CN103623391B - Application of antimicrobial peptide Protegrin-1 for preventing and controlling porcine reproductive and respiratory syndrome - Google Patents
Application of antimicrobial peptide Protegrin-1 for preventing and controlling porcine reproductive and respiratory syndrome Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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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
Technical field
The present invention relates to medical art, be specifically related to the application of antibacterial peptide Protegrin-1 in control pig blue-ear disease.
Background technology
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) also known as pig blue-ear disease, caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome viruse, PRRSV).This disease broke out in the U.S. early than 1987, and spread to Europe subsequently, China was separated to this virus in 1996.At present, according to genome sequence and antigenic specificity, PRRSV is divided into two kinds of genotype, a kind of is the Europe class of representative with Lelystad Virus (LV) strain, the american type that another kind is representative with ATCC VR-2332 strain.In China, PRRSV mainly based on american type, but it is reported and is also separated to Europe class strain.2006, porcine hyperthermia has been broken out in China, serious economic loss is caused to pig industry, afterwards this strain is defined as high pathological form strain (Highly pathogenic porcine reproductive and respiratory syndrome viruse, HP-PRRSV).PRRS mainly causes in-pig miscarriage, stillborn fetus, mummy tire, weak son and each age level pig particularly piglet respiratory symptom, and characteristic pathological changes is interstitial pneumonia, and mortality rate is high, is a kind of global Important Infectious Diseases of high degree in contact.
PRRSV belongs to the many viraleses of Buddhist nun (Nidovirales), Arteriviridae (Arteriviridae), Arterivirus (Arterivirus) member, and electric Microscopic observation virion is spherical in shape or oval, has cyst membrane.Viral genome is a single-stranded positive RNA, total length is about 15Kb, containing 5 ' and 3 ' noncoding region, centre is 10 open reading frame (open reading frame, ORF), wherein ORF2-7 translates viral glycoprotein (glycoprotein GP) GP2a, GP2b, GP3, GP4, GP5, GP5a, M and N protein respectively.Wherein the most important thing is GP5 and N protein, they are not only the key component of virion, and produce important effect in the packaging of virion, maturation, immune evasion and antibody induction.
The prevention and control of PRRSV are the difficult problems in current China and even the world.PRRSV is difficult to prevention and control and is mainly manifested in following several respects: (1) is addicted to phagocytic and immunosuppressive disease, pulmonary alveolar macrophage (the Porcine alveolar macrophages of PRRSV main infection pig, PAMs), PAMs is immunocyte, destroy PAMs, thus destruction body immune system, thus cause immunosuppressant; (2) antigenic variability, current PRRSV variation is very fast, and the use of attenuated vaccine is the reason impelling virus variation, and vaccine does not have cross-protection; (3) antibody dependent strengthens, and the infection of PRRSV can stimulate body to produce antibody, but the antibody of low liter not only can not neutralize virus, has facilitation on the contrary to the propagation of virus; (4) viral persistence infects, and after PRRSV infection, viremia can be detected for a long time in pig body.(5) mixed infection, at present clinically mixed infection particularly porcine circovirus, haemophilus parasuis etc. and the mixed infection of PRRSV make PRRSV prevention and control extremely difficult.
Current PRRSV prevention and control mainly contain inactivated vaccine and attenuated vaccine, and clinically more is attenuated vaccine.Wherein inactivated vaccine has following shortcoming: (1) needs heavy dose of inoculation or application concentrated antigen, and duration of immunity is short, often need strengthen inoculation; (2) local immunity can not be caused, so that the effect of cellular immunization is weak; (3) produce complete immunity and need 2-3 week, be unfavorable for urgent prophylactic immunization and reduce vaccine expense; (4) there is deactivation not thoroughly and the possibility of loose poison.And to there is virulence in attenuated vaccine return by force, recombinate and the danger of latent infection.Therefore vaccine exposes increasing problem in anti-PRRSV processed.
Antibacterial peptide Protegrin-1(PG-1) be from pig leucocyte, be separated the pig derived antimicrobial peptide be made up of 18 amino acid residues obtained, secondary structure is beta sheet, 4 cysteine form 2 disulfide bond and play very important effect to the maintenance of its secondary structure and antimicrobial activity, it is reported, the removal of these 2 disulfide bond can reduce the antimicrobial activity of PG-1 significance.Prove that PG-1 has antimicrobial active, comprising HIV, dengue virus to gram negative bacteria, part gram positive bacteria, fungus and minority togavirus at present.PG-1 is proved to be at present as most potentiality become one of antibiotic substitute.
