CN108938661B - Application of gyrA gene PNA combined antibiotic combined with cell-penetrating peptide in preparation of drug for inhibiting riemerella anatipestifer - Google Patents
Application of gyrA gene PNA combined antibiotic combined with cell-penetrating peptide in preparation of drug for inhibiting riemerella anatipestifer Download PDFInfo
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Abstract
The invention relates to an application of gyrA gene PNA (CPP-PNA-gyrA) combined antibiotic combined with cell-penetrating peptide in preparation of a drug for inhibiting riemerella anatipestifer.
Description
Technical Field
The invention belongs to the field of prevention and treatment of poultry, and relates to application of gyrA gene PNA combined antibiotic combined with cell-penetrating peptide in preparation of a medicine for inhibiting riemerella anatipestifer.
Background
Riemerella Anatipestifer (RA) mainly causes contact infectious diseases of poultry such as ducks, geese, turkeys and the like, is a gram-negative corynebacterium, has no flagella and spores, is most susceptible to ducks of 2-8 weeks old, is distributed worldwide, has high morbidity and mortality and seriously harms the development of the duck breeding industry. At present, on one hand, the prevention and treatment of riemerella anatipestifer are realized by strengthening feeding management and establishing a perfect disinfection system; on the other hand, the antibiotic is adopted for prevention and treatment, and although the antibiotic prevention and treatment is an effective means for preventing and treating the disease, the problem of general drug resistance is caused due to the wide application of the antibiotic. Therefore, it is necessary to provide a prevention and treatment method for solving the problem of antibiotic prevention and treatment resistance.
The DNA-topoisomerase (gyrA) gene encodes DNA topoisomerase, catalyzes the breaking and binding of DNA strands, regulates the topological structure of DNA, and is present in the nucleus. Two topoisomerases mainly exist, and the DNA topoisomerase I changes the topological structure and promotes DNA replication; topoisomerase II packageContains 4 subunits, two α subunits and two β subunits, α subunit is about 105KDa, is coded by gyrA gene and related to DNA replication, β subunit is about 95KDa, is coded by gyrB gene and has ATPase activity, topoisomerase inhibitor can be used as antitumor drug, Peptide Nucleic Acid (PNA) is cooperated with levofloxacin, neomycin and spectinomycin, target gyrase gene, block ribosome function and obviously improve bacteriostatic effect of antibiotic, PNA is combined with penetrating peptide (KFF)3K (K is lysine, F is phenylalanine) targets the gyrA gene, can obviously reduce the mRNA level of the gyrA gene of the Acinetobacter baumannii, the Minimum Inhibitory Concentration (MIC) is 5 mu M, and the Minimum Bactericidal Concentration (MBC) is 10 mu M; PNA binding to transmembrane peptide (KFF) in Streptococcus pyogenes M49 strain3K targets the gyrA gene, and the MIC is between 1.6 and 4.0 mu M; in Klebsiella pneumoniae, PNA binds to transmembrane peptide (KFF)3K targets the gyrA gene and MIC is 20. mu.M. However, the gyrA gene PNA combined with the cell-penetrating peptide and the antibiotic have not been reported to improve the bacteriostatic effect of RA.
Disclosure of Invention
In view of the above, the invention aims to provide an application of gyrA gene PNA combined antibiotic combined with cell-penetrating peptide in preparing a medicament for inhibiting riemerella anatipestifer; the second purpose of the invention is to provide a method for reducing the MIC value of the antibiotic to the riemerella anatipestifer and improving the bacteriostatic effect of the antibiotic to the riemerella anatipestifer.
In order to achieve the purpose, the invention provides the following technical scheme:
1. application of gyrA gene PNA combined antibiotic combined with cell-penetrating peptide in preparation of medicine for resisting riemerella anatipestifer.
Preferably, the antibiotic is a quinolone antibiotic.
Preferably, the antibiotic is ciprofloxacin or norfloxacin.
