sequence of reported Protegrin-1 firstly; then, antimicrobial activity of the antimicrobial peptide Protegrin-1 is proved through an escherichia coli resistant DH5 alpha experiment, and researches on the antivirus activity of the antimicrobial peptide to HP-PRRSV are made through qRT-PCR, Western-Blot, cell supernatant TCID50 detection, IFA and other methods; and further the antivirus mechanism of the antimicrobial peptide is explained in virus adsorption and entrance into cell processes. The antimicrobial peptide Protegrin-1 is expected to be used as a novel medicine for preventing and controlling porcine reproductive and respiratory syndrome.

Description

Application of Antimicrobial Peptide Protegrin-1 in Prevention and Treatment of Pig PRRS

technical field

The invention relates to the technical field of medicine, in particular to the application of antibacterial peptide Protegrin-1 in preventing and treating pig blue-ear disease.

Background technique

Porcine reproductive and respiratory syndrome (PRRS), also known as porcine blue ear disease, is caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). The disease first broke out in the United States in 1987, and then spread to Europe. The virus was isolated in my country in 1996. Currently, PRRSV is divided into two genotypes based on genome sequence and antigenicity differences, one is the European type represented by the Lelystad Virus (LV) strain, and the other is the American type represented by the ATCC VR-2332 strain . In my country, the American type of PRRSV is the main type, but it is reported that European type strains have also been isolated. In 2006, pig hyperthermia broke out in my country, which caused serious economic losses to the pig industry. Later, this strain was defined as Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). PRRS mainly causes abortion in pregnant sows, stillbirth, mummified fetuses, weak piglets and pigs of all ages, especially piglets with respiratory symptoms. The characteristic lesion is interstitial pneumonia, and the mortality rate is extremely high. important infectious diseases.

PRRSV belongs to the order Nidovirales, Arteriviridae, and Arterivirus. The virus particles are spherical or oval with capsules under electron microscope observation. The virus genome is a single-stranded positive-strand RNA with a total length of about 15Kb, containing 5' and 3' non-coding regions, and 10 open reading frames (open reading frames, ORFs) in the middle, of which ORF2-7 translates viral glycoproteins respectively (glycoprotein GP) GP2a, GP2b, GP3, GP4, GP5, GP5a, M and N proteins. The most important ones are GP5 and N proteins, which are not only the main components of virions, but also play an important role in the packaging, maturation, immune evasion and antibody induction of virions.

The prevention and control of PRRSV is a difficult problem in my country and the world at present. The difficulty of prevention and control of PRRSV is mainly manifested in the following aspects: (1) macrophage and immunosuppressive diseases, PRRSV mainly infects alveolar macrophages (Porcine alveolar macrophages, PAMs) of pigs, and PAMs are immune cells that destroy PAMs, Thereby destroying the immune system of the body and causing immunosuppression; (2) antigenic variability, currently PRRSV mutates rapidly, and the use of attenuated vaccines is one of the reasons for the mutation of the virus, and the vaccine has no cross-protection; (3) antibody dependence is enhanced, PRRSV infection will stimulate the body to produce antibodies, but low-titer antibodies not only cannot neutralize the virus, but can promote the proliferation of the virus; (4) The virus is persistently infected. After PRRSV infection, it can be detected in pigs for a long time Viremia. (5) Mixed infection. At present, clinical mixed infection, especially the mixed infection of circovirus, Haemophilus parasuis and PRRSV, makes the prevention and control of PRRSV even more difficult.

