CN112472821A - Application of protein nanoparticles in preparation of VSV (VSV virus) resistant product - Google Patents

Abstract

The invention relates to application of protein nanoparticles in preparation of products for inhibiting VSV virus replication and/or resisting VSV virus, wherein the protein nanoparticles are formed by self-assembly of protein monomers induced by metal ions, and the protein monomers comprise fusion proteins formed by sequentially connecting metallothionein, connecting peptide and glutathione S-transferase in an order from an amino terminal to a carboxyl terminal. The protein nano-particle can remarkably improve the levels of RIG-I, MAVS and p-IRF3 under the condition of virus infection, and promote the release of IFN-beta cell factors, thereby causing more effective natural immune response and remarkably inhibiting the replication of viruses.

Description

Application of protein nanoparticles in preparation of VSV (VSV virus) resistant product

Technical Field

The invention relates to the technical field of bioengineering and medicines, in particular to application of protein nanoparticles in preparation of VSV (VSV virus) resistant products.

Background

Viruses, as a class of extracellular pathogens, require a host to perform a series of vital activities. Typically, the virus and host are able to co-evolve, thereby facilitating the symbiosis of the virus and the host. Infection of a new host by a virus is generally referred to as a sudden infection event, which is not easily identified and analyzed for viral phenotype, but is more pathogenic, and lethal than a symbiotic virus.

Viruses lack the complete system required for propagation and need to be replicated by means of host cells. Viral infection of a host cell typically involves the following six steps: adsorption, invasion, dehulling, biosynthesis, assembly and release. Due to the characteristics of transmission and infection ways, characteristics and mechanisms of viruses, the action mechanisms of antiviral drugs are also divided into various types, such as direct inhibition or killing of viruses, interference of virus adsorption, prevention of virus penetration into cells, inhibition of virus biosynthesis, inhibition of virus release, enhancement of host antiviral ability and the like.

Disclosure of Invention

The invention aims to provide application of protein nanoparticles to preparation of VSV virus replication inhibition and/or VSV virus resistance products, wherein the protein nanoparticles can obviously improve the levels of RIG-I, MAVS and p-IRF3 and promote the release of IFN-beta cytokines under the condition of virus infection, so that a more effective natural immune response is caused, and the virus replication is obviously inhibited.

To this end, in a first aspect of the present invention, there is provided a protein nanoparticle formed by self-assembly of protein monomers induced by metal ions, the protein monomers including a fusion protein composed of metallothionein, a linker peptide, glutathione S-transferase, sequentially linked in order from an amino terminus to a carboxyl terminus.

Further, the metal ion is selected from the group consisting of: fe²⁺、Mn²⁺、Zn²⁺、Cu²⁺、Cr³⁺Preferably Fe²⁺。

Glutathione S-transferases (GSTs) mainly comprise 4 families: A. m, T, P, GSTP1 is preferred in the present invention.

Further, the GSTP1 may be from different species, in particular embodiments, the GSTP1 includes, but is not limited to, GSTP1_ HUMAN, GSTP1_ motion, GSTP1_ RAT, GSTP1_ MACMU, GSTP1_ XENLA, GSTP1_ PIG, GSTP1_ BOVIN, GSTP1_ MESAU, GSTP1_ PONAB, GSTP1_ CRILO, GSTP1_ CRIMI, GSTP1_ CAPHI; preferably GSTP1_ HUMAN, the amino acid sequence of which is set forth in SEQ ID NO: 1 is shown.

Metallothionein (MT) is a protein with low molecular weight, high metal content and rich cysteine, which is commonly present in the biological world.

Furthermore, the metallothionein is MT1, MT2 or MT3, preferably MT 3.

Further, the metallothionein may be from different species, in particular embodiments, the MT1 includes, but is not limited to, MT1A _ HUMAN, MT1G _ HUMAN, MT1_ HUMAN, MT1E _ HUMAN, MT1X _ HUMAN, MT1F _ HUMAN, MT1H _ HUMAN, MT H _ RAT, MT1H _ BOVIN, MT1H _ HUMAN, MT1H _ PIG, MT1H _ HORSE, MT H _ DANRE, MT H _ BOVIN, MT1H _ HUMAN, MT H _ gr, MT H _ CANLF, MT1H _ random, MT1H _ shift, MT1H _ HUMAN, MT1H _ HUMAN, MT1H _ bog, MT1H _ PIG, MT1H _ H, ct _ H _ crystal, ct _ H, ct _ 363672 _ H _ 363672 _ 36363672, tpelm _ H _ 363672, tpelm _ H _ 36363672 _ H _ 363636363672, tpelm _ 363672 _ H _ 36363672 _ 36363636363636363672, skil _ 363672, skil _ 36363636363636363672 _ H _ 363636363636363636363672 _ H _ 36363672, skil _ 3636363672 _ 36363672 _ H _ crystal, skil _ 36;