Preferably, the sequence of the gyrA gene PNA is shown in SEQ ID NO. 1.
Preferably, the sequence of the cell-penetrating peptide is shown as SEQ ID NO. 2.
Preferably, the gyrA gene PNA combined with the cell-penetrating peptide is 5 '-FITC-OO-KFFKFFKFFK-OO-CGGTTGCCCACTCC-3', wherein FITC is a fluorescent label, K is lysine, F is phenylalanine, O is glycine, and OO is an O linker for connecting various groups to enhance solubility.
Preferably, the riemerella anatipestifer is riemerella anatipestifer CH-1 strain, riemerella anatipestifer CH-2 strain and riemerella anatipestifer ATCC11845 strain.
2. A method for reducing MIC value of antibiotics to Riemerella anatipestifer comprises combining cell penetrating peptide (KFF)3The gyrA gene PNA of K (K is lysine and F is phenylalanine) is used in combination with antibiotics.
3. A method for improving antibacterial effect of antibiotics on Riemerella anatipestifer comprises combining penetrating peptide (KFF)3The gyrA gene PNA of K (K is lysine and F is phenylalanine) is used in combination with antibiotics.
The invention has the beneficial effects that: according to the invention, the gyrA gene PNA combined with the cell-penetrating peptide is combined with the antibiotic for the first time, and the result of researching the inhibition effect of the antibiotic on the riemerella anatipestifer is found that the MIC value of different serotype RA can be reduced, and the gyrA gene PNA has a very significant difference in single action with the gyrA gene PNA or the antibiotic, so that the gyrA gene PNA can be used as a medicament for resisting the riemerella anatipestifer.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows OD after different serotypes RA of PNA-gyrA combined with norfloxacin combined with transmembrane peptide600The results of the measurement (a: CPP-PNA-gyrA acts on RA CH-1 strain, b: CPP-PNA-gyrA acts on RA CH-2 strain, and c: CPP-PNA-gyrA acts on ATCC11845 strain).
FIG. 2 shows the results of colony counts after the action of PNA-gyrA bound with transmembrane peptide in combination with norfloxacin on different serotypes RA (a: CPP-PNA-gyrA acting on RA CH-1 strain, b: CPP-PNA-gyrA acting on RA CH-2 strain, c: CPP-PNA-gyrA acting on ATCC11845 strain).
FIG. 3 is a graph showing the effect of PNA-gyrA binding to transmembrane peptide on the transcription level of RA gyrA gene of different serotypes (a: CPP-PNA-gyrA acting on RA CH-1 strain, b: CPP-PNA-gyrA acting on RA CH-2 strain, c: CPP-PNA-gyrA acting on ATCC11845 strain).
FIG. 4 shows the inhibitory effects of PNA-gyrA bound with transmembrane peptide on different serotypes of RA (a: CPP-PNA-gyrA acts on RA CH-1 strain, b: CPP-PNA-gyrA acts on RA CH-2 strain, c: CPP-PNA-gyrA acts on ATCC11845 strain).
FIG. 5 shows the measurement of transcription levels of different serotypes RA 0C-0528 by PNA-gyrA bound to transmembrane peptide (a: CPP-PNA-gyrA acting on strain RA CH-1; b: CPP-PNA-gyrA acting on strain RA CH-2; c: CPP-PNA-gyrA acting on strain ATCC 11845).