At present, there are mainly inactivated vaccines and attenuated vaccines for the prevention and control of PRRSV, and attenuated vaccines are mostly used in clinical practice. Among them, the inactivated vaccine has the following disadvantages: (1) It requires large doses of vaccination or the application of concentrated antigens, and the immunity period is short, often requiring booster vaccination; (2) It cannot cause local immunity, so that the effect of cellular immunity is weak; (3) It produces complete immunity It takes 2-3 weeks, which is not conducive to emergency vaccination and the reduction of vaccine costs; (4) There is a possibility of incomplete inactivation and loose virus. However, the attenuated vaccine has the risk of strong virulence, recombination and potential infection. Therefore, vaccines have exposed more and more problems in the prevention and control of PRRSV. the

Antimicrobial peptide Protegrin-1 (PG-1) is a pig-derived antimicrobial peptide isolated from porcine white blood cells, consisting of 18 amino acid residues, with a secondary structure of β-sheet, 4 cysteines forming 2 disulfides It is reported that the removal of these two disulfide bonds can significantly reduce the antimicrobial activity of PG-1. It has been proven that PG-1 has antimicrobial activity against Gram-negative bacteria, some Gram-positive bacteria, fungi, and a few enveloped viruses, including HIV and dengue virus. PG-1 is currently proven to be one of the most promising alternatives to antibiotics.

Pig blue-ear disease has caused serious economic losses to the pig industry and is highly contagious. Whether antimicrobial peptide PG-1 has antiviral effect on PRRSV virus has not been reported so far. Therefore, the present invention uses PG-1 to study its antiviral effect on PRRSV, in order to find a good anti-PRRSV drug.

Contents of the invention

The purpose of the present invention is to provide the application of antibacterial peptide Protegrin-1 in the preparation of anti-PRRSV virus medicine.

Another object of the present invention is to provide the application of the antimicrobial peptide Protegrin-1 in the preparation of drugs for preventing and treating porcine PRRS.

The above-mentioned purpose of the present invention is achieved through the following technical solutions:

Through experiments, the present invention finds that the antimicrobial peptide Protegrin-1 can significantly inhibit porcine reproductive and respiratory syndrome virus adsorption cells, and provides strong support for enhancing the prevention and control of porcine reproductive and respiratory syndrome virus through drug treatment. Therefore, the present invention provides the application of an antibacterial peptide Protegrin-1 in the preparation of anti-porcine reproductive and respiratory syndrome virus drugs, and at the same time, the present invention provides the application of an antibacterial peptide Protegrin-1 in the preparation of drugs for preventing and treating porcine PRRS virus.

A pharmaceutical preparation against porcine reproductive and respiratory syndrome virus, comprising an effective dose of antibacterial peptide Protegrin-1 and pharmaceutically acceptable auxiliary materials. Preferably, the pharmaceutical preparation is an injection preparation or an oral preparation. More preferably, the injection preparation is a freeze-dried powder injection; the oral preparation is a loose tablet, capsule or granule.

The present invention first chemically synthesizes the bioactive molecule antimicrobial peptide Protegrin-1, its amino acid sequence is shown in SEQ ID NO.1, the 6th and 15th Cys in the amino acid sequence, and the 8th and 13th Cys form 2 pairs of disulfides Bond, C-terminal amidation modification. Then verify its antibacterial activity, further use Marc-145 cells to study its antiviral activity against HP-PRRSV and study the anti-HP-PRRSV mechanism of PG-1 through two processes of virus adsorption and cell entry. The specific experimental design is as follows:

1. First chemically synthesize PG-1, then measure its anti-Escherichia coli activity, and screen out the solvent with the highest activity of PG-1.

2. PG-1 cytotoxicity test. AlamarBlue (purchased from Invitrogen) is used as an indicator of living cell metabolism. Under the mitochondrial enzymatic reduction reaction, a measurable fluorescent metabolite will be produced, and the cell activity can be monitored by measuring its fluorescence intensity. Use a multi-functional microplate reader to read the fluorescence values of excitation light at 540nm and emission light at 590nm, respectively, and make a PG-1 cytotoxicity map.