Pig blue-ear disease causes serious economic loss to pig industry, and infectiousness is extremely strong.Whether antibacterial peptide PG-1 has antivirus action to PRRSV virus, does not also have report so far.Therefore the present invention uses PG-1 to study its antivirus action to PRRSV, to finding a kind of good anti-PRRSV medicine.
Summary of the invention
The object of the present invention is to provide the application of antibacterial peptide Protegrin-1 in the anti-PRRSV virus drugs of preparation.
Another object of the present invention is to provide the application of antibacterial peptide Protegrin-1 in preparation control pig blue-ear disease medicine.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
The present invention found through experiments antibacterial peptide Protegrin-1 can obviously suppress porcine reproductive and respiratory syndrome virus adherent cell, provides powerful support for for strengthening to provide the prevention and control of porcine reproductive and respiratory syndrome virus by Drug therapy.Therefore, the invention provides the application of a kind of antibacterial peptide Protegrin-1 in the anti-porcine reproductive and respiratory syndrome virus medicine of preparation, the invention provides the application of a kind of antibacterial peptide Protegrin-1 in preparation control pig blue-ear disease medicine simultaneously.
A pharmaceutical preparation for anti-porcine reproductive and respiratory syndrome virus, includes the antibacterial peptide Protegrin-1 of effective amount and pharmaceutically acceptable adjuvant.Preferably, described pharmaceutical preparation is ejection preparation or oral formulations.More preferably, described ejection preparation is lyophilized injectable powder; Described oral formulations is discrete piece agent, capsule or granule.
The present invention is chemosynthetic organism bioactive molecule antibacterial peptide Protegrin-1 first, and its aminoacid sequence is for shown in SEQ ID NO.1, and aminoacid sequence the 6th and the 15th Cys, the 8th and the 13rd Cys form 2 pairs of disulfide bond, and C holds amidatioon to modify.Then verify its antibacterial activity, utilize further Marc-145 cell research its to the antiviral activity of HP-PRRSV and by virus absorption onto cell and the anti-HP-PRRSV mechanism entering cell two process study PG-1.Specific experiment design is as follows:
1, first chemosynthesis PG-1, then measures its anti-E. coli Activity, and filters out PG-1 the highest active solvent.
2, PG-1 cell toxicity test.AlamarBlue(is purchased from Invitrogen company) as living cells metabolize indicator, measurable fluorescence metabolite can be produced under mitochondrion lipase-catalyzed, can cytoactive be monitored by measuring its fluorescence intensity.Use multi-functional microplate reader to read 540nm exciting light and 590nm utilizing emitted light fluorescent value respectively, make PG-1 cytotoxicity figure.
3, PG-1 antivirus test.Cultivating Marc-145 cell to degree of converging with the DMEM culture fluid containing 10% hyclone is 60-70%, and discard culture fluid, PBS washes 3 times, will containing HP-PRRSV MOI=0.1(plaque forming unit PFU=0.7 × TCID
50infection multiplicity MOI=virus number/cell number) the DMEM culture fluid of 2% hyclone add 37 DEG C, cell and continue to cultivate 5h, PBS washes 3 times, the DMEM culture fluid of 2% hyclone of the PG-1 containing variable concentrations is added 37 DEG C, cell and cultivates 36h, collects supernatant and is TCID
50detect, carry out qRT-PCR and Western-Blot in addition and detect.
4, PG-1 antiviral indirect immunofluorescence assay (Indirect Immunofluorescent Assay, IFA).Antivirus test method is the same, collecting cell, and 4% paraformaldehyde fixes 10min, PBS washes 10min, 10%Triton-100 perforation 15min, PBS wash 10min, then dilute 1%BSA with PBS and close 30min, add the primary antibodie of anti-PRRSV viral N proteins, room temperature reaction 1h, then add against murine two anti-effect 1h, Hoechst dye core 5min, PBS washes 10min, then at fluorescence microscopy Microscopic observation PG-1 antiviral effect.