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Example 1 PNA design with RA gyrA as target Gene
The nucleotide sequence of the gyrA gene of RA was searched in GenBank (Genbank: CP003787.1), and the gyrA gene obtained by the search was subjected to similarity analysis between strains of RA using BLAST software. A PNA sequence with good binding stability is designed and named as PNA-gyrA, and the nucleic acid sequence of the PNA-gyrA is 5'-cggttgcccactcc-3' (SEQ ID NO. 1). And PNA-gyrA sequence is modified, and in order to increase cell penetrability and solubility, Cell Penetrating Peptide (CPP) [ KFF ] is added3K ] and a solubility-promoting factor O-Linker, wherein the amino acid sequence of the cell-penetrating peptide is as follows: KFFKFFKFFK (SEQ ID NO.2), synthesized by the company pangene, and the sequence for synthesizing PNA (CPP-PNA-gyr) bound to a cell-penetrating peptide was as follows: 5 '-FITC-OO-KFFKFFKFFK-OO-CGGTTGCCCACTCC-3', FITC is a fluorescent label and is used for positioning after PNA (peptide nucleic acid) acts, K is lysine, F is phenylalanine, O is glycine, and OO is an Olinker used for connecting various groups to enhance solubility.
Example 2 study of bacteriostatic effect of CPP-PNA-gyrA on Riemerella anatipestifer
Referring to the standard of operation of antimicrobial drug susceptibility test, the MIC of CPP-PNA-gyrA to RACH-1 strain, RA CH-2 strain and ATCC11845 strain of the tested strains is detected by a microdilution method, and the specific steps are as follows:
(1) preparation of bacterial suspension
RACH-1 strain, RA CH-2 strain, ATCC11845The strain is streaked and inoculated on a TSA agar plate, incubated overnight in an incubator at 37 ℃, a single colony is picked by an inoculating loop the next day, inoculated in 3mL of nutrient broth, shake-cultured in a constant temperature shaking table at 37 ℃ and 220r/min until the logarithmic growth phase (OD)6000.8), the bacterial suspension was diluted with TSB to a bacterial content of 2 × 105CFU/mL。
(2) Dilution of CPP-PNA-gyrA and antibacterial agent
Diluting CPP-PNA-gyrA with sterile water to 50 μ M, and subpackaging for later use; the ofloxacin and the norfloxacin are respectively diluted to 10240 mu g/mL and stored in a refrigerator at the temperature of-20 ℃ for later use.
(3) Sample application
Taking a sterile 96-well plate, marking each row as an experimental group: CPP-PNA-gyrA group, CPP-PNA-gyrA + antibiotic group, antibiotic group; control group: mismatched CPP-PNA group, antibiotic-free group. 160. mu.L of TSB broth was added to well 1 of each row, and 100. mu.L of TSB broth was added to each of the remaining wells, followed by 40. mu.L of CPP-PNA-gyrA, 40. mu.L of antibiotic, 20. mu.L of LCPP-PNA-gyrA + 20. mu.L of antibiotic, and 40. mu.L of TSB broth. After being fully mixed by a row gun, 100 mu L of the mixture is sucked out from the first hole and added into the corresponding 2 nd hole, the mixture is diluted to the 10 th row by two times, and finally 100 mu L of the sucked liquid is discarded. Then 100 mul of the bacterial suspension to be tested prepared above is added into each hole, so that the final bacterial liquid concentration in each hole is 105CFU/mL. Taking a small amount of bacterial suspension to be detected, streaking the bacterial suspension on a TSA agar plate, and carrying out subculture in an incubator at 37 ℃ to check whether the bacterial suspension is polluted or not.
(4) Incubation and result determination
And (3) shaking the 96-well plate added with the bacterial liquid, the antibiotics and the CPP-PNA-gyrA on a micro-oscillator for 1min, fully mixing the materials uniformly, and then placing the mixture in an incubator at 37 ℃ for 12-16 h. And (4) observing by naked eyes, wherein the MIC of the medicament is the lowest medicament concentration of the clear hole, and whether the control group has pollution or not and whether the MIC value of the quality control strain is in a quality control range or not need to be checked.
The results of MIC determination after the action of CPP-PNA-gyrA in combination with ciprofloxacin against different serotypes of RA are shown in Table 1.