3. PG-1 antiviral test. Cultivate Marc-145 cells with DMEM medium containing 10% fetal bovine serum to a confluence of 60-70%, discard the medium, wash with PBS 3 times, and mix HP-PRRSV containing MOI=0.1 (plaque-forming unit PFU= 0.7×TCID ₅₀ , multiplicity of infection (MOI=number of viruses/number of cells) DMEM culture solution of 2% fetal bovine serum was added to the cells at 37°C for 5 hours, washed 3 times with PBS, and 2% fetal bovine serum containing different concentrations of PG-1 The DMEM culture solution of bovine serum was added to the cells and cultured at 37°C for 36 hours, and the supernatant was collected for TCID ₅₀ detection, and qRT-PCR and Western-Blot detection were also performed.

4. PG-1 antiviral indirect immunofluorescent assay (Indirect Immunofluorescent Assay, IFA). The anti-virus test method is the same as above, collect cells, fix with 4% paraformaldehyde for 10 minutes, wash with PBS for 10 minutes, perforate with 10% Triton-100 for 15 minutes, wash with PBS for 10 minutes, then dilute 1% BSA with PBS and block for 30 minutes, add anti-PRRSV virus N protein The primary antibody was reacted at room temperature for 1 hour, then anti-mouse secondary antibody was added for 1 hour, the nucleus was stained with Hoechst for 5 minutes, washed with PBS for 10 minutes, and then the antiviral effect of PG-1 was observed under a fluorescence microscope.

5. Research on the antiviral mechanism of PG-1. (1) Whether PG-1 achieves the purpose of resisting HP-PRRSV by affecting the adsorption of HP-PRRSV to Marc-145 cells. First, wash the 6-well plate with 70% confluence of Marc-145 cells with PBS three times, then inoculate HP-PRRSV at MOI=0.1, add PG-1 with gradient concentration, and incubate at 4°C for 2 hours. After incubation, Wash 3 times with PBS to wash away the virus that is not adsorbed to the cell surface, then culture in a 5% CO ₂ , 37°C incubator for 24 hours, and collect the cells for qRT-PCR and Western-Blot detection of the N gene. (2) Whether PG-1 achieves the purpose of resisting HP-PRRSV by inhibiting HP-PRRSV from entering Marc-145 cells. First, a 6-well plate with 70% confluence of Marc-145 cells was washed 3 times with PBS, then inoculated with HP-PRRSV at MOI=0.1, incubated at 4°C for 2 hours, after incubation, washed 3 times with PBS, and the unadsorbed Wash off the virus on the cell surface, then add PG-1 nutrient solution with gradient concentration, incubate in a 5% CO ₂ , 37°C incubator for 6 hours, wash with PBS 3 times, and replace with fresh nutrient solution containing 2% fetal bovine serum Continue to culture for 24h, collect cells for Western-Blot detection. (3) Whether PG-1 still has antiviral effect after PRRSV enters the cells for 4 hours. First, a 6-well plate with a confluence of Marc-145 cells at 70% was washed 3 times with PBS, then inoculated with HP-PRRSV at MOI=0.1, incubated at 4°C for 2 hours, washed 3 times with PBS, and the virus that was not adsorbed to the cell surface Wash off, culture in 5% CO ₂ , 37°C incubator for 4 hours, wash with PBS 3 times, add 2% fetal bovine serum DMEM medium containing 20, 30, 40 mg/L PG-1 to the cells 37 Incubate at ℃ for 6 hours, wash 3 times with PBS, change to fresh nutrient solution containing 2% fetal bovine serum (excluding PG-1) and continue to culture for 24 hours, discard the nutrient solution, wash 3 times with PBS, collect cells for Western-Blot detection.

6. Statistical analysis. All the above experiments were repeated independently at least three times, and the results were expressed as mean and standard error, and analyzed using Student's t test . All statistical analyzes were performed with P<0.05 as the test standard for significant statistical differences, and the analysis software was SPSS 16.0 and GraphPad Prism 5.

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

The greatest innovation of the present invention is that PG-1 is used for the first time to study its antiviral effect on HP-PRRSV, and it has been proved by various methods that PG-1 has good antiviral effect on HP-PRRSV at the level of Marc-145 cells The results have laid a foundation for the development of new drugs against pig PRRS.