5, PG-1 Antiviral Mechanism research.(1) whether PG-1 adsorbs Marc-145 cell thus the object reaching anti-HP-PRRSV by affecting HP-PRRSV.First, wash 3 times at 6 orifice plate PBS of Marc-145 cell confluency degree 70%, then meet malicious HP-PRRSV with MOI=0.1, and add the PG-1 of Concentraton gradient, hatch 2h, after having hatched for 4 DEG C, wash 3 times with PBS, the Virus eliminating medicine not being adsorbed onto cell surface is washed off, then at 5%CO
2, cultivate 24h in 37 DEG C of incubators, collecting cell is qRT-PCR and Western-Blot to N gene and detects.(2) whether PG-1 is by suppressing HP-PRRSV to enter the object that Marc-145 cell reaches anti-HP-PRRSV.First, wash 3 times at 6 orifice plate PBS of Marc-145 cell confluency degree 70%, then meet malicious HP-PRRSV with MOI=0.1, hatch 2h for 4 DEG C, after having hatched, wash 3 times with PBS, the Virus eliminating medicine not being adsorbed onto cell surface is washed off, then adds the nutritional solution of the PG-1 of Concentraton gradient at 5%CO
2, cultivate 6h in 37 DEG C of incubators, PBS washes 3 times, renews the fresh nutritional solution containing 2% hyclone and continues to cultivate 24h, collecting cell Western-Blot and detect.(3) after PRRSV enters cell 4h, whether PG-1 also has antivirus action.First, wash 3 times at 6 orifice plate PBS of Marc-145 cell confluency degree 70%, then meet malicious HP-PRRSV with MOI=0.1, hatch 2h for 4 DEG C, PBS washes 3 times, is washed off by the Virus eliminating medicine not being adsorbed onto cell surface, 5%CO
2, cultivate 4h in 37 DEG C of incubators, PBS washes 3 times, respectively by be 20 containing concentration, 30, the DMEM culture fluid of 2% hyclone of the PG-1 of 40mg/L adds 37 DEG C, cell and cultivates 6h, PBS washes 3 times, renew the fresh nutritional solution containing 2% hyclone (not containing PG-1) to continue to cultivate 24h, discard nutritional solution, PBS washes 3 times, and collecting cell Western-Blot detects.
6, statistical analysis.All tests at least 3 independent repetitions above, result adopts meansigma methods and standard error to represent, uses
student ' s t testanalyze.All statistical analysiss all adopt with
p<0.05as the touchstone with remarkable significant difference, analysis software is SPSS 16.0 and GraphPad Prism 5.
Compared with prior art, the present invention has following beneficial effect:
The maximum novelty of the present invention is to use first PG-1 to study its antivirus action to HP-PRRSV, and demonstrate PG-1 by multiple method and have good antiviral effect at Marc-145 cellular level to HP-PRRSV, for the newtype drug developing anti-pig blue-ear disease is laid a good foundation.
In order to study the antivirus action of PG-1 further, the present invention is also studied the mechanism of the anti-HP-PRRSV of PG-1.Result shows, the antivirus action of PG-1 occurs in HP-PRRSV adherent cell process.
In order to strengthen the biologic activity of PG-1, the present invention has carried out amidatioon modification to its C end in chemosynthesis PG-1 process.
Accompanying drawing explanation
Fig. 1 is PG-1 anti-bacillus coli DH 5 alpha active figure; DdH in figure
2o:PG-1 is dissolved in the ddH of 0.01% glacial acetic acid
2in O; PBS:PG-1 is dissolved in the PBS of 0.01% glacial acetic acid; 0.2M tris-Hcl 6.8:PG-1 is dissolved in 0.2M tris-Hcl PH=6.8 buffer; 0.2M tris-Hcl 6.8/PBS:PG-1 is dissolved in 0.2M tris-Hcl PH=6.8 PBS buffer; Control:100 μ L concentration is 100mg/L Amp.
Fig. 2 is that AlamarBlue detects PG-1 cytotoxicity figure.
Fig. 3 is the virus titer figure in the Marc-145 cell conditioned medium that infects of HP-PRRSV respectively after variable concentrations PG-1 process.
Fig. 4 is that N gene is relative to the expression figure of reference gene HPRT1 respectively after variable concentrations PG-1 effect 36h.
Fig. 5 be respectively through 20, after 30mg/L PG-1 process 36h, N protein expression figure.
Fig. 6 be respectively through 20,30, after 40mg/L PG-1 process 36h, to the indirect immunofluorescence assay figure of N protein; Green is anti-PRRSV N protein color, and blueness is Hoechst transfect cell core color.
Fig. 7 is in PG-1 Antiviral Mechanism-virus absorption onto cell process, and N gene is relative to the expression figure of internal reference GAPDH.
Fig. 8 is that in PG-1 Antiviral Mechanism-virus absorption onto cell process, N protein Western-Blot detects figure.