TABLE 1 results of MIC determination after CPP-PNA-gyrA combined with ciprofloxacin action on different serotypes RA
The result shows that the CPP-PNA-gyrA acts on RA CH-1 strain, RA CH-2 strain and ATCC11845 strain independently, MIC is more than 256 mug/mL, and no obvious bacteriostatic effect exists; the MIC of ciprofloxacin acting on RACH-1 strain alone is 4 mug/mL, the MIC of ciprofloxacin acting on RACH-2 strain alone is 16 mug/mL, and the MIC of ciprofloxacin acting on ATCC11845 strain alone is lower than 0.5 mug/mL; after the CPP-PNA-gyrA and ciprofloxacin are combined, the MIC for RACH-1 strain is 2 mug/mL, the MIC for RACH-2 strain is 8 mug/mL, and the MIC for ATCC11845 strain is lower than 0.5 mug/mL. The test results show that: after the CPP-PNA-gyrA and ciprofloxacin are acted together, MIC values of RACH-1 strain and RACH-2 strain are reduced, the strain is sensitive to ATCC11845 strain, the difference is not obvious, and meanwhile, non-target CPP-PNA (mismatch CPP-PNA) has no effect.
The results of MIC measurements after the action of CPP-PNA-gyrA in combination with norfloxacin on different serotypes of RA are shown in Table 2.
TABLE 2 results of MIC determination after the action of CPP-PNA-gyrA in combination with norfloxacin on different serotypes of RA
As can be seen from Table 2, the CPP-PNA-gyrA independently acts on RA CH-1 strain, RA CH-2 strain and ATCC11845 strain, MIC is more than 256 mug/mL, and no obvious bacteriostatic action effect exists; the MIC of norfloxacin acting on RA CH-1 strain alone is 8 mug/mL, the MIC of norfloxacin acting on RA CH-2 strain alone is 32 mug/mL, and the MIC of norfloxacin acting on ATCC11845 strain alone is 16 mug/mL; after the CPP-PNA-gyrA and norfloxacin were combined, MIC was 4. mu.g/mL for RA CH-1 strain, 16. mu.g/mL for RA CH-2 strain, and 8. mu.g/mL for ATCC11845 strain. The test results show that: after the CPP-PNA-gyrA and norfloxacin act together, MIC values of RA CH-1 strains, RA CH-2 strains and ATCC11845 strains are all reduced, and meanwhile, non-targeted CPP-PNA (mismatched CPP-PNA) has no effect.
CPP-PNA-gyrA combined with norfloxacin to affect post-OD of different serotypes of RA600Determination of value
Placing the 96-well plate for measuring MIC into a microplate reader for OD600Measurement of (2), Observation of OD600Analyzing the bacteriostatic effect of the CPP-PNA-gyrA. OD after different serotypes RA of CPP-PNA-gyrA combined norfloxacin action600The measurement results of the values are shown in fig. 1 and table 3.
TABLE 3 OD after effect of CPP-PNA-gyrA in combination with norfloxacin on different serotypes RA600Measurement result of (2)
As can be seen from Table 3, the combined action of CPP-PNA-gyrA and norfloxacin resulted in OD of norfloxacin acting on RA CH-1 strain, compared to the sole action of norfloxacin600The value is reduced from 0.36 to 0.08 on average; OD of norfloxacin after acting on RACH-2 strain600The value is reduced from 0.26 to 0.10 on average; OD of norfloxacin after acting on ATCC11845 strain600The value is reduced from 0.35 to 0.11 on average. The test results show that: after the CPP-PNA-gyrA and norfloxacin co-act, the OD was added to RACH-1 strain, RACH-2 strain and ATCC11845 strain600The values are all reduced, and the antibacterial effect is extremely obvious.
Colony count after interaction of CPP-PNA-gyrA with norfloxacin on different serotypes RA
Counting bacterial colonies of the bacterial liquid, taking a hole corresponding to the MIC value, and carrying out 10 times of counting-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8The diluted sample was added 100. mu.L to a cooled agar plate, incubated at 37 ℃ for 24 hours, and then subjected to colony counting. 3 replicates were set for each strain and averaged. The amount of bacteria per mL of sample was the average number of colonies multiplied by the dilution factor and reported as CFU/mL, and the results are shown in FIG. 2 and Table 4.
