CN110200966B - Application of G4 ligand TMPyP4 in preparation of medicine for resisting porcine reproductive and respiratory syndrome virus - Google Patents

Application of G4 ligand TMPyP4 in preparation of medicine for resisting porcine reproductive and respiratory syndrome virus Download PDF

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CN110200966B
CN110200966B CN201910574098.4A CN201910574098A CN110200966B CN 110200966 B CN110200966 B CN 110200966B CN 201910574098 A CN201910574098 A CN 201910574098A CN 110200966 B CN110200966 B CN 110200966B
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tmpyp4
respiratory syndrome
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井汇源
张艳
董望
王金合
曹素芳
孙彦婷
王海花
刑召
包文奇
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Henan University of Animal Husbandry and Economy
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
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Abstract

The invention discloses application of G4 ligand TMPyP4 in preparing a medicament for resisting porcine reproductive and respiratory syndrome virus and in preparing a medicament for preventing and treating porcine reproductive and respiratory syndrome. The invention adopts Real-Time PCR experiment, Western Blot experiment and TCID 50 The effect of tmpypp 4 on PRRSV was verified. Experiments show that TMPyP4 can inhibit the proliferation of PRRSV on PAMs cells, is expected to become a novel medicament for preventing and treating porcine reproductive and respiratory syndrome, and has good application prospect in the prevention and treatment of porcine reproductive and respiratory syndrome.