In order to further study the antiviral effect of PG-1, the present invention also studies the mechanism of PG-1 against HP-PRRSV. The results showed that the antiviral effect of PG-1 occurred in the process of HP-PRRSV adsorption to cells.

In order to enhance the biological activity of PG-1, in the process of chemically synthesizing PG-1, the C-terminus of the present invention is amidated.

Description of drawings

Figure 1 is a diagram of the activity of PG-1 against Escherichia coli DH5α; ddH ₂ O in the figure: PG-1 is dissolved in ddH ₂ O of 0.01% glacial acetic acid; PBS: PG-1 is dissolved in PBS of 0.01% glacial acetic acid; 0.2 M tris-Hcl 6.8: PG-1 dissolved in 0.2M tris-Hcl PH=6.8 buffer; 0.2M tris-Hcl 6.8/PBS: PG-1 dissolved in 0.2M tris-Hcl PH=6.8 PBS buffer; Control: 100μL concentration is 100mg/L Amp.

Figure 2 is a graph showing the cytotoxicity of PG-1 detected by AlamarBlue.

Fig. 3 is a graph showing virus titers in the supernatant of HP-PRRSV-infected Marc-145 cells treated with different concentrations of PG-1.

Fig. 4 is a graph showing the expression level of N gene relative to the internal reference gene HPRT1 after being treated with different concentrations of PG-1 for 36 hours.

Figure 5 is a graph showing the expression level of N protein after being treated with 20 and 30 mg/L PG-1 for 36 hours respectively.

Figure 6 is the indirect immunofluorescence test for N protein after being treated with 20, 30, and 40 mg/L PG-1 for 36 hours; green is the color of anti-PRRSV N protein, and blue is the color of Hoechst-stained cell nuclei.

Fig. 7 is a graph showing the expression level of N gene relative to the internal reference GAPDH during the process of PG-1 antiviral mechanism—viral adsorption to cells.

Fig. 8 is a Western-Blot detection diagram of N protein during the antiviral mechanism of PG-1—the process of virus adsorption to cells.

Fig. 9 is a Western-Blot detection diagram of N protein during the antiviral mechanism of PG-1—the process of virus entering the cell.

Fig. 10 is the antiviral mechanism of PG-1—after the virus enters the cell for 4 hours, the Western-Blot detection diagram of N protein.

Detailed ways

The present invention will be further explained below in conjunction with the accompanying drawings and examples, but the examples do not limit the present invention in any form.

The virus strain used in the embodiment of the present invention is HP-PRRSV (highly pathogenic porcine reproductive and respiratory syndrome virus), which is obtained from the Laboratory of Animal Infectious Diseases, School of Veterinary Medicine, South China Agricultural University. HP-PRRSV is a pathogenic The relatively high abbreviation of porcine reproductive and respiratory syndrome virus does not limit the present invention in any way.

Embodiment 1 PG-1 chemical synthesis and anti-Escherichia coli activity experiment

1. First, search the amino acid sequence of porcine antimicrobial peptide PG-1 through the antimicrobial peptide database (APD) The Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php), as shown in SEQ ID NO.1 showed that the amino acid sequence was sent to Shanghai Gilbetter Biochemical Co., Ltd. (http://glbetter.cn.1688.com/) for synthesis, because the β-sheet structure of PG-1 is crucial to its biological activity, and β- The formation of the folding structure depends on the 2 pairs of disulfide bonds in PG-1, so when synthesizing PG-1, it is necessary to ensure the successful synthesis and accurate positioning of the disulfide bonds. The two pairs of disulfide bonds are the 6th and 15th Cys, and the 8th and 13th Cys, respectively, and in order to enhance the biological activity of PG-1, its C-terminus was modified by amidation. HPLC 95% purity, synthesized 30mg. After synthesis, dilute with 0.01% glacial acetic acid sterilized water to 100mg/L, sterilize by 0.22μm filter, and store in -80℃.