TABLE 4 colony count results of CPP-PNA-gyrA combined with norfloxacin after effect on different serotypes RA
As shown in FIG. 2 and Table 4, the combined action of CPP-PNA-gyrA and norfloxacin resulted in a colony count of 7.1 × 10 after the action of norfloxacin on RA CH-1 strain, as compared to the action of norfloxacin alone7CFU/mL is reduced to 1.6 × 107CFU/mL, the number of colonies after the norfloxacin acts on the RA CH-2 strain is 5.3 × 107CFU/mL is reduced to 1.9 × 107CFU/mL, the number of colonies after norfloxacin action on ATCC11845 strain was 7.0 × 107CFU/mL is reduced to 2.2 × 107CFU/mL. The test results show that: after the CPP-PNA-gyrA and norfloxacin act together, the colony values of RA CH-1 strains, RA CH-2 strains and ATCC11845 strains are all reduced, and the antibacterial effect is very obvious.
Effect of CPP-PNA-gyrA on the transcription level of gyrA Gene
(1) Construction of the Standard Curve
RA CH-1 strain genomic DNA was extracted as a template, and a Real-Time qPCR-specific primer was used to construct a standard curve for RAgyrA (GyrA-F: 5'-acttaccatatctcacgcagg-3' (SEQ ID NO. 3); GyrA-R: 5'-tcgtctcttgtagatgccg-3' (SEQ ID NO. 4)). For the initial concentration of template, first dilute it to an appropriate concentration, then 10 times serial dilution of the concentration, total 6 dilutions, each gradient 4 repeats. The reaction system is 2 mu LDNA template and 10 mu L
PremixExTaqII (TliRNaseH plus), 0.8. mu.L of gyrA upstream primer, 0.8. mu.L of gyrA downstream primer, 6.4. mu.L of RNase Free dH2And O. Reaction procedure: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 3s, annealing at 60 ℃ and extension for 30s, 39 cycles; the reaction was carried out at 65 ℃ for 5 seconds while verifying the specificity of the primers by observing the melting curve and the standard curve. The results showed that the amplification efficiency was 101%, and the method had high specificity and could be used for quantitative detection.
(2) Extraction of Total RNA
After the 96-well plate for measuring MIC has bacteriostatic action for 16-20h, sampling corresponding holes of different RA according to the action of CPP-PNA-gyrA, placing the samples in a 2mL RNA-free enzyme centrifuge tube, carrying out ice bath for 5min, then centrifuging the bacterial liquid, carrying out the program of 3000rpm and 10min, discarding the supernatant, washing with sterile 1 XPBS to remove redundant culture medium, collecting bacterial precipitates, then operating according to the instruction of an RNAiosolplus kit, and finally taking the extracted RNA as a reverse transcription template. The concentration and purity of the extracted total RNA were determined by using a NANODROP2000 ultramicro nucleic acid protein analyzer.
(3) Removal of gDNA from total RNA and reverse transcription
Referring to the instruction of PrimeScriptTMRT reagent Kit with the DNA Eraser (PerfectRealTime) of Takara bioengineering (Dalian) Co., Ltd, firstly removing the genomic DNA in the total RNA, wherein the reaction condition is 42 ℃ and 5 min; then carrying out fluorescent quantitative PCR (polymerase chain reaction) under the reaction conditions of 37 ℃ for 15min and 85 ℃ for 10s to obtain cDNA, and storing the obtained cDNA at-80 ℃.
