CN112209993B

CN112209993B - Antiviral polypeptide and antiviral mask prepared from same

Info

Publication number: CN112209993B
Application number: CN202011098819.8A
Authority: CN
Inventors: 彭菲; 陈印平
Original assignee: Heilongjiang Yinjia Medical Instrument Co ltd
Current assignee: Heilongjiang Yinjia Medical Instrument Group Co.,Ltd.
Priority date: 2020-10-14
Filing date: 2020-10-14
Publication date: 2021-09-14
Anticipated expiration: 2040-10-14
Also published as: CN112209993A

Abstract

The invention relates to an antiviral polypeptide and an antiviral mask prepared from the same. The specific active peptide capable of inhibiting the proliferation of the H1N1 virus is obtained by screening and identifying the specificity of the H1N1 influenza virus, and the peptide is verified to have better effect of inhibiting the virus from spreading in cells and animal models by further verification at a cell level and an animal model level, so that the peptide has excellent application value. And the peptide has no cytotoxicity and is suitable for popularization and application.

Description

Antiviral polypeptide and antiviral mask prepared from same

Technical Field

The invention relates to the field of medical protection, in particular to antiviral polypeptide and an antiviral mask prepared from the same.

Background

In daily life, viruses related to human beings are transmitted through various ways such as aerosol, feces, urine, conjunctiva, mother and baby and the like. A large number of antiviral protective articles are used for isolating and protecting uninfected people and medical staff, and particularly when the medical staff are in contact with blood, clothes, body fluid, secretion and excrement of a patient, the medical protective articles are worn for isolation and protection, so that cross infection is prevented, and the risk of infection is reduced. The medical protective articles comprise medical protective masks, disposable caps or cloth caps, work shoes and socks, work clothes, gloves, protective clothes or isolation clothes, disposable medical surgical masks, protective glasses, shoe covers and the like. Research has shown that textiles play a critical role in the viral infectious chain, extracting large amounts of virus from these medical protective articles, especially medical textile materials. The study of antiviral protective textiles is therefore of particular importance.

In current research, antiviral protective textiles can be achieved by coating antiviral protective materials. The coating antiviral protective material is a reusable coating antiviral protective material which is formed by coating a material with a special chemical structure on the surface of a fabric through processing technologies such as immersion pressing, blade coating and the like so as to block viruses and prevent body fluid and blood with the viruses from entering a human body. However, the isolation suit made of the material has poor air permeability and moisture permeability, so that sweat cannot be discharged, suffocating and air impermeability are caused, even dehydration phenomenon is caused, and physical ability and working efficiency of medical workers are affected. The polyurethane coating material prepared by PARK D has the advantages that when the Quat-12-PU organic solution or the aqueous nano suspension is coated on the surface of a fabric, gram-positive staphylococcus aureus and gram-negative escherichia coli can be killed, and enveloped influenza viruses (excluding non-enveloped poliovirus) can be inactivated. At present, no more efficient antiviral protective textile for biologically controlling viruses is produced.

Among the anti-viral protective textiles, disposable masks are commonly used textiles. The disposable mask mainly adsorbs particles such as bacteria, viruses and the like by virtue of the electrostatic electret of the melt-blown cloth so as to prevent the harmful particles from entering a human body, but the electrostatic electret under the dynamic condition is easy to disappear, once the electrostatic of the electrostatic electret of the melt-blown cloth is dissipated, the blocking effect is greatly reduced, and the disposable mask is made of polypropylene fibers, so that a large amount of waste of the disposable mask can cause serious pollution to the environment; secondly, the raw materials of the disposable mask are difficult to supply under the current situation, and the production of the disposable medical surgical mask is not practical; thirdly, the silver ions have broad-spectrum antibacterial effect, according to the data provided by the relevant data, the silver has the effect of inhibiting 650 bacteria, and the bacteria can be killed within 6 min. Silver has the effect of inhibiting some viruses. However, silver is expensive and consumes non-renewable resources, which is not suitable for large-scale long-term popularization and application.