the MT2 includes but is not limited to MT2_ HUMAN, MT2_ MOUSE, MT2_ BOVIN, MT2_ DANRE, MT2A _ PIG, MT2_ RAT, MT2_ CANLF, MT2_ PONAB, MT2_ CRIGR, MT2_ DROME, MT2_ SHEEP, MT2_ CANGA, MT2_ MACFA, MT2_ CHLAE, MT2_ MESAU, MT2_ CRILO, MT2_ STECO, MT2_ CAEEL, MT2_ COLLI, MT2_ CALSI, MT2_ SCYSE, MT2_ CYPCA, MT2_ YARLI;

the MT3 includes but is not limited to MT3_ BOSMU, MT3_ HUMAN, MT3_ MACFA, MT3_ SHEEP, MT3_ HORSE, MT3_ MOUSE, MT3_ RABIT, MT3_ RAT, MT3_ BOVIN, MT3_ PIG.

Further, the metallothionein is preferably MT3_ HUMAN, and the amino acid sequence of the metallothionein is shown in SEQ ID NO: 2, respectively.

Further, the connecting peptide is a flexible connecting peptide or a rigid connecting peptide; preferably a flexible linker peptide.

Further, the linker peptide is (GGGGS)_nN is an integer of 1 to 4; for example n is 1, 2, 3 or 4. In a preferred embodiment, the linker peptide is GGGGS, the amino acid sequence of which is as set forth in SEQ ID NO: 3, respectively.

Further, the amino acid sequence of the protein monomer is shown as SEQ ID NO: 4, respectively.

In a second aspect of the invention, there is provided the use of said protein nanoparticles for the manufacture of a product for inhibiting VSV virus replication and/or for combating VSV virus.

Further, the product inhibits VSV virus replication and/or is resistant to VSV virus by at least the following pathways:

the protein nanoparticles improve the expression and/or secretion of one or more than two of RIG-I, MAVS, p-IRF3 and IFN-beta cytokines; and/or the presence of a gas in the gas,

the protein nanoparticles enhance the intensity of the innate immune response.

Vesicular stomatitis virus (VSV virus for short) is a single-stranded negative-strand RNA virus that can be transmitted by insects in natural hosts such as cattle, horses, pigs, etc., and belongs to one of the most commonly used viruses in laboratories, and is widely used to simulate human acute cell death caused by viruses; the method is used for quantitative and computational research and the like for researching behaviors such as virus genome replication and transcription. Through related research related to the virus, theoretical guidance is provided for human beings to better face burst novel RNA viruses.

Compared with the prior art, the invention has the following advantages:

the invention provides a novel application of protein nanoparticles GTSP1-MT formed by self-assembly induced by metal ions, the protein nanoparticles have no adverse effect on cell viability, and have remarkable antiviral effect verified by cell level and molecular level experiments, and further, cytokine detection shows that the protein nanoparticles can remarkably improve the levels of RIG-I, MAVS and p-IRF3 under the condition of virus infection, promote the release of IFN-beta cytokines, thereby causing more effective natural immune response and remarkably inhibiting the replication of viruses.

According to the technical scheme of the invention, the protein nanoparticles have potential to be further developed into antiviral drugs, and have good application prospects.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

FIG. 1 shows metallic Fe²⁺Electrophoretogram of GSTP1-MT3 protein expression under induction; protein marker in lane 1 and Fe in lane 2²⁺Inducing the obtained protein nanoparticles GSTP1-MT3 (Fe)²⁺)；

FIG. 2 shows GSTP1-MT3 (Fe)²⁺) A particle size distribution map of the protein nanoparticles;

FIG. 3 shows GSTP1-MT3 (Fe)²⁺) SEM images of protein nanoparticles;

FIG. 4 shows GSTP1-MT3 (Fe)²⁺) Measurement of thermal stability of protein nanoparticles;