Description

Application of G4 ligand TMPyP4 in preparation of medicine for resisting porcine reproductive and respiratory syndrome virus
Technical Field
The invention relates to an application of G4 ligand TMPyP4 in preparation of a medicament for resisting porcine reproductive and respiratory syndrome virus, belonging to the technical field of veterinary medicines.
Background
In the late eighties of the twentieth century, Porcine Reproductive and Respiratory Syndrome (PRRS) was first reported in the united states. The disease is clinically characterized by abortion in pregnant sows, stillbirth, mummy and weak piglets, and respiratory diseases. Because Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is highly pathogenic and is easy to recombine, the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) attracts people to pay attention. PRRSV infected target cells are Porcine Alveolar Macrophages (PAMs), which cause immune function impairment, are prone to mixed infection and secondary infection, and bring serious economic loss to the swine industry worldwide. PRRSV was circulating in China since 1995, representing the strain CH-1a, and is a American type PRRSV-2. Since 2006, highly pathogenic PRRSV (HP-PRRSV) has seen a large pandemic in China, with over two million live pigs infected, representative strains including JXA1, HUN4, etc.
PRRSV is classified as belonging to the order of the nested virus, the family of arteriviruses. PRRSV is a enveloped, single-stranded, positive-stranded RNA virus with a genome of approximately 15kb in length. The process of PRRSV replication first utilizes genomic RNA (grna) as a template to synthesize a negative strand RNA intermediate. Then, using the negative strand RNA intermediate as a template, progeny genomic RNA of the positive strand and a series of subgenomic RNAs (sgmRNAs) were synthesized. These subgenomic RNAs (sgmRNAs) contain open reading frames such as ORF1a, ORF1b, ORF2a, ORF2b, and ORF3-ORF 7. Because the PRRSV nucleic acid sequence is easy to generate variation, the conventional medicament is difficult to form effective protection, and no specific medicament exists for highly pathogenic strains (HP-PRRSV).
The G quadruplex (G4) is a special secondary structure formed by nucleic acid rich in guanine base (G) under certain conditions. In the presence of the G4 ligand, intrachain or interchain G can form a stable G4 structure via a Hoogsteen bond. Studies have shown that G4 structure is associated with the regulation of telomere function and cancer. The G4 ligand-TMPyP 4 can inhibit telomerase activity and inhibit tumor cell proliferation by combining with a G-rich region. The function of TMPyP4 in PRRSV proliferation is not clear at present, and the relevant literature is not disclosed.
Disclosure of Invention
The invention aims to provide the application of a novel substance, namely G4 ligand TMPyP4, in the preparation of a medicament for resisting porcine reproductive and respiratory syndrome virus, aiming at the current situation that PRRSV (porcine reproductive and respiratory syndrome Virus) is lack of current antiviral medicaments, wherein the TMPyP4 has a good antiviral effect on the PRRSV and has a good application prospect in the aspect of PRRS prevention and treatment.
In order to achieve the purpose, the invention adopts the technical scheme that:
the G4 ligand TMPyP4 is applied to the preparation of the medicine for resisting the porcine reproductive and respiratory syndrome virus.
The structural formula of the G4 ligand TMPyP4 is shown below:
Figure BDA0002111630030000021
the porcine reproductive and respiratory syndrome virus is a highly pathogenic PRRSV strain HP-PRRSV.
A medicament against porcine reproductive and respiratory syndrome virus comprising an effective dose of G4 ligand TMPyP 4.
The effective dosage of the G4 ligand TMPyP4 is 10-20. mu.M.
The medicine is spray, injection, tablet, capsule or granule.
The G4 ligand TMPyP4 is applied to the preparation of the medicine for preventing and treating the porcine reproductive and respiratory syndrome.
A medicament for preventing and treating porcine reproductive and respiratory syndrome comprises an effective dose of G4 ligand TMPyP 4.
The effective dosage of the G4 ligand TMPyP4 is 10-20. mu.M.
The medicine is spray, injection, tablet, capsule or granule.
The invention has the beneficial effects that:
the invention discloses application of G4 ligand-TMPyP 4 in preparing a medicament for resisting Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and in preparing a medicament for preventing and treating porcine reproductive and respiratory syndrome. The invention adopts Real-Time PCR experiment, Western Blot experiment and TCID 50 The effect of tmpypp 4 on PRRSV was verified. Experiments show thatOn PAMs cells, TMPyP4 can inhibit the proliferation of PRRSV, is expected to become a novel medicament for preventing and treating porcine reproductive and respiratory syndrome, and has good application prospect in the aspect of preventing and treating the porcine reproductive and respiratory syndrome.
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Figure 1 shows the results of the tmpypp 4 cytotoxicity assay.
Figure 2 is a graph of the inhibitory effect of tmpypp 4 on PRRSV proliferation at the RNA level on PAMs cells.
Figure 3 shows the inhibitory effect of TMPyP4 on PRRSV N protein synthesis on PAMs cells.