2. PG-1 anti-Escherichia coli activity test. Since PG-1 is only active in a weakly acidic environment and has the highest solubility, in order to maximize the antimicrobial activity of PG-1, the chemically synthesized PG-1 powder was dissolved in sterile water containing 0.01% glacial acetic acid, PBS and 0.2M Tris-Hcl in sterilized water and PBS, and then study its antibacterial activity against Escherichia coli DH5α, and screen out the solvent with the highest PG-1 activity.

The experimental method of PG-1 anti-Escherichia coli activity adopts the standard agar hole diffusion method. Mix 10 μL of E.coli DH5α with 30 mL of LB solid medium at 55°C and coat the plate. After it solidifies, punch holes with a sterilized hole punch with a diameter of 3 mm, add 100 μL of PG-1, and add An equal volume of dissolved PG-1 solvent was used as a negative control, and 2 μL of Amp (100 mg/mL) was used as a positive control. Cultivate in a 37°C incubator for 12 hours, take out and measure the diameter of the inhibition zone when an obvious inhibition zone can be seen.

Add 200 μL of the diluted PG-1 to each well, and set up ampicillin positive control and 0.01% glacial acetic acid negative control at the same time, see Figure 1. It can be seen from Figure 1 that when PG-1 is dissolved in 0.01% glacial acetic acid sterilized water, the bacteriostatic zone is the largest, and the bacteriostatic zone is slightly smaller in 0.01% glacial acetic acid PBS, but in 0.2M Tris-Hcl sterilization There is almost no zone of inhibition when dissolved in water, so Figure 1 shows that in order to make PG-1 have the best antibacterial effect on Escherichia coli DH5α, PG-1 must be dissolved in sterilized water with 0.01% glacial acetic acid.

3. After screening out the solvent with the highest activity of PG-1, dilute the PG-1 powder to a final concentration of 100 mg/L, filter and sterilize at 0.22 μm, aliquot and store at -80°C.

Embodiment 2 PG-1 cytotoxicity test

AlamarBlue (purchased from Invitrogen) is used as an indicator of living cell metabolism. Under the mitochondrial enzymatic reduction reaction, a measurable fluorescent metabolite will be produced, and the cell activity can be monitored by measuring its fluorescence intensity. Cultivate Marc-145 cells with DMEM medium containing 10% fetal bovine serum to 60-70%, discard the medium, add nutrient solution containing PG-1 times dilution for 36 hours, set PBS control group, and then add 10 % (V/V) ratio AlamarBlue was continued to incubate for 3 hours, and the fluorescence values of excitation light at 540nm and emission light at 590nm were read using a multi-functional microplate reader to make a PG-1 cytotoxicity graph (see Figure 2). Taking the cell activity of the PBS control group as 100%, the fluorescence value of the cells treated with PG-1 in multiple dilutions is higher than the fluorescence value of the PBS control group, which is the relative cell activity of PG-1 at different concentrations. It can be seen from Figure 2 that when PG-1 1 When the concentration is 40mg/L, it has no toxicity to Marc-145 cells, and the cell viability is 100%. This concentration is the maximum concentration of the subsequent test.

Embodiment 3 PG-1 antiviral test

1. Cultivate Marc-145 cells in a 6-well plate containing DMEM medium containing 10% fetal bovine serum until the cell confluence reaches 70%, discard the culture medium, wash 3 times with PBS, and inoculate HP-PRRSV with MOI=0.1 , continue to culture at 37°C for 5h in DMEM medium with 2% fetal bovine serum.

2. Wash 3 times with PBS, add 2% fetal bovine serum DMEM medium with a concentration of 20, 30, 40 mg/L PG-1 to the cells and incubate at 37°C for 36 hours, set up a PBS control group, wash 3 times with PBS, collect The supernatant was tested for TCID ₅₀ , see accompanying drawing 3. It can be seen from Fig. 3 that PG-1 can significantly reduce the virus yield in the cell supernatant and inhibit the release of the virus into the cell supernatant; in addition, add 400 μL Trizol to each well to improve RNA was detected by qRT-PCR, see Figure 4. It can be seen from Figure 4 that at the transcriptional level, PG-1 can significantly reduce the expression level of N gene.