FIG. 5 shows GSTP1-MT3 (Fe) at various concentrations²⁺) Effect on raw264.7 cell viability;

FIG. 6 shows fluorescence detection results of different treated Raw264.7 cells after 0, 6 and 12 hours of infection with VSV; wherein (A) is Mock treated group, (B) is 0.025mg/mL GSTP1 treated group, and (C) is 0.025mg/mL GSTP1-MT3 (Fe)²⁺) A treatment group;

FIG. 7 shows the results of FACS analysis of different treated Raw264.7 cells infected with VSV virus at 0, 6 and 12 hours;

FIG. 8 shows the results of cytokine detection 12 hours after infection of different treated Raw264.7 cells with VSV;

FIG. 9 shows the Western Blot results of different treated Raw264.7 cells infected with VSV virus 12 hours later;

FIG. 10 shows fluorescence measurements of differently treated PM cells after 12 hours of infection with VSV virus;

FIG. 11 shows the results of FACS analysis of differently treated PM cells 12 hours after infection with VSV virus;

FIG. 12 shows fluorescence measurements of differently treated THP-1 cells after 12 hours of infection with VSV virus;

FIG. 13 shows the Western Blot assay of different treated THP-1 cells 12 hours after infection with VSV virus.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Example 1 amino acid sequence of protein nanoparticles GSTP1-MT3

Based on previous experimental studies, the amino acid sequence of protein nanoparticles GSTP1-MT3 is constructed in the example, GSTP1-MT3 mainly consists of GSTP1 and MT3, wherein MT3 is at the N end of the amino acid sequence, GSTP1 is at the C end of the amino acid sequence, and MT3 and GSTP1 are coupled through GGGGS sequences.

GSTP1 amino acid sequence:

MPPYTVVYFPVRGRCAALRMLLADQGQSWKEEVVTVETWQEGSLKASCLYGQLPKFQDGDLTLYQSNTILRHLGRTLGLYGKDQQEAALVDMVNDGVEDLRCKYISLIYTNYEAGKDDYVKALPGQLKPFETLLSQNQGGKTFIVGDQISFADYNLLDLLLIHEVLAPGCLDAFPLLSAYVGRLSARPKLKAFLASPEYVNLPINGNGKQ(SEQ ID NO：1)；

MT3 amino acid sequence:

MDPETCPCPSGGSCTCADSCKCEGCKCTSCKKSCCSCCPAECEKCAKDCVCKGGEAAEAEAEKCSCCQ(SEQ ID NO：2)；

linker between GSTP1 and MT 3:

GGGGS(SEQ ID NO：3)。

the complete GSTP1-MT3 amino acid sequence is shown as SEQ ID NO: 4, the molecular weight is 30.566kDa, and the isoelectric point PI is 5.14; the coding nucleotide sequence of GSTP1-MT3 is shown as SEQ ID NO: 5, respectively.

Example 2 expression vector construction of protein nanoparticles GSTP1-MT3

In order to express the protein nanoparticle GSTP1-MT3 in recombinant cells, a prokaryotic expression vector is constructed in the embodiment, pET-28a (+) is selected as the prokaryotic expression vector, and enzyme cutting sites HindIII and NdeI on the prokaryotic expression vector are utilized to express the protein nanoparticle GSTP1-MT3 in the recombinant cells, wherein the expression vector is shown in SEQ ID NO: and 5, connecting the nucleotide sequence of coding GSTP1-MT3 into pET-28a (+), and successfully obtaining a recombinant expression vector pET-28a (+) -GSTP1-MT3 after enzyme digestion, electrophoresis and monoclonal sequencing detection.

Example 3 expression and purification of protein nanoparticles GSTP1-MT3

In the embodiment, escherichia coli is used as a host bacterium for recombinant expression, and the specific steps are as follows:

1. plasmid transformation

mu.L of 42 ng/. mu.L of pET-28a (+) -GSTP1-MT3 plasmid was added to 20. mu.L of BL21(DE3) competent cells, premixed on ice for 15-30min, then heated in a 42 ℃ water bath for 90s, followed by another 10min on ice. 800. mu.L of non-resistant LB medium was added, incubated at 37 ℃ for 1h at 220rpm, then centrifuged at 3500rpm for 10min, 600. mu.L of the supernatant was removed, and the remaining 200. mu.L was mixed well and used.