Figure 4 is a graph of the inhibitory effect of tmpypp 4 on PRRSV titre on PAMs cells.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention, but the present invention is not limited thereto in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Statistical analysis of the following examples of the invention: all experiments were repeated at least 3 times independently, with results expressed as mean and standard error, using one-way analysis of variance and t-test analysis. All statistical analyses used P <0.01 as a test standard with very significant statistical differences, the analysis software was GraphPad Prism 5.
Example 1: TMPyP4 cytotoxicity assay
1X 10 per hole 4 Each of the PAMs cells was seeded in 96-well plates in RPMI-1640 medium containing 10% (v/v) fetal bovine serum. After 24h the medium was discarded and replaced with 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M and 80. mu.M TMPyP4 containing maintenance medium (RPMI-1640 medium containing 2% (v/v) fetal bovine serum) at 200. mu.l per well, in 3 replicates per concentration. A DMSO cell control (i.e., 0. mu.M TMPyP 4) supplemented with a 2% (v/v) DMSO-containing maintenance solution was also set. 37 ℃ and 5% CO 2 After further culturing for 3 days in the incubator, 20. mu.l of MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide) solution (dissolved in PBS) with the concentration of 5mg/ml is added into each hole, and the incubatorAfter 4h incubation. The culture supernatant was aspirated off the wells, 150. mu.l DMSO was added to each well, and the blue crystals of the MTT reaction were fully dissolved. The 570nm wavelength is selected, the absorbance value of each hole is measured on an enzyme linked immunosorbent, and the result is recorded. The cell activity of the DMSO cell control group is taken as 100%, and the reading of the cells of the TMPyP4 treatment group with different concentrations is compared with the reading of the DMSO cell control group to obtain the relative cell activity.
The results are shown in FIG. 1: at concentrations below 20 μ M TMPyP4, it was not toxic to PAMs cells. Even when the concentration of TMPyP4 is higher than 40. mu.M and reaches 80. mu.M, the toxicity to cells is very low, and the cell activity reaches more than 90%.
Example 2: inhibitory Effect of TMPyP4 on PRRSV proliferation at RNA level on PAMs cells
PAMs cells were cultured in six-well plates in RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin and streptomycin. After 24h the medium was discarded and PRRSV (HP-PRRSV strain) with MOI ═ 0.1 was inoculated. Using RPMI-1640 medium (200. mu.l per well) containing 2% (v/v) fetal bovine serum and 1% (v/v) penicillin and streptomycin, at 5% CO 2 Culturing for 2h at 37 ℃ in a constant-temperature incubator. The medium was discarded, washed twice with PBS, and cultured in RPMI-1640 medium (200. mu.l per well) containing TMPyP4, 2% (v/v) fetal bovine serum at concentrations of 0. mu.M, 5. mu.M, 10. mu.M, and 20. mu.M, respectively, in 5% CO 2 Culturing at 37 deg.C for 36 h. The six-well plate was repeatedly frozen and thawed at-80 ℃ and 4 ℃ for 3 times to fully lyse the cells, each lysate was collected, total RNA was extracted, and reverse transcription was immediately performed. A reverse transcription Kit (Transcriptor First Strand cDNA Synthesis Kit) was purchased from Roche.
Step one, a reaction system: 1.0 μ L oligo (dT) 18 Primer, 1. mu.g total RNA, 12.0. mu.L RNase-free H 2 And O. Reaction conditions are as follows: 10min at 65 ℃.
Step two, reaction system: 4.0 μ L of 5 XT Buffer, 2.0 μ L of Deoxynucleotide Mix, 0.5 μ L of Protector RNase Inhibitor, 0.5 μ L of Transcriptor Reverse Transcriptase. And mixing the above system with the first reaction system. Reaction conditions are as follows: 30min at 55 ℃ and 5min at 85 ℃.
And (3) performing Real-Time PCR amplification by using the cDNA obtained by reverse transcription as a template and beta-actin as an internal reference gene and using primers to detect the copy number of the N gene of the PRRSV. Real-time PCR reagents (SYBR qPCR Mix) were purchased from ABI. Reaction system: 5.0. mu.L of 2 XSSYBR qPCR Mix, 4.0. mu.L of RNase-free H 2 O, 0.25. mu.L of forward primer (10. mu.M), 0.25. mu.L of reverse primer (10. mu.M), 0.5. mu.L of cDNA. Reaction conditions are as follows: the pre-denaturation reaction was carried out at 95 ℃ for 10min and then 40 cycles were performed. The cycling reactions were 94 ℃ for 10s, 56 ℃ for 15s, and 72 ℃ for 15 s.
The upstream and downstream primer sequences of the N gene are as follows:
N-F：5’-AAACCAGTCCAGAGGCAA-3’(SEQ ID NO.1)
N-R：5’-CGGCAAACTAAACTCCACA-3’(SEQ ID NO.2)
the upstream and downstream primer sequences of the beta-actin gene are as follows:
β-actin-F：5’-TGAGAACAGCTGCATCCACTT-3’(SEQ ID NO.3)
β-actin-R：5’-CGAAGGCAGCTCGGAGTT-3’(SEQ ID NO.4)
the test results are shown in figure 2, and show that the TMPyP4 of the invention has obvious inhibition effect on RNA synthesis of PRRSV on PAMs cells in the concentration range of 10-20 mu M.
Example 3: inhibitory Effect of TMPyP4 on PRRSV N protein Synthesis on PAMs cells
