CN117919378A - Application of gnat banna host defensin peptide Siba3 - Google Patents
Application of gnat banna host defensin peptide Siba3 Download PDFInfo
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- CN117919378A CN117919378A CN202311801101.4A CN202311801101A CN117919378A CN 117919378 A CN117919378 A CN 117919378A CN 202311801101 A CN202311801101 A CN 202311801101A CN 117919378 A CN117919378 A CN 117919378A
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Classifications
-
- 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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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses an application of a gnat host defensin peptide Siba. The application of the Banana gnat host defense peptide Siba in preparing a drug for resisting Zika virus infection diseases; the amino acid sequence of the gnat host defense peptide Siba is as follows: KINKQKIKNGAKKALGVASKVAPVVAAFAR-NH 2. The invention takes host defense peptide Siba from gnat banna as a research object, and aims to elucidate the role of Siba3 in ZIKV infection. We have found that Siba can significantly inhibit ZIKV infection in vitro and also can significantly inhibit ZIKV infection in vivo, and has both prevention and treatment effects. Our results show that Siba can directly act on ZIKV, destroy viral envelope, induce viral genome to leak, thereby inactivating viral particles; siba3 can also reduce the susceptibility of host cells to Zika virus. The combination of the molecular weight is small, the structure is simple, and therefore, the method has wide development and application prospects.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of a gnat banna host defensin peptide Siba.
Background
Zika virus (ZIKV) is a single-stranded positive strand RNA virus belonging to the flaviviridae family, enveloped. The main transmission mode of ZIKV is mosquito medium transmission, and is mainly transmitted through aedes bites. In addition, ZIKV can also be transmitted by sexual pathways, maternal and infant vertical transmission and transfusion pathways.
ZIKV infection can cause a variety of serious neurological and reproductive diseases that can lead to severe complications such as guillain-barren syndrome, neonatal "microcephaly", fetal abortion or death, male sterility, etc. In addition, ZIKV infection can also cause blindness and a variety of ocular abnormalities, including retinal spots, subluxation of the lens, and optic neuritis. However, there is currently no clinically safe and effective vaccine or drug to prevent and treat ZIKV infection. Therefore, there is a need to develop safe and effective medicaments for preventing and treating ZIKV infections.
Disclosure of Invention
The invention aims at providing an application of a Banana gnat host defense peptide Siba.
The purpose of the invention is realized in such a way that the application of the Banana gnat host defense peptide Siba3 in the preparation of the anti-Zika virus infection disease medicine; the amino acid sequence of the gnat host defense peptide Siba is as follows: KINKQKIKNGAKKALGVASKVAPVVAAFAR-NH 2.
Host defense peptides (host DEFENSE PEPTIDES, HDPs), including antimicrobial peptides (antimicrobial peptides, AMPs), are a class of naturally occurring immune small molecule polypeptides encoded by specific genes of biological cells, widely expressed in animals and plants. The antibacterial peptide is taken as an important effector molecule of natural immunity of vertebrates, can directly kill bacteria and regulate the immune response of organisms. In recent years, more and more researches show that the polypeptide has broad-spectrum efficient antibacterial action and immune regulation action, and can also play an antiviral role by destroying viral envelope, regulating host antiviral immune response and the like. Thus, antimicrobial peptides have become an important source for screening antiviral polypeptides.
The invention takes host defense peptide Siba (the amino acid sequence is KINKQKIKNGAKKALGVASKVAPVVAAFAR-NH 2) from gnat in Banana as a research object, and aims to elucidate the role of Siba3 in ZIKV infection. We have found that Siba can significantly inhibit ZIKV infection in vitro and also can significantly inhibit ZIKV infection in vivo, and has both prevention and treatment effects. Our results show that Siba can directly act on ZIKV, destroy viral envelope, induce viral genome to leak, thereby inactivating viral particles; siba3 can also reduce the susceptibility of host cells to Zika virus. The combination of the molecular weight is small, the structure is simple, and therefore, the method has wide development and application prospects.
Drawings
FIG. 1 is a schematic representation of a cytotoxicity assay of the Banana gnat host defensin peptide Siba according to the present invention;
FIG. 2 is a schematic representation of cytopathic effects of the inhibition of ZIKV infection by the Banana gnat host defensin peptide Siba of the present invention;
Fig. 3 is a graph showing the percentage of the pongnat host defensin Siba3 inhibiting the Vero cytopathic effect induced by ZIKV infection as described herein, p <0.01, p <0.001;
fig. 4 is a schematic diagram showing that the clamp's nano-rope host defensin peptide Siba3 according to the present invention inhibits the RNA synthesis of ZIKV in Vero cells, p <0.01, p <0.001;
FIG. 5 is a schematic representation of the reduction of viral titres in ZIKV infected Vero cell supernatants by Banana gnat host defenses peptide Siba according to the present invention, p <0.01, p <0.001;
Fig. 6 is a schematic diagram showing a functional study of the present invention of the pongnat host defensin Siba against a constant ZIKV infection in C57BL/6 mice with p <0.01 and p <0.001;
Fig. 7 is a schematic diagram showing the functional study of the tsunami host defense peptide Siba of the present invention for a consistent ZIKV infection in pregnant mice, where p <0.01 and p <0.001.