2. Resistance screening

The 200. mu.L of the bacterial solution remaining in step 1 was added to an agarose plate containing kanamycin, incubated at 37 ℃ for 2 hours in an incubator, and the plate was inverted and incubated overnight.

3. Monoclonal selection

Individual colonies were picked from the plate obtained by the culture in step 2, added to 10mL of LB medium containing kanamycin, and cultured at 37 ℃ and 220rpm for 10 hours, with the solution becoming gradually cloudy.

4. Protein induced expression

Adding 10mL of the bacterial solution obtained in step 3 into 1L of LB medium containing kanamycin, culturing at 37 ℃ and 220rpm for 4h, and adding 1mL of 0.1mol/L IPTG (so that I isPTG in the final concentration of 0.1mmol/L) and 0.3mmol/L of metal ion Fe in the culture medium²⁺The induction expression was continued overnight, centrifugation was carried out at 4000rpm for 20min at 4 ℃ and the supernatant was discarded, 20mL of GST resuspension (pH 8.0, 50mM Tris/HCl, 100mM NaCl, 60 mM. beta. -Mercaptoethanol) was added and sonicated (30% power, SCIENTZ, JY 92-IIN), ultracentrifugation was carried out at 12000rpm at 4 ℃ and the supernatant was collected and filtered using a 0.22 μm filter for further purification.

5. Protein purification

Purification was performed using the AKTA purification system, first equilibrated with 5 column volumes (5V) of PBS, then the supernatant from step 4 was bound to a GST column using AKTA, and after washing was continued with 5V column volumes of PBS until the baseline leveled off, elution was performed using GST eluent (pH 8.0, 10mmol/L GSH, 50mM Tris/HCl, 100mM NaCl, 60mM β -mercaptethanol) and collected. The collected proteins were subjected to GSH removal using a 10kDa ultrafiltration tube and finally to filtration using a 0.22 μm filter. Short-term storage at 4 deg.C (such as long-term storage at-80 deg.C, which is required to be in 10% glycerol).

As shown in FIG. 1, via Fe²⁺Inducing protein nanoparticles GSTP1-MT3 (Fe) with the molecular weight of about 30kDa^{2 +}). Protein nanoparticles GSTP1-MT3 (Fe)²⁺) The particle size distribution of (A) is shown in FIG. 2, GSTP1-MT3 (Fe)²⁺) The SEM image of the protein nanoparticles is shown in figure 3. As can be seen from FIGS. 2 and 3, GSTP1-MT3 is derived from Fe²⁺After induction, nano particles with uniform size distribution are formed. GSTP1-MT3 (Fe) prepared in this example²⁺) Used in the following examples.

Example 4GSTP1-MT3 (Fe)²⁺) Thermal stability of protein nanoparticles

(1) GSTP1-MT3(Fe2+) nanoparticles were formulated in PBS as 15mM stock solution with absorbance a280 of 0.702;

(2) mixing 5mL of 200 Xdye Orange with 45mL of the sample stock solution obtained in step (1);

(3) the mixture prepared in step (2) was added to an opaque 8-row PCR tube and placed in a LightCycler 96 instrument with a detector for melting temperature (Tm value). The procedure used was 37 ℃ to 98 ℃, 2.2 ℃ rise per minute, three organisms per sample in parallel.

GSTP1-MT3(Fe²⁺) The results of the thermal stability measurements of the protein nanoparticles are shown in FIG. 4.

Example 5GSTP1-MT3 (Fe)²⁺) Effect on Raw264.7 cell viability

(1) 2 plates were added Raw264.7 cells (10)⁵Individual cells/well), 37 ℃ 5% CO₂Culturing overnight;

(2) 0. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL GSTP1-MT3 (Fe) was prepared with high-glucose DMEM (containing diabody and 10% FBS), respectively²⁺) For standby;

(3) removing cell supernatant with pipette gun, and adding GSTP1-MT3 (Fe) at different concentrations²⁺)，37℃、5％CO₂Incubation for 24 hours (each group concentration contains 4 replicates);

(4) adding MTT into a high-sugar DMEM medium to enable the final concentration to be 0.5mg/mL for later use;

(5) removing the supernatant from step (3), and slowly adding the MTT culture solution from step (4) at 37 deg.C and 5% CO₂Continuing to culture for 4 hours;

(6) the cell supernatant was slowly removed, 1mL of DMSO was added to each well, and the wells were incubated at 37 ℃ for 30 minutes;

(7) cool for 10 minutes at room temperature, then transfer to 96-well plates (6 technical replicates per sample);

(8)OD₄₅₀the results of measurement of absorbance and calculation of cell viability are shown in FIG. 5, and GSTP1-MT3 (Fe) is shown in FIG. 5²⁺) Has no adverse effect on the cell viability of Raw264.7.