Fig. 9 is that in PG-1 Antiviral Mechanism-cell entry cell processes, N protein Western-Blot detects figure.
Figure 10 is that after PG-1 Antiviral Mechanism-cell entry cell 4h, N protein Western-Blot detects figure.
Detailed description of the invention
Explain the present invention further below in conjunction with Figure of description and embodiment, but embodiment does not limit in any form to the present invention.
Strain used in the embodiment of the present invention is HP-PRRSV(highly pathogenic PRRSV), this strain is obtained by College of Veterinary Medicine, South China Agricultural University's zoonosis laboratory, HP-PRRSV is the abbreviation of pathogenic higher porcine reproductive and respiratory syndrome virus, does not do any restriction to the present invention.
embodiment 1 PG-1 chemosynthesis and Chinese People's Anti-Japanese Military and Political College's enterobacteria activity experiment
1, first the aminoacid sequence of pig derived antimicrobial peptide PG-1 is searched by antibacterial peptide data base (APD) The Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php), as shown in SEQ ID NO.1, aminoacid sequence is sent to the biochemical company limited (http://glbetter.cn.1688.com/) of Shanghai gill to synthesize, because the beta sheet structure of PG-1 is most important to its biologic activity, and the formation of beta sheet structure depends on 2 pairs of disulfide bond in PG-1, therefore must guarantee that disulfide bond synthesizes successfully and accurately locates when synthesizing PG-1.The 2 pairs of disulfide bond are the 6th and the 15th Cys, the 8th and the 13rd Cys respectively, and in order to strengthen the biologic activity of PG-1, hold amidatioon to modify to its C.HPLC 95% purity, synthesis 30mg.After synthesis, 0.01% glacial acetic acid aquesterilisa is diluted to 100mg/L, 0.22 μm of filtration sterilization, subpackage-80 DEG C preservation.
2, PG-1 Chinese People's Anti-Japanese Military and Political College enterobacteria activity experiment.Because PG-1 just has activity and dissolubility is the highest under weak acid environment, in order to make PG-1 antimicrobial activity the highest, the PG-1 powder of chemosynthesis is dissolved in respectively containing in the aquesterilisa of 0.01% glacial acetic acid, the aquesterilisa of PBS and 0.2M Tris-Hcl and PBS, and then study its antibacterial activity to bacillus coli DH 5 alpha, filter out PG-1 the highest active solvent.
PG-1 Chinese People's Anti-Japanese Military and Political College enterobacteria activity experiment method adopts standard agar hole diffusion method.Will
e.colithe LB solid medium 30mL of DH5 α 10 μ L and 55 DEG C mixes rear spread plate, after it solidifies, it is the card punch punching of the sterilizing of 3mm with diameter, and add 100 μ L PG-1, adding isopyknic dissolving PG-1 solvent as negative control, Amp(100mg/mL simultaneously) 2 μ L are as positive control.Cultivate 12h in 37 DEG C of incubators, take out when obvious inhibition zone can be seen and measure antibacterial circle diameter.
Every hole adds the good PG-1 of the above-mentioned dilution of 200 μ L, sets up the negative control of ampicillin positive control and 0.01% glacial acetic acid simultaneously, sees accompanying drawing 1.As shown in Figure 1, when PG-1 dissolves in the aquesterilisa of 0.01% glacial acetic acid, inhibition zone is maximum, in the PBS of 0.01% glacial acetic acid, inhibition zone is slightly little, but almost there is no inhibition zone when dissolving in the aquesterilisa of 0.2M Tris-Hcl, therefore illustrated by Fig. 1, in order to make PG-1 best to bacillus coli DH 5 alpha fungistatic effect, PG-1 must be dissolved in the aquesterilisa of 0.01% glacial acetic acid.
3, after PG-1 the highest active solvent is sent as an envoy in screening, PG-1 powder being diluted to final concentration is 100mg/L, 0.22 μm of filtration sterilization, subpackage, and in-80 DEG C of preservations.