Detailed Description
The invention is further described below with reference to examples and figures, but is not limited in any way, and any alterations or substitutions based on the teachings of the invention are within the scope of the invention.
The application of the Banana gnat host defense peptide Siba3 disclosed by the invention is the application of the Banana gnat host defense peptide Siba3 in preparing a drug for resisting Zika virus infection diseases; the amino acid sequence of the gnat host defense peptide Siba is as follows: KINKQKIKNGAKKALGVASKVAPVVAAFAR-NH 2.
The invention is further illustrated by the following examples:
Example 1
Siba3 cytotoxicity.
We first examined Siba for toxicity to Vero cells. After the Vero cells are spread in a 96-well plate and are adhered, a series of Siba with double dilution concentration is added into the Vero cells by a double dilution method, so that the final concentration of Siba is 200, 100, 50, 25, 12.5 and 6.25mg/ml in sequence, and 3 compound wells are arranged in each concentration. Vero cells were incubated for a further 48 hours after Siba had been added, then the viability of the Vero cells was checked using a cell viability check kit (cell counting kit, CCK-8), 10 CCK-8 reagent ml was added to each well, incubated for 2-4 hours, and the absorbance at 450nm was checked using a microplate reader.
As shown in FIG. 1, the results of CCK-8 detection show that Siba3 still does not generate cytotoxicity to Vero cells at concentrations as high as 200 mg/ml, indicating that the newly synthesized batch of Siba3 is not cytotoxic to Vero cells in the several concentration ranges examined. This result is consistent with the result of Siba3 of our previous synthetic batch. Therefore, in the following cell antiviral experiments, siba was used at a concentration of less than 200 mg/ml.
Example 2
Siba3 inhibition of ZIKV infection-induced Vero cytopathy
ZIKV infection can induce Vero cytopathy. We evaluated Siba on the effect of ZIKV infection-induced Vero cytopathy. After plating Vero cells in 24-well plates and attaching cells, ZIKV (PRVABC 59) with a multiplicity of infection (multiplicity of infection, MOI) of 1 is added, 10, 20, 40 mg/ml of Siba are added, incubation is carried out for 2 hours at 37 ℃, virus particles which are not adsorbed in the culture supernatant are discarded, PBS is used for washing 3 times, DMEM culture medium containing 2% FBS is added, the same dose of Siba3 is added, and after continuous culture is carried out for 48 hours, cytopathic effect is observed under a microscope.
As shown in FIG. 2, the Vero cells of the PBS-treated group were significantly diseased after ZIKV infection compared to the normal control group (Sham) cells, while Siba of 10, 20, 40 mg/ml each inhibited ZIKV infection-induced Vero cytopathy and exhibited a dose-dependent effect.
We next examined the percent inhibition of Siba on ZIKV infection-induced Vero cytopathic effect (CPE) by CCK-8. We spread Vero cells in 96-well plates, after adherence, add ZIKV with MOI of 1, add Siba with MOI of 10, 20, 40 mg/ml, after incubation for 2 hours at 37 ℃, discard unadsorbed virus particles in culture supernatant, wash 3 times with PBS, add DMEM medium containing 2% FBS, and supplement Siba3 with the same dose, after continuing to culture for 48 hours, detect survival rate of Vero cells with CCK-8 kit, add CCK-8 reagent of 10 ml per well, after incubation for 2-4 hours, detect light absorption value at 450 nm with enzyme marker. The experiment was set up with 3 duplicate wells.
The results are shown in FIG. 3, and compared with uninfected Vero cells, after ZIKV is added, the PBS-treated Vero cells have obvious lesions, and the lesion rate reaches 77.1%. Compared with the Vero cells treated by PBS, the lesion rates of the Vero cells treated by Siba3 are obviously reduced, and the lesion rates of the Vero cells treated by Siba of 10, 20 and 40 mg/ml are respectively 45.7, 21.7 and 11.4 percent. This result is consistent with the inhibition of Vero cell lesions induced by ZIKV infection by Siba observed with an optical microscope, again indicating that Siba3 can inhibit lesions of Vero cells induced by ZIKV infection.