The addition of antiviral substances to masks is one of the major directions of current research. For example, the mask is added with a medicine core layer or a medicine feeding film to realize the antiviral effect, and the added antiviral substances comprise western medicines and Chinese herbal medicines. Inactivating viruses by adding at least one fine particle of platinum iodide, palladium iodide, silver iodide, copper iodide or copper thiocyanate as in RU2549065C 2; the patent CN205390400U mask comprises a mask body, wherein a filtering and sterilizing core is arranged inside the mask body, the sterilizing core consists of a filter screen, an adsorption layer and a sterilizing layer, sterilizing particles are adhered to the surface of the sterilizing layer, and the sterilizing particles are penicillin particles; the interlayer of the drug core of patent CN110742339A is soaked with extract of flos forsythiae; in patent CN109832691A, the middle layer is soaked with mixed Chinese medicinal decoction of flos Lonicerae, Glycyrrhrizae radix, Coptidis rhizoma, flos Chrysanthemi, etc. However, the above method is complicated or expensive in preparation and is not suitable for large-scale popularization.

Influenza is an acute respiratory disease with fever caused by Influenza virus (Influenza virus), and can be divided into 3 subtypes, namely subtype A, subtype B and subtype C, and the current different subtypes and increasing drug-resistant strains pose serious threats to human health. anti-Influenza a virus (IVA) polypeptides are mainly classified into 3 major groups, first, Influenza virus invasion blocking peptides, such as flu interacting with Influenza virus hemagglutinin, block virus binding to host cells and prevent membrane fusion; secondly, some peptides can disrupt the viral envelope and thus inactivate the virus, such as melittin; still other polypeptides may inhibit influenza virus replication, such as polypeptides derived from PB 1. However, the research is still relatively few at present, and the development of polypeptides with stable properties, smaller molecular mass and high targeting property is the direction of the research on the antiviral polypeptides at present.

Disclosure of Invention

The invention provides an antiviral polypeptide capable of specifically and targetedly killing influenza A virus, which is an improvement aiming at the prior art.

An anti-influenza virus mask comprises an outer filter layer, an antibacterial layer, a bacteriolysis layer, an antiviral layer and an inner filter layer, wherein the inner filter layer is a layer contacting with skin, and is characterized in that antiviral active peptide is soaked on an interlayer of an inner drug core.

Further, the amino acid sequence of the antiviral active peptide is shown as SEQ ID NO: 1 is shown.

The activity inhibitory peptide specific to the H1N1 influenza virus is obtained by screening the peptide library and is used as an antiviral agent, so that the mask has proper humidity and temperature to excite the biological activity of the peptide in the using process, and can effectively exert antiviral performance.

Furthermore, the mask of the invention can also sequentially comprise three functional layers from outside to inside, namely an antibacterial layer, a bacteriolysis layer and an antiviral layer, bacteria and viruses entering the mask from outside firstly pass through the antibacterial layer to kill most of bacteria with thinner capsules, simultaneously damage the residual bacteria and viruses, then thoroughly kill the residual bacteria through the bacteriolysis layer, simultaneously further damage the protein shell of the damaged viruses, and finally carry out bioactive dissolution on the residual viruses through antiviral peptides in the antiviral layer. Therefore, by the three functional layers in a specific order, excellent antibacterial and antiviral properties can be achieved.

As a preferred technical scheme, the antibacterial and antiviral mask sequentially comprises an outer filter layer, an antibacterial layer, a bacteriolysis layer, an antiviral layer and an inner filter layer from outside to inside, wherein the inner filter layer is a layer in contact with the skin.

Preferably, the antibiotic layer includes an inorganic antibiotic agent.

Preferably, the inorganic antibacterial agent comprises any one of nano zinc oxide, nano titanium dioxide, bismuth tungstate, nano silver sol or Ag @ C core-shell structured nanoparticles or a combination of at least two of them, and typical but non-limiting examples of the combination are: the nano-silver/core-shell-structured nanoparticles, and the like, preferably Ag/silver/core-shell-structured nanoparticles.

Preferably, the bacteriolytic layer comprises lysozyme.

Preferably, the lysozyme is egg white lysozyme.

Furthermore, the inner filter layer is prepared from a skin affinity material.

Advantageous effects

According to the invention, 3 specific active peptides capable of inhibiting the proliferation of the H1N1 virus are obtained by screening and identifying the specificity of the H1N1 influenza virus, and the peptides are verified to have better effect of inhibiting the virus from spreading in cells and animal models by further verification at a cell level and an animal model level, so that the peptide has excellent application value. And the peptide has no cytotoxicity and is suitable for popularization and application.

