CN108938661A - The application in the drug for inhibiting Riemerellosis Anatipestifer is being prepared in conjunction with the gyrA gene PNA joint antibiotic of cell-penetrating peptide - Google Patents

The application in the drug for inhibiting Riemerellosis Anatipestifer is being prepared in conjunction with the gyrA gene PNA joint antibiotic of cell-penetrating peptide Download PDF

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CN108938661A
CN108938661A CN201810845490.3A CN201810845490A CN108938661A CN 108938661 A CN108938661 A CN 108938661A CN 201810845490 A CN201810845490 A CN 201810845490A CN 108938661 A CN108938661 A CN 108938661A
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gyra
pna
plants
cpp
antibiotic
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CN108938661B (en
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程安春
吴莉萍
邱浩
汪铭书
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Sichuan Agricultural University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4704—2-Quinolinones, e.g. carbostyril
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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

Abstract

The present invention relates to a kind of application of the gyrA gene PNA (CPP-PNA-gyrA) of the combination cell-penetrating peptide joint antibiotic in the drug that preparation inhibits Riemerellosis Anatipestifer, by CPP-PNA-gyrA and antibiotic to Riemerellosis Anatipestifer collective effect after, one times is reduced to the MIC of Riemerellosis Anatipestifer, illustrate that CPP-PNA-gyrA can enhance the antibacterial action of antibiotic, therefore the drug that anti-Riemerellosis Anatipestifer is made can be compounded with antibiotic.

Description

Inhibit to write from memory in pest of duck in preparation in conjunction with the gyrA gene PNA joint antibiotic of cell-penetrating peptide Application in the drug of Salmonella
Technical field
The invention belongs to birds prevention and control field, it is related to pressing down in conjunction with the gyrA gene PNA joint antibiotic of cell-penetrating peptide in preparation Application in the drug of Riemerellosis Anatipestifer processed.
Background technique
Riemerellosis Anatipestifer (Riemerella anatipestifer, RA) mainly causes connecing for the birds such as duck, goose, turkey Sexually transmitted disease is touched, is a kind of Gram negative rods bacillus, it is in generation that atrichia, without gemma is the most susceptible with the duck of 2-8 week old Criticality distribution, morbidity and mortality are higher, seriously endanger the development of duck culturing industry.Riemerellosis Anatipestifer prevention and treatment at present is on the one hand By reinforcing feeding management, perfect antisepsis system is established；On the other hand it is prevented and treated using antibiotic, although antibiotic prevention and treatment is anti- The effective means of the disease is controlled, but due to the extensive use of antibiotic, the problem of universal drug resistance occurs.Therefore, it is necessary to provide A kind of control method solving antibiotic prevention and treatment resistance problems.
DNA- topoisomerase (gyrA) gene coding DNA topoisomerase, catalytic dna chain break and combination, regulation The topological structure of DNA, is present in nucleus.Two kinds of topoisomerases are primarily present, DNA topoisomerase I changes topology knot Structure promotes DNA replication dna；Topoisomerase II includes 4 subunits, two α subunits and two β subunits.α subunit about 105KDa is GyrA coded by said gene is related to DNA replication dna；β subunit about 95KDa is gyrB coded by said gene, has atpase activity, topology Isomerase inhibitors can be used as anti-tumor drug.Peptide nucleic acid (PNA) cooperates with lavo-ofloxacin, ovobiocin and grand mould Element targets gyrase gene, hinders ribose body function, significantly improves the fungistatic effect of antibiotic；PNA combination cell-penetrating peptide (KFF)3K (K is lysine, and F is phenylalanine) targets gyrA gene, can obviously lower Acinetobacter bauamnnii gyrA gene mRNA levels, Minimal inhibitory concentration (Minimum inhibitory Concentration, MIC) is 5 μM, minimum bactericidal concentration (Minimum Bactericidal Concentration, MBC) it is 10 μM；In streptococcus pyogenes M49 bacterial strain, PNA combination cell-penetrating peptide (KFF)3K targets gyrA gene, and MIC is between 1.6-4.0 μM；In Klebsiella Pneumoniae, PNA combination cell-penetrating peptide (KFF)3K target To gyrA gene, MIC is 20 μM.But the fungistatic effect of RA is improved not in conjunction with the gyrA gene PNA of cell-penetrating peptide joint antibiotic It appears in the newspapers.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of gyrA gene PNA of combination cell-penetrating peptide joint antibiotic to exist Preparation inhibits the application in the drug of Riemerellosis Anatipestifer；The second object of the present invention is to provide a kind of reduction antibiotic to duck Write from memory in epidemic disease Salmonella MIC value, improve antibiotic to the method for Riemerellosis Anatipestifer fungistatic effect.