Example 6GSTP1-MT3 (Fe)²⁺) anti-VSV effect in Raw264.7 cells

(1) Raw264.7 cells (10) were added to 6-well plates⁶Individual cells/well), 37 ℃ 5% CO₂Culturing overnight;

(2) 3 clean 50mL centrifuge tubes were prepared and labeled Mock, 0.025mg/mL GSTP1, 0.025mg/mL GSTP1-MT3 (Fe)²⁺)；

Remarking: GSTP1 andGSTP1-MT3(Fe²⁺) All had been endotoxin removed (LPS)<0.5EU/mL), the same as in the following examples;

add 10mL DMEM to the centrifuge tube labeled Mock, 9.868mL DMEM, 132. mu.L 1.9mg/mL GSTP1 to the centrifuge tube labeled 0.025mg/mL GSTP1, and 0.025mg/mL GSTP1-MT3 (Fe)²⁺) The centrifuge tube was charged with 9.900mL of DMEM, 100. mu.L of 2.48mg/mL GSTP1-MT3 (Fe)²⁺) (ii) a Uniformly mixing the liquid in the centrifugal tube for later use;

(3) taking out the cells in the step (1), removing the culture medium, adding 500 mu L of DPBS to the cells each time, and washing the cells for three times;

remarking: the 6-well plate is not suitable for removing all of the culture medium in 6 wells at once, and 1mL of DPBS is removed and added to 2 wells on average immediately after removing the culture medium in 2 wells (2 wells in one 6-well plate) with the tip.

(4) 2mL of media containing different drug concentrations (Mock, 0.025mg/mL GSTP1, 0.025mg/mL GSTP1-MT3 (Fe) were added to 6-well plates, respectively²⁺))，37℃、5％CO₂Culturing for 6 hours;

(5) after the same amount of VSV- (GFP) virus (which can express GFP green fluorescent protein and has no difference in structure, function and property from natural VSV virus and comes from Chenying jade laboratory of Beijing university medical department) is added, the change of fluorescence is observed at 0 hour, 6 hours and 12 hours respectively, and FACS analysis is carried out at the same time; the results of fluorescence detection are shown in FIG. 6, and the results of FACS analysis are shown in FIG. 7, from which FIGS. 6-7, GSTP1-MT3 (Fe)²⁺) Effectively inhibits the proliferation of VSV virus and has obvious effect of resisting VSV virus.

Example 7 detection of cytokine changes during infection of Raw264.7 cells by VSV Virus

(1) Raw264.7 cells (10) were added to 6-well plates⁶Individual cells/well), 37 ℃ 5% CO₂Culturing overnight;

(2) taking out the cells in the step (1), removing the culture medium, adding 500 mu L of DPBS to the cells each time, and washing the cells for three times;

(3) dividing the cells treated in step (2) into A, B, C, D four groups, adding 2mL DMEM into group A and group BAdding 2mL of GSTP1-MT3 (Fe) to group C and group D²⁺) The DMEM medium of (1); 37 ℃ and 5% CO₂Culturing for 6 hours;

(4) the same amount of VSV- (GFP) Virus was added to groups B and D, and the cells of each group were cultured for an additional 12 hours to detect the amount of the cytokine IFN-. beta.in a Human Anti-Virus Response (Biolegend 740349) kit, each of which was repeated 3 times independently.

The results of the detection of the cytokine IFN-. beta.are shown in FIG. 8, and it can be seen from FIG. 8 that GSTP1-MT3 (Fe) was added²⁺) And then, the expression of a cytokine IFN-beta in the process of infecting Raw264.7 cells by the VSV virus is obviously improved. This revealed at least from one perspective GSTP1-MT3 (Fe)²⁺) The mechanism of inhibiting viral replication: the results in FIG. 8 show that GSTP1-MT3 (Fe) was added²⁺) And in the case of viral infection, a more effective innate immune response is elicited, thereby significantly inhibiting viral replication.