Drawings

FIG. 1 is a graph showing the binding results of phage display peptide libraries after each round of screening

FIG. 2 is a graph showing the results of the titer of H1N1-16 polypeptide inhibiting H1N1 subtype influenza virus at chick embryo level

FIG. 3 is a graph showing the results of the cell viability of the polypeptide H1N1-16 acting on virus-infected cells at the stage of cell infection

FIG. 4 is a graph showing the results of cytotoxicity test of polypeptide H1N1-16

FIG. 5H1N1 mRNA expression level results

Detailed Description

To further illustrate the objects, aspects and advantages of the present invention, we shall now describe the invention with reference to the following specific examples, which are only for better illustrating the patent of the present invention and are not intended to limit the scope of the present invention. All other embodiments that can be obtained by a person skilled in the art without making any inventive step based on the examples of the present invention belong to the protection scope of the present invention.

Example 1 screening of phage random 7 peptide library for anti-H1N 1 viral polypeptides

Random peptide libraries were screened against purified influenza virus particles H1N1(H1N1 Beijing 262/95, purchased from PROSpect, cat # IHA-002) as target molecules.

The viral particles were diluted in coating solution, coated on an ELISA plate overnight at 4 ℃ and blocked with 5% BSA. The virus coating amount is decreased sequentially and respectively to 10 mug/mL, 5 mug/mL, 2 mug/mL and 1 mug/mL, and the total of 4 rounds of screening are carried out, and the phage amount added in each round is 2.0 multiplied by 10¹¹PFU. The concentration of Tween-20 in the first round of washing step was 0.1%, and that in the remaining three rounds was 0.5%. Before each round of screening, the screened peptide library is placed in an enzyme-labeled plate hole which only contains 5% BSA and does not contain virus particles, and is incubated for 1h and then is combined with target virus particles, so that nonspecific short peptides combined with the BSA in the peptide library are deducted. The rest of the manipulations were performed according to the random peptide library instructions. Phage input and output were determined for each round of screeningBody titers to calculate recovery results are shown in table 1.

TABLE 1 Virus particle dose per round and phage recovery

As can be seen from Table 1, the recovery of the phage-displayed peptide library did not increase after 4 rounds of selection, indicating that the enrichment was maximal and thus the selection was stopped.

Meanwhile, by taking the virus particle H1N1 as a target molecule, the ELISA identifies the affinity of the phage display peptide eluted in each round with the respective virus particle. The ELISA method was performed by coating an ELISA plate with influenza H1N1 at a concentration of 5. mu.g/mL, and taking an equal amount (2.0X 10) of each phage clone picked out¹¹PFU) were tested by ELISA. Meanwhile, the original phage display peptide library which is not screened is used as a negative control, a virus-free coated hole is used as a blank control, and phage clones with OD450nm values 3 times higher than that of the negative control are judged to be positive. The results are shown in FIG. 1. After 4 rounds of screening, the binding force of the phage display peptide library is not increased any more, indicating that the enrichment effect is the highest, and thus the screening is stopped.

After 4 rounds of screening, 74 clones were picked each from the phage library H1N1 against the target molecule H1N 1. Each clone is subjected to ELISA detection and influenza virus inhibition activity detection after amplification, wherein the ELISA detection OD450nm value is positive when being more than 3 times of that of a negative control; the activity test uses H1N1 cultured by MDCK cells as stock solution of influenza virus, and performs antiviral activity test on each detected phage clone and NA inhibition activity test by using a NA test method conventional in the field, and the activity test is positive when the inhibition activity is higher than 80% and the inhibition activity has a dose-dependent relationship. The detection result shows that 48 positive clones which can resist viruses and inhibit NA are selected from 74 clones. These 48 clones were subjected to rapid extraction of phage single-stranded DNA by NaI method according to the instructions of phage random peptide library kit. Using this as a template, DNA fragments of the PCR amplified phage display polypeptides were submitted for sequencing. The amino acid sequences of the phage display polypeptides were deduced from the determined DNA sequences, 3 of which were conserved sequences common to multiple clones, and the results are shown in Table 2. These 3 polypeptides were most fully enriched during the screening process and most likely played an important role in neuraminidase binding and enzyme activity inhibition, so these 3 polypeptides were selected for chemical synthesis for subsequent cell and animal experiments.