In order to achieve the above objectives, the invention provides the following technical scheme:
1, combine antibiotic answering in the drug for preparing anti-Riemerellosis Anatipestifer in conjunction with the gyrA gene PNA of cell-penetrating peptide With.
Preferably, the antibiotic is quinolone antibiotics.
Preferably, the antibiotic is Ciprofloxacin or Norfloxacin.
Preferably, the sequence of the gyrA gene PNA is as shown in SEQ ID NO.1.
Preferably, the sequence of the cell-penetrating peptide is as shown in SEQ ID NO.2.
Preferably, the gyrA gene PNA of the combination cell-penetrating peptide is 5 '-FITC-OO-KFFKFFKFFK-OO- CGGTTGCCCACTCC-3 ', wherein FITC is fluorescent marker, and K is lysine, and F is phenylalanine, and O is glycine, OO be for Connecting each group enhances deliquescent O linker.
Preferably, the Riemerellosis Anatipestifer is Riemerellosis Anatipestifer CH-1 plants, in CH-2 plants of Riemerellosis Anatipestifer, pest of duck Silent 11845 plants of Salmonella ATCC.
2, a kind of reduction antibiotic will combine cell-penetrating peptide (KFF) to the method for Riemerellosis Anatipestifer MIC value3(K is to rely ammonia to K Acid, F are phenylalanines) gyrA gene PNA and Antibiotic combination use.
3, antibiotic is improved to the method for Riemerellosis Anatipestifer fungistatic effect, will combine cell-penetrating peptide (KFF)3(K is to rely ammonia to K Acid, F are phenylalanines) gyrA gene PNA and Antibiotic combination use.
The beneficial effects of the present invention are: the present invention will combine the gyrA gene PNA and Antibiotic combination of cell-penetrating peptide for the first time Using, research antibiotic to the inhibitory effect of Riemerellosis Anatipestifer as a result, it has been found that, can reduce the MIC to different serotypes RA Value, and have extremely significant difference with gyrA gene PNA or antibiotic independent role, therefore can be used as the medicine of anti-Riemerellosis Anatipestifer Object uses.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out Illustrate:
Fig. 1 is the PNA-gyrA in conjunction with cell-penetrating peptide in conjunction with OD after Norfloxacin effect different serotypes RA600Measurement result (a:CPP-PNA-gyrA acts on CH-1 plants of RA；B:CPP-PNA-gyrA acts on CH-2 plants of RA；C:CPP-PNA-gyrA makees For 11845 plants of ATCC).
Fig. 2 is the PNA-gyrA in conjunction with cell-penetrating peptide in conjunction with bacterium colony count results after Norfloxacin effect different serotypes RA (a:CPP-PNA-gyrA acts on CH-1 plants of RA；B:CPP-PNA-gyrA acts on CH-2 plants of RA；C:CPP-PNA-gyrA makees For 11845 plants of ATCC).
Fig. 3 is the PNA-gyrA in conjunction with cell-penetrating peptide to the influence (a:CPP- of different serotypes RA gyrA gene transcription level PNA-gyrA acts on CH-1 plants of RA, and b:CPP-PNA-gyrA acts on CH-2 plants of RA, and c:CPP-PNA-gyrA is acted on 11845 plants of ATCC).
Fig. 4 is the PNA-gyrA in conjunction with cell-penetrating peptide to different serotypes RA bacteriostasis effect (a:CPP-PNA-gyrA work For CH-1 plants of RA；B:CPP-PNA-gyrA acts on CH-2 plants of RA；C:CPP-PNA-gyrA acts on ATCC 11845 Strain).
