CN112546239B

CN112546239B - Use of protein nanoparticles for preparing antiviral products

Info

Publication number: CN112546239B
Application number: CN202011308430.1A
Authority: CN
Inventors: 朱新杰; 林坚; 赵磊
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2022-09-02
Anticipated expiration: 2040-11-19
Also published as: CN112546239A

Abstract

The invention relates to application of protein nanoparticles in preparing products for inhibiting virus replication and/or resisting viruses, 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. Under the condition of virus infection, the protein nano-particles can obviously improve the levels of RIG-I, MAVS and p-IRF3 and promote the release of IFN-beta cell factors, thereby causing more effective natural immune response and obviously inhibiting the replication of viruses.

Description

Use of protein nanoparticles for preparing antiviral products

Technical Field

The invention relates to the technical field of bioengineering and medicines, in particular to application of protein nanoparticles in preparation of antiviral 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 compared to a commensal virus, and has greater pathogenicity, and lethality.

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 products for inhibiting virus replication and/or resisting viruses, 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 more effective natural immune response is caused, and 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: fe ²⁺ 、Mn ²⁺ 、Zn ²⁺ 、Cu ²⁺ 、Cr ³⁺ Preferably Fe ²⁺ 。

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 _ H, skil _ 363672 _ H _ crystal, skil _ H _ 363672 _ H, skil _ H _ 3636363636363636363672 _ H, skil _ crystal, skil _ 363672, skil _ crystal, skip _ H _ 3636363672 _ H, skip _ 363636363672 _ H _ crystal, a _ H _ 363672 _ crystal, a _ H _ crystal, a _ 36363636363636363672 _ H _ 363672 _ H, a _ H _ 363636363672 _ crystal, a _ 3636363672 _ H _ 3636363672 _ crystal, a _ H, a _ crystal, a _ H _ crystal, a _ H _ crystal _ H _ 36363672 _ H, a _ H _ crystal _ H, a _ H _ crystal _;

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 flexible connecting peptide or rigid connecting peptide; preferably a flexible linker peptide.

Further, the linker peptide is (GGGGS) _n N 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 preparation of a product inhibiting viral replication and/or anti-viral.

Further, the virus is an RNA virus.

Further, the virus is a double-stranded RNA virus or a single-stranded RNA virus.

Further, the virus is a single stranded RNA virus, e.g., a single stranded negative stranded RNA virus, a single stranded positive stranded RNA virus.

In a specific embodiment, the single-stranded negative-strand RNA virus is vesicular stomatitis virus (VSV virus) or H1N1 virus.

Further, 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.

The VSV is a single-stranded negative strand RNA virus which can be transmitted by insects in natural hosts such as cows, horses, pigs and the like, belongs to one of the most commonly used viruses in laboratories, and is widely used for simulating 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. In the development of antiviral drugs, studies on the mechanism of VSV virus are often performed as a model.

The invention researches by taking VSV virus as an example, and discovers a molecular mechanism for resisting virus of the protein nanoparticle, namely the protein nanoparticle can obviously improve the levels of RIG-I, MAVS and p-IRF3 of cells and promote the release of IFN-beta cell factors under the condition of virus infection, thereby causing more effective natural immune response and obviously inhibiting the replication of the virus. According to the molecular mechanism, the replication characteristics of the single-stranded negative strand RNA virus are combined, and the protein nanoparticles can realize remarkable replication inhibition effect on the single-stranded negative strand RNA virus through the molecular mechanism. On the basis, the invention further verifies the effect of the protein nanoparticles on inhibiting the replication of the H1N1 virus, and experiments prove that the protein nanoparticles have excellent inhibiting effect on the replication of the H1N1 virus.

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 Fe ²⁺ Electrophoretogram of GSTP1-MT3 protein expression under induction; protein marker in lane 1 and Fe in lane 2 ²⁺ 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 concentrations ²⁺ ) 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, and (B) is 0.025mg/mL GSTP1 treatedGroup (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 the fluorescence detection results of Raw264.7 cells treated differently after 18 hours of infection with H1N1 virus;

FIG. 11 shows the results of quantifying the mean fluorescence intensity of the fluorescence signals in FIG. 10.