Table 2 identifies the resulting 7 peptide sequences

Example 2 Effect of H1N1-16 Polypeptides on inhibiting H1N1 subtype influenza Virus at chick embryo level

The polypeptide H1N 1-1650 mu L with the concentration of 1000 mu mol/L is respectively mixed with 50 mu L H1N1 with the hemagglutination unit of 8, the mixture is incubated for 1H at room temperature and then injected into the allantoic cavity of a chicken embryo for virus titer determination, PBS is taken as a negative control, and FIG. 2 shows that the polypeptide H1N1-16 has better antiviral activity in the chicken embryo, and the antiviral activity of the polypeptide on influenza virus can reduce the virus titer to 25 percent, so that the polypeptide has better effect.

EXAMPLE 3 verification of the Effect of the polypeptide H1N1-16 on the stage of Virus-infected cells

In order to preliminarily explore the stage of the polypeptide H1N1-16 acting on virus infected cells, the polypeptide H1N1-16 diluted to 5 concentrations (0, 1, 10, 100 and 1000 mu mol/L) is mixed with H1N1 of 100TCID50 uniformly before cell inoculation, then the mixture is incubated for 1H at room temperature, inoculated into a 96-well plate and cultured for 48H, and the cell survival rate is detected by a CCK8 method; as shown in FIG. 3, the survival rate of cells was gradually increased in the virus group treated with the polypeptide H1N1-16 before the inoculation as the concentration of the polypeptide was increased; the peptide group added after the virus inoculation also had a tendency to increase gradually compared with the negative control group (0. mu. mol/L), but the tendency to increase was not evident in the virus group before the virus inoculation. However, it has been also demonstrated that the polypeptide acts not only on the adhesion stage of target cells infected with influenza virus but also inhibits influenza virus replication to exert antiviral action.

EXAMPLE 4 cytotoxicity assay of polypeptide H1N1-16

To evaluate the toxicity of the H1N1-16 polypeptide on cells, the toxicity of H1N1-16 on MDCK cells at various concentrations was determined by the CCK8 method, H1N1-16 still showed no significant cytotoxicity up to 1000. mu. mol/L as shown in FIG. 4 (FIG. 4).

Example 5 Effect of H1N1-16 Polypeptides on mouse models

The mice are divided into blank control group, model control group, western medicine treatment group and polypeptide treatment group according to a random number table, and each group contains 8 mice. After adaptive feeding for 1 week, except a blank control group, all the mice of the other groups are subjected to ether light anesthesia and then subjected to H1N1 influenza virus mouse lung adaptive strain nasal drip treatment, wherein each mouse is subjected to 50 mu L/mouse and continuously treated for 3 days, and an influenza model is established.

Methods of administration drug dosages are determined by reference to equivalent dose ratios converted from human and animal intermediate surface areas. After the molding is finished, the oseltamivir phosphate (the dose is 22.75 mg.kg < -1 >) is administered to the western medicine treatment group for intragastric administration, and the equal volume of normal saline is administered to the blank control group and the model control group for intragastric administration. The stomach was gavaged for 4 days, 2 times daily. The polypeptide treatment group is administered with H1N1-16 polypeptide (dosage is 20 mg. kg-1) for intragastric administration, and the blank control group and the model control group are administered with equal volume of physiological saline for intragastric administration. The stomach was gavaged for 4 days, 2 times daily.

(1) After the administration, the patient was killed by removing the neck, and the lung was dissected and subjected to a virus hemagglutination test. Taking lung tissues, grinding and homogenizing, taking 25 mu L of supernatant, selecting a row of holes on a micro reaction plate, diluting from the 1 st hole to the 8 th hole according to a multiple dilution method, adding 25 mu L of 1.2% chicken erythrocyte suspension into each hole, fully mixing uniformly, standing at room temperature for 45min, and observing a reaction result. The results are shown in Table 3. The lung tissue homogenate dilution of the model control group is 1: 2-1: 256 to cause hemagglutination, and no reaction is caused at the beginning of 1: 512, which indicates that the hemagglutination price is 1: 256. The hemagglutination of the western medicine treatment group viruses causes hemagglutination reaction at the dilution of 1: 2-1: 16, and no reaction is caused at the beginning of 1: 32, which indicates that the hemagglutination value is 1: 16. The hemagglutination of polypeptide therapy group virus is 1: 2-1: 8, no reaction is started at 1: 16, which shows that the hemagglutination is 1: 8. The polypeptide treatment can reduce the hemagglutination titer of the mouse lung influenza virus.

TABLE 3 comparison of hemagglutination titers of the pulmonary influenza viruses in groups of mice

-uncoagulated, + coagulated.