Fig. 5 is the PNA-gyrA in conjunction with cell-penetrating peptide to the detection (a:CPP- of different serotypes RA0C_0528 transcriptional level PNA-gyrA acts on CH-1 plants of RA；B:CPP-PNA-gyrA acts on RA CH-2；C:CPP-PNA-gyrA acts on ATCC 11845 plants).
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.
Embodiment 1 is designed by the PNA of target gene of RA gyrA
The gyrA gene nucleotide series (Genbank:CP003787.1) of RA are retrieved in GenBank, it is soft using BLAST The gyrA gene that retrieval obtains is carried out similarity analysis between each bacterial strain of RA by part.The preferable PNA sequence of combination stability is designed, It is named as PNA-gyrA, nucleic acid sequence 5 '-cggttgcccactcc-3 ' (SEQ ID NO.1).And to PNA-gyrA sequence It is modified, in order to increase its Cell permeable and solubility, is added cell-penetrating peptide (CPP) [(KFF)3K] and solubility promote because Sub- O-Linker, cell-penetrating peptide amino acid sequence are as follows, and: KFFKFFKFFK (SEQ ID NO.2) is closed by panagene company At PNA (CPP-PNA-gyr) composition sequence in conjunction with cell-penetrating peptide is as follows: 5 '-FITC-OO-KFFKFFKFFK-OO- CGGTTGCCCACTCC-3 ', FITC are fluorescent marker, and for the positioning after PNA effect, K is lysine, and F is phenylalanine, O For glycine, OO is to enhance deliquescent Olinker for connecting each group.
Embodiment 2, CPP-PNA-gyrA study the inhibitory effect of Riemerellosis Anatipestifer
Referring to sensitivity testing to antibacterials operation standard, with micro-dilution method detection CPP-PNA-gyrA to strain subject RACH-1 plants, the MIC of CH-2 plants of RA, 11845 plants of ATCC, it is described that specific step is as follows:
(1) prepared by bacteria suspension
11845 plants of RACH-1 plants, CH-2 plants of RA, ATCC streak inoculations are incubated in TSA agar plate, 37 DEG C of incubators It educates overnight, secondary daily oese picking single colonie is inoculated in 3mL nutrient broth, is shaken in 37 DEG C, 220r/min constant-temperature table Culture, until logarithmic growth phase (OD600=0.8) after, bacterium solution is diluted to bacteria containing amount 2 × 10 with TSB5CFU/mL。
(2) dilution of CPP-PNA-gyrA and antibacterials
CPP-PNA-gyrA is diluted to 50 μM with sterile water, is dispensed spare；Ofloxacin, Norfloxacin are diluted to respectively 10240 μ g/mL, -20 DEG C of refrigerators save backup.
(3) it is loaded
Sterile 96 orifice plate is taken, every row is respectively labeled as experimental group: CPP-PNA-gyrA group, CPP-PNA-gyrA+ antibiotic Group, antibiotic group；Control group: mispairing CPP-PNA group is free of antibiotic group.Add 160 μ L TSB meat soups in every the 1st hole of row, remaining Each hole is separately added into 100 μ L TSB meat soups, sequentially adds 40 μ L CPP-PNA-gyrA, 40 μ L antibiotic, 20 μ LCPP-PNA- GyrA+20 μ L antibiotic, 40 μ L TSB meat soups.After being mixed well with the volley of rifle fire, 100 μ L are sucked out from the first hole and are added to corresponding the In 2 holes, twice of doubling dilution discards 100 μ L liquid being finally sucked out to the 10th column.Then the 100 above-mentioned preparations of μ L are added in every hole Bacteria suspension to be measured make the final bacterial concentration 10 in every hole5CFU/mL.A small amount of bacteria suspension to be measured is taken, TSA agar plate is lined On, secondary culture is carried out in 37 DEG C of incubators, is used to check bacterium solution with the presence or absence of pollution.
(4) it is incubated for and result judges
96 orifice plates of bacterium solution, antibiotic, CPP-PNA-gyrA will have been added to shake 1min on micro oscillator, it is sufficiently mixed It is even, it is subsequently placed in 12-16h in 37 DEG C of incubators.By visually being observed, the MIC of drug is that the minimum drug in limpid hole is dense Degree also needs to check control group with the presence or absence of pollution, and whether the MIC value of Quality-control strains is in Quality Control range.