3. As above, inoculate HP-PRRSV with MOI=0.1, continue culturing in DMEM culture solution with 2% fetal bovine serum at 37°C for 5 hours, wash with PBS three times, and inoculate with 20, 30, and 40 mg/L PG-1 respectively 2% fetal bovine serum DMEM culture solution was added to the cells and cultured at 37°C for 36 hours, and a PBS control group was set up. 1 mL of supernatant was collected for TCID ₅₀ detection, the remaining culture solution was discarded, washed 3 times with PBS, digested with 0.25% trypsin, and lysed cells , protein concentration, Western-Blot detection, see Figure 5, as can be seen from Figure 5, at the level of protein translation, PG-1 can significantly reduce the expression level of N protein. Therefore, this experiment proves that PG-1 has a good anti-HP-PRRSV effect from many aspects.

4. PG-1 antiviral indirect immunofluorescent assay (Indirect Immunofluorescent Assay, IFA). Culture Marc-145 cells in 12-well plates containing 10% fetal bovine serum in DMEM until the cell confluence reaches 70%, discard the culture medium, wash with PBS for 3 times, inoculate HP-PRRSV at MOI=0.1, and Continue culturing in DMEM medium with 2% fetal bovine serum at 37°C for 5 hours, wash with PBS three times, add DMEM medium with 2% fetal bovine serum at a concentration of 20, 30, and 40 mg/L PG-1 to the cells and incubate at 37°C 36 hours, and a PBS control group was set up, washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 minutes, washed with PBS for 10 minutes, perforated with 10% Triton-100 for 15 minutes, washed with PBS for 10 minutes, and then diluted with 1% BSA in PBS for 30 minutes. Anti-PRRSV N Protein primary antibody at room temperature for 1 hour, anti-mouse secondary antibody for 1 hour, Hoechst stained nuclei for 5 minutes, washed with PBS for 10 minutes, carried out IFA detection, and observed the antiviral effect of PG-1 under a fluorescence microscope, see attached drawing 6, as shown in Figure 6, PG- 1 When the concentration was 20, 30, and 40 mg/L, the fluorescence value of anti-PRRSV N protein was significantly lower than that of the PBS control group, indicating that PRRSV N protein was almost not expressed in cells, which meant that PG-1 had obvious antiviral effect, and when PG- 1 When the concentration is 40mg/L, the antiviral effect is the most obvious.

Example 4 PG-1 Antiviral Mechanism Research—HP-PRRSV Adsorption Inhibition Test

1. Wash the 6-well plate with 70% confluence of Marc-145 cells with PBS for 3 times, then inoculate HP-PRRSV with MOI=0.1, and add 20, 30, 40mg/L PG-1 respectively, at 4°C Incubate for 2h.

2. Wash with PBS 3 times to remove the virus that is not adsorbed on the cell surface, then continue to culture in a 5% CO ₂ , 37°C incubator for 24 hours, discard the culture medium, wash with PBS 3 times, collect the cells, add 400 μL Trizol was used to extract RNA and perform qRT-PCR detection on the N gene, see Figure 7; cells were lysed, protein concentration was measured, and Western-Blot was detected, see Figure 8. The results show that PG-1 can significantly reduce the expression levels of N gene and N protein, and with the increase of PG-1 concentration, the inhibitory effect is more obvious. When the PG-1 concentration is 20mg/L, HP-PRRSV N protein The expression level cannot be detected, indicating that PG-1 has a good antiviral effect during the process of HP-PRRSV adsorption to cells.

Example 5 PG-1 Antiviral Mechanism Research—HP-PRRSV Entry Inhibition Test

1. Wash a 6-well plate with a confluence of 70% Marc-145 cells three times with PBS, then inoculate with HP-PRRSV at MOI=0.1, and incubate at 4°C for 2 hours.