Example 3
Siba3 inhibition of viral RNA synthesis
Vero cells were plated in 12-well plates, after adherence, ZIKV with MOI of 1 was added, simultaneously Siba with MOI of 10, 20, 40 mg/ml was added, incubated at 37℃for 2 hours, unadsorbed virus particles in the culture supernatant were discarded, washed 3 times with PBS, then DMEM medium containing 2% FBS was added and supplemented with Siba3 at the same dose, after further incubation for 48 hours, the supernatant was discarded, total RNA of the cells was extracted with Trizol, and cDNA was synthesized in one strand using a reverse transcription kit, and RNA synthesis of the virus was detected by real-time fluorescent quantitative PCR (Q-PCR).
As shown in fig. 4, the level of ZIKV RNA synthesis in Vero cells in PBS-treated group was significantly increased after infection compared to uninfected Vero cells (Sham), whereas Siba3 could significantly inhibit viral RNA synthesis in Vero cells, and had dose-dependent effects of 85.3, 89.9, 93.0% after treatment with Siba3 at 10, 20, 40 mg/ml, respectively, in Vero cells.
Example 4
Siba3 reduces viral titres in ZIKV infected Vero cell supernatants
Spreading Vero cells in a 12-well plate, after adherence, adding ZIKV with MOI of 1, adding Siba with MOI of 10, 20 and 40 mg/ml, incubating at 37 ℃ for 2 hours, discarding unadsorbed virus particles in the culture supernatant, washing 3 times with PBS, adding DMEM culture medium containing 2% FBS, supplementing Siba3 with the same dose, continuing to culture for 48 hours, taking the supernatant, adding the supernatant into freshly plated Vero cells, and detecting the virus titer in the supernatant by using a virus plaque experiment.
As shown in FIG. 5, the virus titer in the supernatant of the Vero cells of the PBS-treated group after ZIKV addition was 3.3X10 5/ml, and after 10, 20, 40, mg/ml of Siba3, the virus titer in the supernatant of the Vero cells was 3.3X10 4/ml、3.3×105/ml、7.5×102/ml、1.7×102/ml, respectively, which revealed that the virus titer in the supernatant of the Vero cells after Siba3 addition was significantly reduced and had a dose-dependent effect.
Example 5
Functional study of Siba on inhibition of ZIKV infection in C57BL/6 mice
We infected male C57BL/6 mice with ZIKV of 10 6 PFU by tail vein injection, and after 2 h, siba3 (10 mg/kg) was tail vein injected into ZIKV infected mice. 4 days after infection, C57BL/6 mice were sacrificed by cervical dislocation, and the heart, liver, spleen, lung, kidney, brain, and the viral RNA levels in each tissue of the mice were examined by Q-PCR.
The results are shown in fig. 6, where the level of viral RNA in heart, liver, spleen, lung, kidney and brain was significantly reduced in Siba 3-treated mice compared to PBS-treated mice, indicating that Siba3 can inhibit ZIKV infection in C57BL/6 mice.
Example 6
Siba3 functional study of inhibiting ZIKV infection in pregnant mice
We infected C57BL/6 pregnant mice with ZIKV of 10 6 PFU by tail vein injection, and after 2h, siba3 (10 mg/kg) was injected by tail vein injection into ZIKV infected pregnant mice. 3 days after infection, C57BL/6 pregnant mice were sacrificed by cervical dislocation, the fetal mice and placenta were removed, and viral RNA levels in the fetal mice and placenta tissues were detected by Q-PCR.
The results are shown in fig. 7, where the levels of viral RNA in the placenta and fetal head of Siba 3-treated pregnant mice were significantly reduced compared to PBS-treated mice, indicating that Siba3 can inhibit ZIKV infection in C57BL/6 pregnant mice.
Claims (1)
1. The application of the gnat banna host defense peptide Siba is characterized in that the application of the gnat banna host defense peptide Siba3 in preparing a drug for resisting Zika virus infection diseases; the amino acid sequence of the gnat host defense peptide Siba is as follows: KINKQKIKNGAKKALGVASKVAPVVAAFAR-NH2.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311801101.4A CN117919378A (en) | 2023-12-26 | 2023-12-26 | Application of gnat banna host defensin peptide Siba3 |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311801101.4A CN117919378A (en) | 2023-12-26 | 2023-12-26 | Application of gnat banna host defensin peptide Siba3 |
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| CN117919378A true CN117919378A (en) | 2024-04-26 |
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| CN202311801101.4A Pending CN117919378A (en) | 2023-12-26 | 2023-12-26 | Application of gnat banna host defensin peptide Siba3 |
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| CN (1) | CN117919378A (en) |
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