A.CPP-PNA-gyrA is as shown in table 1 in conjunction with the measurement result of MIC after Ciprofloxacin effect different serotypes RA.
Table 1, CPP-PNA-gyrA act on the measurement result of MIC after different serotypes RA in conjunction with Ciprofloxacin
The results show that CPP-PNA-gyrA acts solely on CH-1 plants of RA, CH-2 plants of RA, 11845 plants of ATCC, MIC is equal Greater than 256 μ g/mL, without obvious bacteriostasis effect；The MIC that Ciprofloxacin acts solely on RACH-1 plants is 4 μ g/mL, cyclopropyl The MIC that Sha Xing acts solely on RACH-2 plants is 16 μ g/mL, and the MIC that Ciprofloxacin acts solely on 11845 plants of ATCC is lower than 0.5μg/mL；It is 2 μ g/mL to RACH-1 plant of MIC, to RACH-2 plants after CPP-PNA-gyrA and Ciprofloxacin collective effect MIC be 8 μ g/mL, to 11845 plants of ATCC of MIC be lower than 0.5 μ g/mL.The above test results show that: CPP-PNA-gyrA with After Ciprofloxacin collective effect, the MIC value of RACH-1 plants, RACH-2 plants is reduced, to 11845 plants of sensitivities of ATCC, difference is not Significantly, meanwhile, non-targeted CPP-PNA (mispairing CPP-PNA) is without function and effect.
CPP-PNA-gyrA is as shown in table 2 in conjunction with the measurement result of MIC after Norfloxacin effect different serotypes RA.
Table 2, CPP-PNA-gyrA act on the measurement result of MIC after different serotypes RA in conjunction with Norfloxacin
As shown in Table 2, CPP-PNA-gyrA acts solely on CH-1 plants of RA, CH-2 plants of RA, 11845 plants of ATCC, MIC 256 μ g/mL are all larger than, without obvious bacteriostasis effect；The MIC that Norfloxacin acts solely on CH-1 plants of RA is 8 μ g/mL, promise The MIC that Flucloxacillin acts solely on CH-2 plants of RA is 32 μ g/mL, and the MIC that Norfloxacin acts solely on ATCC11845 plants is 16μg/mL；It is 4 μ g/mL to CH-1 plant of RA of MIC, to CH-2 plants of RA after CPP-PNA-gyrA and Norfloxacin collective effect MIC be 16 μ g/mL, to 11845 plants of ATCC of MIC be 8 μ g/mL.The above test results show that: CPP-PNA-gyrA and promise After Flucloxacillin collective effect, CH-2 plants, ATCC11845 plants CH-1 plants of RA, RA MIC values are declined, meanwhile, non-targeted CPP- PNA (mispairing CPP-PNA) is without function and effect.
B.CPP-PNA-gyrA is in conjunction with OD after Norfloxacin effect different serotypes RA600The measurement of value
96 orifice plates of above-mentioned survey MIC are put into microplate reader and carry out OD600Measurement, observe OD600Variation, analyze CPP- The bacteriostasis effect of PNA-gyrA.CPP-PNA-gyrA is in conjunction with OD after Norfloxacin effect different serotypes RA600The measurement of value As a result as shown in Fig. 1 and table 3.
Table 3.CPP-PNA-gyrA is in conjunction with OD after Norfloxacin effect different serotypes RA600Measurement result
As shown in Table 3, both CPP-PNA-gyrA and Norfloxacin collective effect are compared to Norfloxacin independent role, promise Flucloxacillin is to the OD after CH-1 plants of RA effects600Value is average to be reduced to 0.08 by 0.36；After Norfloxacin is to RACH-2 plants of effects OD600Value is average to be reduced to 0.10 by 0.26；Norfloxacin is to the OD after 11845 plants of ATCC effects600Value is average to be subtracted by 0.35 Less to 0.11.The above test results show that: after CPP-PNA-gyrA and Norfloxacin collective effect, to RACH-1 plants, RACH-2 11845 plants of strain, ATCC OD600Value declines, and has extremely significant bacteriostasis effect.