(4) Real-Time qPCR reaction
The qPCR reaction was carried out using the fluorescent quantitative specific primers for the gyrA gene and the internal reference gene, recA, using cDNA as a template (recA-F: 5'-tgaaactaggtgatggtacg-3' (SEQ ID NO. 5); recA-R: 5'-cttaggataaccgcctactc-3' (SEQ ID NO. 6)). The reaction program is pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 3s, annealing at 60 ℃ and extension for 30s, 39 cycles; reaction at 65 ℃ for 5 s. Using recA gene as an internal reference, the relative transcription levels of RA CH-1, RA CH-2 and ATCC11845 strains were analyzed by means of Bio-RadcFX manager (version3.0) software. Each sample to be tested was set to 4 replicates and three independent replicates were performed, statistical significance of the test data was analyzed using unpaired t-test, plotted using Graphpad prism7.0 software, and the results are shown in fig. 3 and table 5.
TABLE 5 Effect of CPP-PNA-gyrA on transcription levels of different serotype RA gyrA genes
The results showed that the transcription level of the gyrA gene of the RA CH-1 strain was determined at 3.1. mu.M for CPP-PNA-gyrA2 times of the down regulation is carried out, 5 times of the down regulation is carried out when the concentration is 12.5 mu M, and the difference is very obvious; the CPP-PNA-gyrA is regulated down 2 times to the gyrA gene transcription level of the RA CH-2 strain at 6.2 mu M and regulated down 3 times at 12.5 mu M, and the difference is very obvious; CPP-PNA-gyrA downregulates the gyrA gene transcription level of ATCC11845 strain by 4 times when the concentration is 3.1-12.5. mu.M, and the difference is very obvious. The results show that the CPP-PNA-gyrA can down-regulate the transcription levels of the gyrA genes of RA CH-1 strains, RA CH-2 strains and ATCC11845 strains when the concentration is more than 6.2 mu M, and the difference is very obvious. But probably due to the bacterial liquid content 10 used in MIC test5CFU/mL, too high concentration, although to the target gene level down-regulated, but not reach bacteriostatic or bactericidal concentration, so alone does not have obvious effect.
Effect of CPP-PNA-gyrA on inhibiting riemerella anatipestifer
The concentration of CPP-PNA-gyrA is set to be 20 mu M, RA CH-1 strain, RA CH-2 strain and ATCC11845 strain are respectively diluted to 2000CFU/mL and 200CFU/mL, norfloxacin is adopted as a positive control, the specific operation is the same as that of the MIC test, then colony counting is carried out, the result is observed after 24 hours, whether the CPP-PNA-gyrA used alone has the antibacterial effect after the bacteria liquid content is reduced is detected, and the result is shown in figure 4 and table 6.
TABLE 6 inhibitory effect of CPP-PNA-gyrA on different serotypes of RA
The result shows that when the content of RA bacterial liquid is 2000CFU/mL and 200CFU/mL, the influence of CPP-PNA-gyrA on the growth of RA CH-1 strain is small and can be ignored; there was little effect on RA CH-2 strain and ATCC11845 strain. The above results indicate that CPP-PNA-gyrA alone acts on RA CH-1, RA CH-2 and ATCC11845 strains without exerting bacteriostatic effect even when the bacterial liquid content is low.
Detection of transcription level of RA gyrA downstream gene RA 0C-0528 by CPP-PNA-gyrA
The structural repeat lipid (TPR) protein is regulated by DNA binding sites on the genome. This protein occurs in only one transmembrane histidine kinase and DNA binding reactor, and the interaction of TPR protein regulates PEP-CTERM protein expression. The position downstream of gyrA on the genome, transcribed in the same direction. The nucleotide sequence similarity of the TPR protein (RA0C _0528) of RA is more than 94%, and the protein level similarity reaches 99.33%, so that the influence of gyrA on the expression of downstream genes is determined by observing the transcription level of the downstream RA0C _0528 gene of gyrA, and whether the effect of CPP-PNA-gyrA on RA is the result of targeting gyrA is judged.