Example 8 verification of GSTP1-MT3 (Fe) at the protein level²⁺) anti-VSV effects in Raw264.7 cells

(1) Raw264.7 cells (10) were added to 6-well plates⁶Individual cells/well), 37 ℃ 5% CO₂Culturing overnight;

(2) cell supernatants were removed and 2mL DMEM medium, DMEM medium with 0.025mg/mL GSTP1, DMEM medium with 0.025mg/mL GSTP1-MT3 (Fe)²⁺) The DMEM medium of (1); 37 ℃ and 5% CO₂Culturing for 6 hours;

(3) after the same amount of VSV-GFP virus was added to each 6-well plate, the mixture was incubated at 37 ℃ with 5% CO₂Continuing to culture for 12 hours;

(4) removing the culture medium, and reversely buckling the 6-hole plate on the absorbent paper to ensure that the absorbent paper absorbs the culture solution;

(5) 200. mu.L of neutral RIPA lysate containing protease inhibitor and phosphatase inhibitor was added to each petri dish and left on ice for 10 minutes;

(6) gently scrape cells with a cell scraper, and aspirate the liquid into a 1.5mL centrifuge tube; carrying out ultrasonic treatment for 10 seconds, and immediately placing on ice after the ultrasonic treatment is finished; centrifuging at 12000rpm for 20min at 4 deg.C;

(7) transferring the centrifuged supernatant into a 1.5mL centrifuge tube;

(8) protein quantification using BCA;

(9) loading (10. mu.g of sample per well), running gel by electrophoresis (80V,30 min; 120V, 90 min);

(10) transferring the membrane (70V,70 min), sealing with 5% skimmed milk for 1 hr;

(11) immune response

a) Taking out the membrane from the sealing solution, and performing 1 × TBST 5min for 3 times;

b) placing the membrane protein face up in a tank, adding primary antibody, standing at 4 deg.C overnight, and washing with 1 × TBST for the next time on a decolorizing table at room temperature for 10min each time;

c) preparing the second antibody diluent and contacting with the membrane by the same method, incubating at room temperature for 90 min, and preparing with 1

TBST was washed three times on a decolorizing shaker at room temperature, 5 minutes each time;

(12) chemiluminescence, development, and fixation

a) Mixing equal amounts (350. mu.L each) of the two reagents A and B in a 1.5mL centrifuge tube in equal volume;

b) the 1 × developer was uniformly spread on the PVDF film and imaged in the imaging system, and the imaging result is shown in fig. 9.

Example 9GSTP1-MT3 (Fe)²⁺) anti-VSV effect in peritoneal macrophages

(1) Macrophages (PM) from the abdominal cavity of C57 mice were transferred to 6-well plates at 37 ℃ with 5% CO₂Culturing overnight;

(2) the next day, cell supernatants were removed and cells were washed 3 times with PBS;

(3) 2mL of high-sugar DMEM medium containing 0mg/mL, 0.025mg/mL GSTP1, and 0.025mg/mL GSTP1-MT3(Fe2+) was added to each well, and these were labeled A, B, C and group D in this order;

(4)37℃、5％CO₂culturing for 6 hours;

(5) the same amount of VSV-GFP virus was added to B, C and group D, respectively, and the mixture was incubated at 37 ℃ with 5% CO₂Continuing to culture for 12 hours;

(6) GFP fluorescence was observed under a fluorescence microscope; the results of fluorescence detection are shown in FIG. 10;

(7) the cells were washed with PBS, subsequently blown off with PBS and analyzed by FACS assay; the results of FACS analysis are shown in FIG. 11.

Example 10GSTP1-MT3 (Fe)²⁺) anti-VSV effect in monocyte macrophages

(1) Placing mononuclear macrophage (THP-1) in 1640 medium containing 12% FBS;

(2) adding PMA 30ng/mL at 37 deg.C and 5% CO₂Culturing for 48 hours, and inducing THP-1 cells to adhere to the wall;

(3) the cell supernatant was removed and the cells were washed 3 times with PBS;

(4) 2mL of 1640 medium containing 12% FBS, 0mg/mL, 0.025mg/mL GSTP1, 0.025mg/mL GSTP1-MT3(Fe2+), respectively, were added to each well, and labeled A, B, C and group D in that order;

(5)37℃、5％CO₂culturing for 6 hours;

(6) the same amount of VSV-GFP virus was added to B, C and group D, respectively, and the mixture was incubated at 37 ℃ with 5% CO₂Continuing to culture for 12 hours;

(7) GFP fluorescence was observed under a fluorescence microscope, and the results of fluorescence detection are shown in FIG. 12.