C.CPP-PNA-gyrA acts on the bacterium colony counting after different serotypes RA in conjunction with Norfloxacin
Above-mentioned bacterium solution is subjected to bacterium colony counting, the corresponding hole of MIC value is taken to carry out 10-1、10-2、10-3、10-4、10-5、10-6、 10-7、10-8It dilutes again, 100 μ L is taken to instill the agar plate cooled down, 37 DEG C are incubated for for 24 hours, then carry out bacterium colony counting.Each 3 repetitions are arranged in bacterial strain, are averaged.Bacterium amount in every milliliter of sample liquid is average colony number multiplied by extension rate, and unit is denoted as CFU/mL, as a result as shown in Fig. 2 and table 4.
Table 4.CPP-PNA-gyrA is in conjunction with bacterium colony count results after Norfloxacin effect different serotypes RA
As a result as shown in Fig. 2 and table 4, both CPP-PNA-gyrA and Norfloxacin collective effect are compared to Norfloxacin list Solely effect, Norfloxacin is to the clump count after CH-1 plants of RA effects by 7.1 × 107CFU/mL is reduced to 1.6 × 107CFU/mL； Norfloxacin is to the clump count after CH-2 plants of RA effects by 5.3 × 107CFU/mL is reduced to 1.9 × 107CFU/mL；Norfloxacin To the clump count after 11845 plants of ATCC effects by 7.0 × 107CFU/mL is reduced to 2.2 × 107CFU/mL.The above test result Show: after CPP-PNA-gyrA and Norfloxacin collective effect, to 11845 plants of CH-1 plants of RA, CH-2 plants of RA, ATCC bacterium colonies Numerical value declines, and has extremely significant bacteriostasis effect.
Influence of the D.CPP-PNA-gyrA to gyrA gene transcription level
(1) building of standard curve
RA CH-1 pnca gene group DNA is extracted as template, constructs RA with Real-Time qPCR specific primer Standard curve (the GyrA-F:5 '-acttaccatatctcacgcagg-3 ' (SEQ ID NO.3) of gyrA；GyrA-R:5 '- tcgtctcttgtagatgccg-3'(SEQ ID NO.4)).For the initial concentration of template, it is diluted to one first and is fitted Suitable concentration, then 10 times of serial dilution concentration, amount to 6 dilutions, 4 repetitions of each gradient.Reaction system is 2 μ L DNA profiling, 10 μ LPremixExTaqII (TliRNaseH Plus), 0.8 μ L gyrA upstream primer, 0.8 μ L GyrA downstream primer, 6.4 μ L RNase Free dH2O.Response procedures: 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 3s, 60 DEG C of annealing And extend 30s, 39 circulations；65 DEG C of reaction 5s, while by observation melting curve and standard curve to verify the special of primer Property.The results show that amplification efficiency is 101%, there is very high specificity, can be used in quantitative detection.
(2) extraction of total serum IgE
After surveying the 96 orifice plate bacteriostasis 16-20h of MIC, the corresponding aperture of different RA is acted on according to CPP-PNA-gyrA Sampling, loaded on 2mL without in RNA enzyme centrifuge tube, then bacterium solution is centrifuged, program 3000rpm, 10min by ice bath 5min, Supernatant is abandoned, is cleaned with sterile 1 × PBS, to remove extra culture medium, bacterial precipitation is collected, is then tried according to RNAisoPlus Agent box specification is operated, finally, using the RNA of extraction as reverse transcription template.With NANODROP2000 ultramicron nucleic acid egg The concentration and purity of the total serum IgE of white analyzer Detection and Extraction.
(3) gDNA and the reverse transcription in total serum IgE are removed
Specific steps are referring to treasured bioengineering (Dalian) Co., Ltd PrimeScriptTMRT reagent Kit with GDNA Eraser (PerfectRealTime) specification, first removal total serum IgE in genomic DNA, reaction condition be 42 DEG C, 5min；Then quantitative fluorescent PCR is carried out, reaction condition is 37 DEG C of reaction 15min, 85 DEG C of reaction 10s obtain cDNA, will be obtained CDNA be stored in -80 DEG C.