PAMs cells were cultured in six-well plates in RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin and streptomycin. After 24h the medium was discarded and PRRSV (HP-PRRSV strain) with MOI ═ 0.1 was inoculated. Using RPMI-1640 medium (200. mu.l per well) containing 2% (v/v) fetal bovine serum and 1% (v/v) penicillin and streptomycin, at 5% CO 2 Culturing for 2h at 37 ℃ in a constant-temperature incubator. The medium was discarded, washed twice with PBS, and cultured in RPMI-1640 medium (200. mu.l per well) containing TMPyP4, 2% (v/v) fetal bovine serum at concentrations of 0. mu.M, 5. mu.M, 10. mu.M, and 20. mu.M, respectively, in 5% CO 2 Culturing at 37 deg.C for 36 h. The 6-well plate medium was discarded, washed twice with pre-cooled PBS, 120. mu.l of cell lysate was added to each well, shaken well, allowed to stand on ice for 5min, and cells were scraped off with a cell scraper for Western Blot. Determining eggs in a sampleWhite concentration for SDS-PAGE.
SDS-PAGE electrophoresis: the loading was determined by protein concentration, 20. mu.g of sample per well. And (3) switching on a power supply, adjusting the voltage to 80V, adjusting the voltage to 120V when the bromophenol blue indicator runs to the separation gel, and ending electrophoresis when the bromophenol blue indicator runs to the bottom of the separation gel (about 2 h).
Film transfer: the SDS-PAGE gel was directly subjected to charge Transfer of the protein to a PVDF membrane using Mini Trans-Blot Electrophoretic Transfer Cell Transfer apparatus from Bio-RAD. The specific operation is as follows: a small amount of a transmembrane buffer was added to a tray, and the filter paper and sponge were soaked therein. PVDF membrane was also transferred to the transfer membrane buffer after 2min immersion in methanol. Sequentially laying sponge and filter paper on a glass plate, removing bubbles by using a glass rod, lightly putting the gel after electrophoresis on the filter paper, removing the bubbles by using the glass rod, sticking a PVDF film, laying the filter paper and the sponge, and removing the bubbles by using the glass rod. The whole is transferred to a transfer film clip. Rotating the film for 3-4h under the voltage of 40V, and putting an ice bag around the film.
PVDF membrane was spread flat in 5% (w/v) BSA containing blocking solution with tweezers and incubated for 2h on a shaker. TBST washing 3 times, each time for 5 min. The washing solution was decanted off, the primary antibody solution (diluted 1: 500 PRRSV N protein monoclonal antibody, diluted 1: 2000 internal reference beta-actin) was added and incubated on a shaker for 4 h. TBST washing 3 times, each for 5 min. The rinse was decanted off and secondary antibody solution (FITC labeled goat anti-mouse secondary antibody 1: 3000 diluted) was added and incubated for 2 h. And (3) putting the PVDF membrane into a chemiluminescence substrate mixed solution for lucifugal color development, and imaging by using a chemiluminescence imaging system.
The test results are shown in figure 3, and show that the TMPyP4 of the invention has obvious inhibition effect on the synthesis of N protein of PRRSV on PAMs cells in the concentration range of 5-20 mu M and shows dose dependence. At a concentration of 20. mu.M, no N protein synthesis could be detected with TMPyP 4.
Example 4: inhibitory Effect of TMPyP4 on PRRSV titers on PAMs cells
PAMs cells were cultured in six-well plates in RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin and streptomycin. After 24h the medium was discarded and the PRRSV (HP-PRR) with an MOI of 0.1 was addedSV strain). Using RPMI-1640 medium (200. mu.l per well) containing 2% (v/v) fetal bovine serum and 1% (v/v) penicillin and streptomycin, at 5% CO 2 Culturing for 2h in a constant temperature incubator at 37 ℃. The medium was discarded, washed twice with PBS, and cultured in RPMI-1640 medium (200. mu.l per well) containing TMPyP4, 2% (v/v) fetal bovine serum at concentrations of 0. mu.M, 5. mu.M, 10. mu.M, and 20. mu.M, respectively, in 5% CO 2 Culturing at 37 deg.C for 36 h. Repeatedly freezing and thawing six-hole plate at-80 deg.C and 4 deg.C for 3 times to fully lyse cells, collecting each lysate, and treating with TCID 50 The method detects the PRRSV titer.
TCID 50 The detection method comprises the following steps: the samples to be tested were serially diluted 10-fold in 1.5mL EP tubes using DMEM medium, i.e., 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 And the gun head needs to be replaced when liquid is transferred in the dilution process. Each dilution of the virus solution was inoculated into 96-well cell microplates in columns 1-10, 8 wells each, and 100. mu.L each. Columns 11-12 were controlled by adding an equal amount of DMEM medium. MARC-145 cells in 96-well plates had confluent monolayers. Placing 96-well cell micro-culture plate at 37 deg.C and 5% CO 2 The cells were cultured in an incubator for 72h, and the number of lesion holes produced by the cells was recorded. Calculating TCID of virus to be detected according to Reed-Muench method 50 The value is obtained.
The test results are shown in figure 4, and show that the TMPyP4 has obvious inhibition effect on the proliferation titer of PRRSV on PAMs cells in the concentration range of 10-20 mu M.
Sequence listing
<110> Henan animal husbandry economic school
Application of <120> G4 ligand TMPyP4 in preparation of drugs for resisting porcine reproductive and respiratory syndrome virus
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence ()
<400> 1
aaaccagtcc agaggcaa 18
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence ()
<400> 2
cggcaaacta aactccaca 19
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence ()
<400> 3
tgagaacagc tgcatccact t 21
<210> 4
<211> 18
<212> DNA
<213> Artificial sequence ()
<400> 4
cgaaggcagc tcggagtt 18