embodiment 2 PG-1 cell toxicity test
AlamarBlue(is purchased from Invitrogen company) as living cells metabolize indicator, measurable fluorescence metabolite can be produced under mitochondrion lipase-catalyzed, can cytoactive be monitored by measuring its fluorescence intensity.Marc-145 cell is cultivated to 60-70% with the DMEM culture fluid containing 10% hyclone, discard culture fluid, add the nutritional solution effect 36h containing PG-1 doubling dilution, setting PBS matched group, then 10%(V/V is added) ratio AlamarBlue continuation cultivation 3h, use multi-functional microplate reader to read 540nm exciting light and 590nm utilizing emitted light fluorescent value respectively, make PG-1 cytotoxicity figure (see accompanying drawing 2).Active in 100% using PBS cellular control unit, under the fluorescent value of the cell of the PG-1 process of doubling dilution is variable concentrations than upper PBS matched group fluorescent value, PG-1 versus cell is active, as shown in Figure 2, when PG-1 concentration is 40mg/L, it does not have toxicity to Marc-145 cell, cytoactive 100%, this concentration is the Cmax of later tests.
embodiment 3 PG-1 antivirus test
When 1, cultivating Marc-145 cell to cell confluency degree 70% in 6 orifice plates of the DMEM culture medium containing 10% hyclone, discard culture fluid, PBS washes 3 times, meets malicious HP-PRRSV with MOI=0.1, and in the DMEM culture fluid of 2% hyclone, 37 DEG C are continued to cultivate 5h.
2, PBS washes 3 times, be 20 respectively by concentration, 30, the DMEM culture fluid of 2% hyclone of 40mg/L PG-1 adds 37 DEG C, cell and cultivates 36h, and establish PBS matched group, PBS washes 3 times, collects supernatant and is TCID
50detect, see accompanying drawing 3, as shown in Figure 3, PG-1 can reduce the viral yield in cell conditioned medium significance, suppresses virus to be discharged in cell conditioned medium; Every hole adds 400 μ L Trizol in addition, carries RNA, is qRT-PCR and detects, see accompanying drawing 4, and as shown in Figure 4, on transcriptional level, PG-1 can reduce N gene expression dose in significance ground.
3, the samely malicious HP-PRRSV is met with MOI=0.1, in the DMEM culture fluid of 2% hyclone, 37 DEG C are continued to cultivate 5h, PBS washes 3 times, be 20 respectively by concentration, 30, the DMEM culture fluid of 2% hyclone of 40mg/L PG-1 adds 37 DEG C, cell and cultivates 36h, and establish PBS matched group, collect 1mL supernatant and be TCID
50detect, discard remaining culture liq, PBS washes 3 times, 0.25% trypsinization, cell lysis, surveys protein concentration, and Western-Blot detects, and see accompanying drawing 5, as shown in Figure 5, in protein translation level, PG-1 can significantly reduce N protein expression.Therefore this experiment proves that PG-1 has good anti-HP-PRRSV effect from many aspects.
4, PG-1 antiviral indirect immunofluorescence assay (Indirect Immunofluorescent Assay, IFA).When cultivating Marc-145 cell to cell confluency degree 70% in 12 orifice plates of the DMEM culture medium containing 10% hyclone, discard culture fluid, PBS washes 3 times, malicious HP-PRRSV is met with MOI=0.1, in the DMEM culture fluid of 2% hyclone, 37 DEG C are continued to cultivate 5h, PBS washes 3 times, be 20 by concentration respectively, 30, the DMEM culture fluid of 2% hyclone of 40mg/L PG-1 adds 37 DEG C, cell and cultivates 36h, and establish PBS matched group, PBS washes 3 times, 4% paraformaldehyde fixes 10min, PBS washes 10min, 10%Triton-100 bores a hole 15min, PBS washes 10min, then dilute 1%BSA with PBS and close 30min, anti-PRRSV N protein primary antibodie room temperature 1h, against murine two anti-effect 1h, Hoechst contaminates core 5min, PBS washes 10min, carry out IFA detection, at fluorescence microscopy Microscopic observation PG-1 antiviral effect, see accompanying drawing 6, as shown in Figure 6, PG-1 concentration is 20, 30, during 40mg/L, anti-PRRSV N protein fluorescent value is starkly lower than PBS matched group, show PRRSV N protein almost not at cells, namely illustrate that PG-1 antiviral effect is obvious, and when PG-1 concentration is 40mg/L, antiviral effect is the most obvious.
embodiment 4 PG-1 Antiviral Mechanism research-HP-PRRSV adsorbs inhibition test
1, wash 3 times at 6 orifice plate PBS of Marc-145 cell confluency degree 70%, then meet malicious HP-PRRSV with MOI=0.1, and add respectively concentration be 20,30,40mg/L PG-1, hatch 2h for 4 DEG C.