(4) Real-Time qPCR reacts
Using cDNA as template, qPCR is carried out with the fluorescent quantitation specific primer of reference gene recA with gyrA gene and is reacted (recA-F:5'-tgaaactaggtgatggtacg-3'(SEQ ID NO.5)；recA-R:5'- cttaggataaccgcctactc-3'(SEQID NO.6)).Response procedures are 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 3s, 60 DEG C It anneals and extends 30s, 39 circulations；65 DEG C of reaction 5s.Using recA gene as internal reference, with Bio-RadCFX manager (version3.0) software analyzes the relative transcript levels of RA CH-1 plants, CH-2 plants of RA, 11845 plants of ATCC.Each to test sample 4 repetitions are arranged in product, carry out independent repeated trials three times, and the statistical significance of test data is divided using unpaired t- inspection Analysis, is mapped, as a result as shown in fig. 3 and table 5 using Graphpad Prism7.0 software.
Influence of the table 5.CPP-PNA-gyrA to different serotypes RA gyrA gene transcription level
The results show that CPP-PNA-gyrA has lowered 2 times to CH-1 plants of RA of gyrA gene transcription level at 3.1 μM, 5 times are lowered at 12.5 μM, difference is extremely significant；GyrA genetic transcription of the CPP-PNA-gyrA at 6.2 μM to CH-2 plants of RA 2 times of horizontal down-regulation, 3 times are lowered at 12.5 μM, difference is extremely significant；CPP-PNA-gyrA is at 3.1 μM -12.5 μM to ATCC 11845 plants of gyrA gene transcription level has lowered 4 times, and difference is extremely significant.These results suggest that CPP-PNA-gyrA is at 6.2 μM 11845 plants of CH-1 plants of RA, CH-2 plants of RA, ATCC gyrA gene transcription levels can be lowered when above, difference pole Significantly.But bacteria used thereby liquid hold-up 10 may be tested due to MIC5CFU/mL, concentration is too high, although to target gene level under It adjusts, but antibacterial or bacteriocidal concentration has not yet been reached, so having no positive effect when independent role.
E.CPP-PNA-gyrA is to Riemerellosis Anatipestifer bacteriostasis effect
CPP-PNA-gyrA concentration is set to 20 μM, and CH-1 plants of RA, CH-2 plants of RA, 11845 plants of ATCC are diluted to respectively 2000CFU/mL, 200CFU/mL, positive control use Norfloxacin, then the same MIC test of concrete operations carries out bacterium colony meter Number is observed afterwards for 24 hours as a result, CPP-PNA-gyrA is used alone whether have fungistatic effect after detection reduces bacterium solution content, as a result As shown in Fig. 4 and table 6.
Table 6, CPP-PNA-gyrA are to different serotypes RA bacteriostasis effect
The results show that CPP-PNA-gyrA is to RA CH-1 when RA bacterium solution content is 2000CFU/mL and 200CFU/mL Strain growth effect is smaller, can be ignored；CH-2 plants of RA are had little effect with 11845 plants of ATCC.Result above table It is bright, even if CPP-PNA-gyrA in the lower situation of bacterium solution content, acts solely on CH-1 plants of RA, CH-2 plants of RA and ATCC 11845 plants, fungistatic effect is also not achieved.
Detection of the F.CPP-PNA-gyrA to RA gyrA downstream gene RA0C_0528 transcriptional level
Structure repeats rouge (TPR) albumen, is regulated and controled in the genome by DNA binding site.This albumen occurs only On one cross-film histidine kinase and DNA binding reactor, the interaction of TPR albumen regulates and controls PEP-CTERM protein expression.? Position is in the downstream gyrA on genome, transcribes in the same direction.The similitude of TPR albumen (RA0C_0528) nucleotide sequence of RA exists 94% or more, protein level similitude is up to 99.33%, so true by the observation downstream gyrA RA0C_0528 gene transcription level Determine influence of the gyrA to downstream gene expression, judges whether CPP-PNA-gyrA is the result for targeting gyrA to the effect of RA.