Claims (4)

  1. Use of G4 ligand TMPyP4 for the manufacture of a medicament against porcine reproductive and respiratory syndrome virus, wherein the medicament comprises an effective dose of 10-20 μ M of G4 ligand TMPyP 4; the medicine is spray, injection, tablet, capsule or granule.
  2. 2. The use according to claim 1, wherein the G4 ligand TMPyP4 has the formula:
    Figure DEST_PATH_IMAGE002
    。
  3. 3. the use according to claim 1, wherein the porcine reproductive and respiratory syndrome virus is the highly pathogenic PRRSV strain HP-PRRSV.
  4. The application of G4 ligand TMPyP4 in preparing a medicament for preventing and treating porcine reproductive and respiratory syndrome, which is characterized in that the medicament comprises G4 ligand TMPyP4 with the effective dose of 10-20 mu M; the medicine is spray, injection, tablet, capsule or granule.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008006085A3 (en) * 2006-07-07 2009-04-09 Univ Washington Compositions and methods for predicting inhibitors of protein targets

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008006085A3 (en) * 2006-07-07 2009-04-09 Univ Washington Compositions and methods for predicting inhibitors of protein targets

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"A highly conserved G-rich consensus sequence in hepatitis C virus core gene represents a new anti-hepatitis C target;Shao-Ru Wang et al.，;《Sci Adv》;20160430(第2期);第1-12页,尤其是第3页右栏最后一段 *
DexD/h-Box helicase 36 signaling via Myeloid Differentiation Primary response gene 88 contributes to NF-kappa B activation to Type 2 Porcine reproductive and respiratory syndrome Virus infection;Jing,HY et al.,;《Frontiers in Immunology》;20171023(第8期);第1-9页，尤其是摘要和第2页左栏第3段 *
阳离子卟啉对膀胱肿瘤细胞端粒酶活性抑制作用的研究;邱学德等;《昆明医学院学报》;20101231(第4期);第15-19页，尤其是第16页右栏1.3MTT测定细胞抑制率 *

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