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 selection

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 liquid obtained in step 3 into 1L of LB culture medium containing kanamycin, culturing at 37 ℃ and 220rpm for 4h, and then adding 1mL of 0.1mol/L IPTG (so that the final concentration of IPTG in the culture medium is 0.1mmol/L) and 0.3mmol/L of metal ion Fe ²⁺ The expression was further induced overnight, centrifuged at 4000rpm at 4 ℃ for 20min, the supernatant was discarded, 20mL of GST resuspension (pH 8.0, 50mM Tris/HCl, 100mM NaCl, 60 mM. beta. -Mercaptoethanol) was added for ultrasonication (30% power, SCIENTZ, JY 92-IIN), ultracentrifuged at 12000rpm at 4 ℃, 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 Fe ²⁺ Inducing protein nanoparticles GSTP1-MT3 (Fe) capable of obtaining a 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 Fe ²⁺ After induction, nano particles with uniform size distribution are formed. GSTP1-MT3 (Fe) prepared in this example ²⁺ ) 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(Fe ²⁺ ) 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) ⁵ Individual cells/well), 37 ℃ 5% CO ₂ Culturing 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), respectively ²⁺ ) For standby;

(3) removing cell supernatant with pipette gun, and adding GSTP1-MT3 (Fe) at different concentrations ²⁺ )，37℃、5％CO ₂ Incubation for 24 hours (each group concentration contains 4 replicates);

(4) adding MTT into a high-glucose DMEM culture 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% CO ₂ Continuing 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)OD ₄₅₀ the results of measurement of absorbance and calculation of cell viability are shown in FIG. 5, and GSTP1-MT3 (Fe) is shown in FIG. 5 ²⁺ ) Has no adverse effect on the cell viability of Raw264.7.

Example 6GSTP1-MT3 (Fe) ²⁺ ) anti-VSV Virus Effect of

(1) Raw264.7 cells (10) were added to 6-well plates ⁶ Cell/well), 37 ℃ 5% CO ₂ Culturing overnight;

(2) preparing 3 clean50mL centrifuge tubes, labeled Mock, 0.025mg/mL GSTP1, 0.025mg/mL GSTP1-MT3 (Fe) ²⁺ )；

Remarking: GSTP1 and GSTP1-MT3 (Fe) ²⁺ ) 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⁺ ) Into the centrifuge tube, 9.900mL of DMEM and 100. mu.L of 2.48mg/mL of 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 medium in 6 wells at once, and 1mL of DPBS is removed and added to an average of 2 wells immediately after removing the medium from 2 wells (2 wells in one 6-well plate) with the tip of the pipette.

(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, respectively ²⁺ ))，37℃、5％CO ₂ Culturing 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, the results of FACS analysis are shown in FIG. 7, and GSTP1-MT3 (Fe) is shown in FIGS. 6-7 ²⁺ ) Effectively inhibits the proliferation of VSV virus and has obvious effect of resisting VSV virus.

Example 7GSTP1-MT3 (Fe) ²⁺ ) Influencing cytokine changes during VSV virus infection of cells

(1) Raw264.7 cells (10) were added to 6-well plates ⁶ Individual cells/well), 37 ℃ 5% CO ₂ Culturing 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 medium to group A and group B, and adding 2mL GSTP1-MT3 (Fe) containing 0.025mg/mL to group C and group D ²⁺ ) The DMEM medium of (1); 37 ℃ and 5% CO ₂ Culturing 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 added ²⁺ ) 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⁺ ) Mechanism of inhibition of replication of mononegavirales: the results in FIG. 8 show that GSTP1-MT3 (Fe) was added ² ⁺ ) And in the case of viral infection, a more effective innate immune response is elicited, thereby significantly inhibiting viral replication.