(1) the bent building of mark
RA CH-1 pnca gene group DNA is extracted as template, with Real-Time qPCR specific primer (RA0C_0528- F:5 '-gatacatcaagagaaccctcct-3 ' (SEQ ID NO.7)；RA0C_0528-R:5 '- Caaatactctcaaccagtagca-3 ' (SEQID NO.8)) construct standard curve.For the initial concentration of template, first It is diluted to a suitable concentration, then 10 times of serial dilution concentration, amounts to 6 dilutions, 4 weights of each gradient It is multiple.Reaction system be 2 μ L templates,PremixExTaqII (TliRNaseH Plus), 0.8 μ L upstream primer, 0.8 μ L downstream primer, 6.4 μ L RNaseFree dH2O.Response procedures are 95 DEG C of initial denaturation 30s；95 DEG C of denaturation 3s, 60 DEG C move back It fights and extends 30s, 39 circulations；65 DEG C of reaction 5s.The specificity for verifying primer by observation amplification curve simultaneously, obtains Amplification efficiency is 103%, has very high specificity, can be used in quantitative detection.
It quantifies primer with RA0C_0528-F and RA0C_0528-R to be detected, and using recA as internal reference, testing result As shown in Fig. 5 and table 7.
The detection of table 7, CPP-PNA-gyrA to different serotypes RA0C_0528 transcriptional level
The results show that CPP-PNA-gyrA 4h, 8h, 12h, 16h, 20h, for 24 hours when to RA gyrA downstream gene RA0C_ For 0528 transcriptional level without influence, bacterial content is 2000CFU/mL and 200CFU/mL.Also illustrate that CPP-PNA-gyrA is special simultaneously Inhibit the transcription of gyrA gene anisotropicly, and antibacterial by enhancing antibiotic with quinolone antibiotics Norfloxacin collective effect Function and effect.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Sequence table
<110>Sichuan Agricultural University
<120>in conjunction with gyrA gene PNA joint antibiotic the answering in the drug that preparation inhibits Riemerellosis Anatipestifer of cell-penetrating peptide With
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cggttgccca ctcc 14
<210> 2
<211> 10
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys
1 5 10
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
acttaccata tctcacgcag g 21
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
acttaccata tctcacgcag 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tgaaactagg tgatggtacg 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
cttaggataa ccgcctactc 20
<210> 7
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gatacatcaa gagaaccctc ct 22
<210> 8
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
caaatactct caaccagtag ca 22

Claims (8)

1. combining application of the gyrA gene PNA joint antibiotic of cell-penetrating peptide in the drug that preparation inhibits Riemerellosis Anatipestifer.
2. application according to claim 1, it is characterised in that: the antibiotic is quinolone antibiotics.
3. application according to claim 1, it is characterised in that: the antibiotic is Ciprofloxacin or Norfloxacin.
4. application according to claim 1, it is characterised in that: the sequence of the gyrA gene PNA such as SEQ ID NO.1 institute Show.
5. application according to claim 1, it is characterised in that: the sequence of the cell-penetrating peptide is as shown in SEQ ID NO.2.
6. application according to claim 1, it is characterised in that: the gyrA gene PNA of the combination cell-penetrating peptide is 5 '- FITC-OO-KFFKFFKFFK-OO-CGGTTGCCCACTCC-3 ', wherein FITC is fluorescent marker, and K is lysine, and F is phenylpropyl alcohol Propylhomoserin, O are glycine, and OO enhances deliquescent O linker for connecting each group.
7. application according to claim 1, it is characterised in that: the Riemerellosis Anatipestifer is Riemerellosis Anatipestifer CH-1 Strain, CH-2 plants of Riemerellosis Anatipestifer, 11845 plants of Riemerellosis Anatipestifer ATCC.
8. a kind of antibiotic that reduces is to the method for Riemerellosis Anatipestifer MIC value, it is characterised in that: the gyrA base of cell-penetrating peptide will be combined Because PNA and Antibiotic combination are used.
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CN110468186A (en) * 2019-09-11 2019-11-19 福建省农业科学院畜牧兽医研究所 Whether a kind of 11 type Riemerellosis Anatipestifers of identification are to the drug resistant method of fluoroquinolones
CN110564876A (en) * 2019-09-11 2019-12-13 福建省农业科学院畜牧兽医研究所 Method for identifying whether Riemerella anatipestifer type 1 is resistant to fluoroquinolone drugs

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