2. Wash with PBS for 3 times to wash off the virus not adsorbed on the cell surface, then add 20, 30, and 40 mg/L PG-1 respectively, and incubate in a 5% CO ₂ , 37°C incubator for 6 hours.

3. Wash 3 times with PBS, change to fresh nutrient solution containing 2% fetal bovine serum and continue culturing for 24 hours, discard the nutrient solution, wash 3 times with PBS, collect cells for Western-Blot detection, see Figure 9, as can be seen from Figure 9, During the process of HP-PRRSV entering the cells, the antiviral effect of PG-1 was not obvious, only when the concentration of PG-1 was 40mg/L, the expression level of N protein decreased, indicating that PG-1 was involved in the process of HP-PRRSV entering the cells Antiviral effect is not obvious.

Example 6 PG-1 Antiviral Mechanism Research—Inhibition Test after HP-PRRSV Entered 4h

1. Wash the 6-well plate with 70% confluence of Marc-145 cells with PBS 3 times, then inoculate HP-PRRSV at MOI=0.1, incubate at 4°C for 2 hours, wash 3 times with PBS, and remove the virus that is not adsorbed to the cell surface Wash off, and incubate in a 5% CO ₂ , 37°C incubator for 4 hours.

2. Wash 3 times with PBS, add DMEM medium with 2% fetal bovine serum at a concentration of 20, 30, and 40 mg/L PG-1 to the cells and incubate at 37°C for 6 hours.

3. Wash 3 times with PBS, change to fresh nutrient solution containing 2% fetal bovine serum and continue culturing for 24 hours, discard the nutrient solution, wash 3 times with PBS, collect cells for Western-Blot detection, see Figure 10, the results show that when HP -PRRSV entered the cells at 37°C for 4 hours and then treated with different concentrations of PG-1. The antiviral effect of PG-1 was not obvious. Only when the concentration of PG-1 was 40mg/L, there was an antiviral effect, indicating that the virus entered the cells. After 4 hours, the antiviral effect of PG-1 was not obvious.

sequence listing

SEQUENCE LISTING

the

<110> Sun Yat-Sen University

the

<120> Application of Antimicrobial Peptide Protegrin-1 in Prevention and Treatment of Pig PRRS

the

<130>

the

<160> 1

the

<170> PatentIn version 3.3

the

<210> 1

<211> 18

<212> PRT

<213> Amino acid sequence of antimicrobial peptide Protegrin-1

the

<400> 1

the

Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val

1 5 10 15

the

the

Gly Arg

``

the

the

the

Claims (8)

1. the application of antibacterial peptide Protegrin-1 in the anti-PRRSV virus drugs of preparation, is characterized in that, the aminoacid sequence of described Protegrin-1 is as shown in SEQ ID NO.1.

2. the application of antibacterial peptide Protegrin-1 in preparation control pig blue-ear disease medicine, is characterized in that, the aminoacid sequence of described Protegrin-1 is as shown in SEQ ID NO.1.

3. according to application described in claim 1 or 2, it is characterized in that, the secondary structure of described Protegrin-1 is beta sheet, the 6th and the 15th Cys of its aminoacid sequence, the 8th and 2 pairs of disulfide bond of the 13rd Cys formation, and the amidatioon of C end is modified.

4. according to application described in claim 1 or 2, it is characterized in that, described antibacterial peptide Protegrin-1 using dosage scope is 20 mg/L ~ 40mg/L.

5. a pharmaceutical preparation for anti-porcine reproductive and respiratory syndrome virus, is characterized in that, comprises antibacterial peptide Protegrin-1 and the pharmaceutically acceptable adjuvant of effective dose.

6. pharmaceutical preparation according to claim 5, is characterized in that, described pharmaceutical preparation is ejection preparation or oral formulations.

7. pharmaceutical preparation according to claim 6, is characterized in that, described ejection preparation is lyophilized injectable powder.

8. pharmaceutical preparation according to claim 6, is characterized in that, described oral formulations is loose tablet, capsule or granule.

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