(1) Construction of Standard protocols
RA CH-1 strain genomic DNA was extracted as a template, and a Real-Time qPCR specific primer (RA 0C-0528-F: 5'-gatacatcaagagaaccctcct-3' (SEQ ID NO. 7); RA 0C-0528-R: 5'-caaatactctcaaccagtagca-3' (SEQ ID NO.8)) was used to construct a standard curve. For the initial concentration of template, first dilute it to an appropriate concentration, then 10 times serial dilution of the concentration, total 6 dilutions, each gradient 4 repeats. The reaction system is 2 mu L of template,
PremixExTaqII (TliRNaseH plus), 0.8. mu.L of the forward primer, 0.8. mu.L of the reverse primer, 6.4. mu.L of RNaseFreedH2And O. The reaction program is pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 3s, annealing at 60 ℃ and extension for 30s, 39 cycles; reaction at 65 ℃ for 5 s. Meanwhile, the specificity of the primer is verified by observing an amplification curve, the obtained amplification efficiency is 103%, and the method has high specificity and can be used for quantitative detection.
The quantitative primers RA 0C-0528-F and RA 0C-0528-R were used for detection, and recA was used as an internal reference, and the detection results are shown in FIG. 5 and Table 7.
TABLE 7 detection of transcript levels of different serotypes RA0C _0528 by CPP-PNA-gyrA
The results show that CPP-PNA-gyrA has no influence on the transcription level of RA gyrA downstream gene RA0C _0528 at 4h, 8h, 12h, 16h, 20h and 24h, and the bacterial content is 2000CFU/mL and 200 CFU/mL. Meanwhile, the CPP-PNA-gyrA is also shown to specifically inhibit the transcription of gyrA gene and enhance the antibacterial effect of antibiotics through the combined action with quinolone antibiotics norfloxacin.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Sequence listing
<110> Sichuan university of agriculture
Application of gyrA gene PNA combined antibiotic combined with cell-penetrating peptide in preparation of drug for inhibiting riemerella anatipestifer
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<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 (5)
1. Binding of cell-penetrating peptidesgyrAThe application of the gene PNA and antibiotic combination in preparing the drug for inhibiting the riemerella anatipestifer comprises the following steps: characterized in that the antibiotic is ciprofloxacin or norfloxacin.
2. Use according to claim 1, characterized in that: the above-mentionedgyrAThe sequence of the gene PNA is shown in SEQ ID NO. 1.
3. Use according to claim 1, characterized in that: the sequence of the cell-penetrating peptide is shown in SEQ ID NO. 2.
4. Use according to claim 1, characterized in that: said binding of cell-penetrating peptidesgyrAThe gene PNA is 5 '-FITC-OO-KFFKFFKFFK-OO-CGGTTGCCCACTCC-3', wherein FITC is a fluorescent label, K is lysine, F is phenylalanine, O is glycine, and OO is used for connecting O linker with groups to enhance solubility.
5. Use according to claim 1, characterized in that: the riemerella anatipestifer is riemerella anatipestifer CH-1 strain, riemerella anatipestifer CH-2 strain and riemerella anatipestiferATCC 11845And (4) strain.
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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 |
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| CN105176884A (en) * | 2015-10-19 | 2015-12-23 | 山东省农业科学院家禽研究所 | Method for mixed culture and re-separation of riemerella anatipestifer and escherichia coli |
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| CN105176884A (en) * | 2015-10-19 | 2015-12-23 | 山东省农业科学院家禽研究所 | Method for mixed culture and re-separation of riemerella anatipestifer and escherichia coli |
Non-Patent Citations (2)
| Title |
|---|
| Inhibition of Growth and Gene Expression by PNA-peptide Conjugates in Streptococcus pyogenes;Nadja Patenge 等;《Molecular Therapy—Nucleic Acids》;20131105;第2卷;1-9 * |
| γPNA一种新型高效的肽核酸;邱浩等;《中国生物工程杂志》;20180215;第38卷(第02期);75-81 * |
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