Example 11 verification of GSTP1-MT3 (Fe) at the protein level²⁺) anti-VSV effect in monocyte macrophages

The B, C, D cell cultures obtained by the culture in step (6) of example 9 were collected and analyzed by Western Blot analysis according to the procedures from steps (4) to (12) of example 7, and the results are shown in FIG. 13.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Sequence listing

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210 215 220

Ala Asp Tyr Asn Leu Leu Asp Leu Leu Leu Ile His Glu Val Leu Ala

225 230 235 240

Pro Gly Cys Leu Asp Ala Phe Pro Leu Leu Ser Ala Tyr Val Gly Arg

245 250 255

Leu Ser Ala Arg Pro Lys Leu Lys Ala Phe Leu Ala Ser Pro Glu Tyr

260 265 270

Val Asn Leu Pro Ile Asn Gly Asn Gly Lys Gln

275 280

<210> 5

<211> 852

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggaccccg agacctgccc ctgtcccagc ggaggaagct gcacctgcgc cgactcctgc 60

aagtgcgagg gctgcaagtg caccagctgc aagaagagct gctgcagctg ctgccccgcc 120

gaatgcgaaa aatgtgccaa ggactgcgtg tgtaaggggg gcgaggccgc cgaggctgag 180

gctgagaagt gctcctgctg ccaaggcgga ggcggcagca tgccccctta taccgtggtg 240

tacttccccg tgagaggcag atgcgccgcc ctgagaatgc tgctggccga ccaaggccaa 300

agttggaagg aggaggtggt cacagtggag acatggcagg agggcagtct gaaggcttcc 360

tgtctgtatg gccagctgcc caaattccaa gacggggatc tgaccctgta ccagagcaac 420

accatactga gacatctggg ccggacactg ggtctctatg ggaaggatca gcaggaggcc 480

gccctggtgg acatggtcaa cgacggagtg gaggacctga gatgcaagta catcagcctg 540

atctacacaa actacgaggc tggcaaagat gattacgtga aagcactgcc cggacagctg 600

aaacctttcg agaccctgct gtctcagaac cagggcggca agaccttcat cgtgggcgac 660

cagatcagct tcgcagatta caacctgctg gacctgctgc tgattcatga ggttctggcc 720

cccggctgtc tcgacgcctt cccactgctc tctgcttacg tgggccggct gagcgccaga 780

cccaagctca aggccttcct ggcctccccc gagtacgtga acctgcccat caacggaaac 840

ggcaagcaat aa 852

Claims (9)

1. Use of a protein nanoparticle for the manufacture of a product for inhibiting VSV virus replication and/or combating VSV virus;

the protein nanoparticles are formed by self-assembly of protein monomers through induction of metal ions, and the protein monomers comprise fusion proteins formed by sequentially connecting metallothionein, connecting peptide and glutathione-S-transferase from an amino terminal to a carboxyl terminal.

2. The use according to claim 1, wherein the metal ion is selected from the group consisting of: fe²⁺、Mn²⁺、Zn²⁺、Cu²⁺、Cr³⁺Preferably Fe²⁺。

3. The use of claim 1, wherein the glutathione-s-transferase is GSTP 1;

preferably, the amino acid sequence of the glutathione s-transferase is as shown in SEQ ID NO: 1 is shown.

4. The use according to claim 1, wherein the metallothionein is MT1, MT2 or MT 3.

5. The use according to claim 4, wherein the metallothionein is MT 3;

preferably, the amino acid sequence of the metallothionein is shown as SEQ ID NO: 2, respectively.

6. The use of claim 1, wherein the linker peptide is a flexible linker peptide or a rigid linker peptide; preferably a flexible linker peptide.

7. The transit use of claim 6, wherein the linker peptide is (GGGGS)_nN is an integer of 1 to 4;

preferably, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO: 3, respectively.

8. The use of claim 1, wherein the amino acid sequence of the protein monomer is as set forth in SEQ ID NO: 4, respectively.

9. Use according to claim 1, wherein the product inhibits viral replication and/or is antiviral by at least:

the protein nanoparticles improve the expression and/or secretion of one or more than two of RIG-I, MAVS, p-IRF3 and IFN-beta cytokines; and/or the presence of a gas in the gas,

the protein nanoparticles enhance the intensity of the innate immune response.

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