2, PBS washes 3 times, washes, the Virus eliminating medicine not being adsorbed onto cell surface then at 5%CO off
2, continue to cultivate 24h in 37 DEG C of incubators, discard culture fluid, PBS washes 3 times, collecting cell, and every hole adds 400 μ L Trizol, carries RNA, is qRT-PCR and detects, see accompanying drawing 7 to N gene; Cell lysis, surveys protein concentration, and Western-Blot detects, and sees accompanying drawing 8.Result shows that PG-1 can reduce N gene and N protein expression significance, and along with the rising of PG-1 concentration, inhibition is more obvious, when PG-1 concentration is 20mg/L, HP-PRRSV N protein expression can't detect, illustrate that PG-1 has good antiviral effect in HP-PRRSV adherent cell process.
embodiment 5 PG-1 Antiviral Mechanism research-HP-PRRSV enters inhibition test
1, wash 3 times at 6 orifice plate PBS of Marc-145 cell confluency degree 70%, then meet malicious HP-PRRSV with MOI=0.1, hatch 2h for 4 DEG C.
2, PBS washes 3 times, is washed off by the Virus eliminating medicine not being adsorbed onto cell surface, then add respectively concentration be 20,30,40mg/L PG-1, at 5%CO
2, cultivate 6h in 37 DEG C of incubators.
3, PBS washes 3 times, renew the fresh nutritional solution containing 2% hyclone to continue to cultivate 24h, discard nutritional solution, PBS washes 3 times, collecting cell Western-Blot detects, see accompanying drawing 9, as shown in Figure 9, enter in cell processes at HP-PRRSV, PG-1 antiviral effect is not obvious, only have when PG-1 concentration is 40mg/L, N protein expression declines to some extent, illustrates that PG-1 enters antivirus action in cell processes at HP-PRRSV not obvious.
embodiment 6 PG-1 Antiviral Mechanism research-HP-PRRSV enters inhibition test after 4h
1, wash 3 times at 6 orifice plate PBS of Marc-145 cell confluency degree 70%, then meet malicious HP-PRRSV with MOI=0.1, hatch 2h for 4 DEG C, PBS washes 3 times, is washed off by the Virus eliminating medicine not being adsorbed onto cell surface, 5%CO
2, cultivate 4h in 37 DEG C of incubators.
2, PBS washes 3 times, be 20 respectively by concentration, 30, the DMEM culture fluid of 2% hyclone of 40mg/L PG-1 adds 37 DEG C, cell and cultivates 6h.
3, PBS washes 3 times, renews the fresh nutritional solution containing 2% hyclone and continues to cultivate 24h, discard nutritional solution, PBS washes 3 times, collecting cell Western-Blot detects, and see accompanying drawing 10, result shows, again with variable concentrations PG-1 process after HP-PRRSV enters cell 4h at 37 DEG C, PG-1 antiviral effect is not obvious, only has when PG-1 concentration is 40mg/L, just has antiviral effect, illustrate that PG-1 antiviral effect is not obvious after cell entry cell 4h.
Sequence table
SEQUENCE LISTING
<110> Zhongshan University
The application of <120> antibacterial peptide Protegrin-1 in control pig blue-ear disease
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> PRT
<213> antibacterial peptide Protegrin-1 aminoacid sequence
<400> 1
Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val
1 5 10 15
Gly Arg
Claims (1)
1. the application of antibacterial peptide Protegrin-1 in the anti-PRRSV virus drugs of preparation, it is characterized in that, the aminoacid sequence of described Protegrin-1 is as shown in SEQ ID NO.1;
Described PRRSV virus is high pathological form strain HP-PRRSV.
2. the application of antibacterial peptide Protegrin-1 in preparation control pig blue-ear disease medicine, it is characterized in that, the aminoacid sequence of described Protegrin-1 is as shown in SEQ ID NO.1;
Described pig blue-ear disease is the pig blue-ear disease caused by high pathological form strain HP-PRRSV.
3. apply according to claim 1 or 2, it is characterized in that, the secondary structure of described Protegrin-1 is beta sheet, and its aminoacid sequence the 6th and the 15th Cys, the 8th and the 13rd Cys form 2 pairs of disulfide bond, and C holds amidatioon modification.
4. apply according to claim 1 or 2, it is characterized in that, described antibacterial peptide Protegrin-1 using dosage scope is 20 mg/L ~ 40mg/L.
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| CN108558998B (en) * | 2018-02-27 | 2020-07-28 | 深圳市前海金卓生物技术有限公司 | Preparation and application of recombinant yeast preparation co-expressed by pig interleukin 4/6 and fused pig antibacterial peptide |
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