Example 8 Studies of GSTP1-MT3 (Fe) at the protein level ²⁺ ) Against viral action

(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% CO ₂ Culturing for 6 hours;

(3) after adding an equal amount of VSV-GFP virus to each 6-well plate, the reaction was carried out at 37 ℃ with 5% CO ₂ Continuing 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) adding 200 mu L of neutral RIPA lysate containing protease inhibitor and phosphatase inhibitor into the culture dish, and placing 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 a second antibody diluent in the same way, contacting the second antibody diluent with the membrane, incubating the membrane for 90 minutes at room temperature, and washing the membrane for three times by using a 1 xTBST decolorizing shaking table at room temperature, wherein each time lasts for 5 minutes;

(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-H1N 1 virus effect in Raw264.7 cells

Raw264.7 cells were added to a cell culture dish at 37 ℃ with 5% CO ₂ Culturing overnight; subsequently, the control group and the experimental group were set, and GSTP1-MT3 (Fe) was added to the experimental group at a final concentration of 0.025mg/mL ²⁺ ) For the control group, an equal volume of PBS was added; incubation was continued for 6 hours, then an equal amount of H1N1(WSN) virus was added to each well for 18 hours of infection; cell supernatants were removed and washed 3 times with PBS; fixation with 4% paraformaldehyde for 15 minutes at 37 ℃ (in an incubator); 0.1% Triton x-100 permeabilization for 10 minutes (room temperature); 3% BSA (PBS) blocking solution was blocked at room temperature for 20 min; incubation primary anti [ anti-influenza A Virus Nucleoprotein antibody (C43), diluted 1:200]Overnight at 4 ℃; washing with PBS for 5min for 3 times; blocking was continued at blocking chamber temperature for 30min and secondary antibody [ Donkey anti-Mouse IgG (H + L) Ready probes with Alexa fluorescein 488 was added ^TM Second Antibody Alexa Fluro 488, diluted 1:1000]1 hour at 37 ℃; PBS wash 3 times, each for 5 minutes, and observe under fluorescence microscope. The results of fluorescence detection are shown in FIG. 10, and mean fluorescence intensity quantification (MFI) was performed on the fluorescence signals in FIG. 10, and the results are shown in FIG. 11 (3 independent replicates for each group).

As can be seen from FIGS. 10-11, GSTP1-MT3 (Fe) ²⁺ ) Effectively inhibits the proliferation of the H1N1 virus and has obvious effect of resisting the H1N1 virus.

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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Cys Val Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu Ala Glu Lys Cys

50 55 60

Ser Cys Cys Gln Gly Gly Gly Gly Ser Met Pro Pro Tyr Thr Val Val

65 70 75 80

Tyr Phe Pro Val Arg Gly Arg Cys Ala Ala Leu Arg Met Leu Leu Ala

85 90 95

Asp Gln Gly Gln Ser Trp Lys Glu Glu Val Val Thr Val Glu Thr Trp

100 105 110

Gln Glu Gly Ser Leu Lys Ala Ser Cys Leu Tyr Gly Gln Leu Pro Lys

115 120 125

Phe Gln Asp Gly Asp Leu Thr Leu Tyr Gln Ser Asn Thr Ile Leu Arg

130 135 140

His Leu Gly Arg Thr Leu Gly Leu Tyr Gly Lys Asp Gln Gln Glu Ala

145 150 155 160

Ala Leu Val Asp Met Val Asn Asp Gly Val Glu Asp Leu Arg Cys Lys

165 170 175

Tyr Ile Ser Leu Ile Tyr Thr Asn Tyr Glu Ala Gly Lys Asp Asp Tyr

180 185 190

Val Lys Ala Leu Pro Gly Gln Leu Lys Pro Phe Glu Thr Leu Leu Ser

195 200 205

Gln Asn Gln Gly Gly Lys Thr Phe Ile Val Gly Asp Gln Ile Ser Phe

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

1. Use of a protein nanoparticle for the preparation of a product for inhibiting viral replication and/or for combating viruses;

the protein nano-particles are prepared by processing protein monomers through metal ions Fe ²⁺ Induced to self-assemble, the protein monomer comprises a fusion protein formed by sequentially connecting metallothionein, connecting peptide and glutathione-S-transferase from an amino terminal to a carboxyl terminal;

the amino acid sequence of the glutathione-S-transferase is shown as SEQ ID NO: 1 is shown in the specification; the amino acid sequence of the metallothionein is shown as SEQ ID NO: 2 is shown in the specification; the connecting peptide is (GGGGS) _n N is an integer of 1 to 4; the virus is H1N1 virus.

2. The use of claim 1, wherein the amino acid sequence of the linker peptide is as set forth in SEQ ID NO: 3, respectively.

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

4. 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-b cytokines; and/or the presence of a gas in the gas,

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