(2) RT-PCR detection of mouse lung tissue H1N1 mRNA expression level mouse lung tissue is extracted, TRIzol kit is used to extract total RNA of tissue, nucleic acid protein quantitative analyzer is used to determine absorbance values with wavelength of 260nm and 280nm, and RNA purity is judged according to D (260)/D (280) value. And (3) carrying out reverse transcription on the total RNA with qualified purity to synthesize cDNA, taking the cDNA as a template, and carrying out PCR detection by adopting a SYBR method. The 2- Δ Ct method calculated the relative expression level of H1N1 mRNA in lung tissue. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 3 min; 30s at 94 ℃, 30s at 55 ℃ and 30s at 72 ℃ and repeated 35 times. The primer sequence is as follows: upstream, 5'-ctgagaagcagatactgggc-3'; downstream, 5 '-ctgcattgtctccgaagaaat 3'. The length of the amplified fragment is 340 bp. GAPDH primer sequence: upstream, 5'-TGATGACATCAAGAAGGTGGTGAAG-3'; downstream, 5'-TCCTTGGAGGCCATGTAGGCAT-3'. The length of the amplified fragment is 340 bp. Figure 5 results show that: compared with a blank control group, the lung tissue H1N1 mRNA expression level of the mouse of the model control group is increased (P < 0.05). The high expression of the lung tissue virus nucleic acid of the mouse in the model control group is shown, so that the success of the model construction is determined. After treatment, compared with a model control group, the expression level of the H1N1 mRNA of the lung tissue of the mice in the traditional Chinese medicine treatment group and the polypeptide treatment group is reduced (P <0.05), and the expression level of the H1N1 mRNA of the lung tissue of the mice in the polypeptide treatment group is lower than that of the Western medicine treatment group and is only 200(P < 0.05). The polypeptide can effectively reduce the high expression of influenza mouse lung tissue virus nucleic acid, and the treatment effect is better than that of oseltamivir.

EXAMPLE 6 preparation of anti-H1N 1 mask

(1) Preparation of polypeptide solution: dissolving the antiviral active peptide polypeptide in normal saline to prepare 2g/100ml polypeptide solution for later use;

(2) preparation of an antiviral layer: adding 1 part (by weight) of polyamine and guanidinium polymer, 4 parts (by weight) of polypropylene special material and 95 parts (by weight) of polypropylene ethylene resin into raw materials for manufacturing gauze, mixing and linking at a high temperature of 500 ℃ for 45 minutes, pressing and granulating to obtain antiviral matrix resin, processing the resin to prepare 50 nm-diameter nano fibers, then processing the nano fibers into sheets, immersing the nano fibers into the prepared polypeptide solution at a temperature of 55 ℃ for 2 hours, and then drying;

(3) preparing the mask: taking a non-woven fabric sheet material to be laminated on two surfaces of the prepared antiviral layer to be respectively used as an outer filter layer and an inner filter layer, repeatedly laminating 2 non-woven fabric sheets, and then performing high-temperature compression molding, wherein the non-woven fabric sheets are provided with regularly formed through holes which are in staggered configuration and avoid direct penetration of air flow, and the diameter of each through hole is smaller than 1 um; the two ends of the mask are hung on ears.

(4) Mask performance test

And (4) performing a comparative test on the mask prepared in the step (3) and a commercially available N95 mask. A ZR-1000A type respirator Virus Filtration Efficiency (VFE) detector is used for performance detection, a YY/T1497-2016 medical protective respirator material virus filtration efficiency evaluation phage test method is adopted, a double-air-path simultaneous comparison sampling method is adopted, sampling accuracy is improved, and detection results show that the virus filtration efficiency of the invention reaches 99.52% which is higher than 97.3% of that of N95. The mask prepared by the invention has a better virus isolation function and has a better isolation effect than N95.

Sequence listing

<110> Beijing Yuehao science and technology development Co., Ltd

<120> an antiviral polypeptide and an antiviral mask prepared therefrom

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<170> SIPOSequenceListing 1.0

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<213> Artificial Sequence (Artificial Sequence)

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Arg Ile Trp Arg His Met

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<213> Artificial Sequence (Artificial Sequence)

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Arg Trp Tyr Arg Pro Asn Gln

1 5

<210> 3

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<213> Artificial Sequence (Artificial Sequence)

<400> 3

Lys His Trp Pro His Leu

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Claims

1. An antiviral active peptide characterized by: the amino acid sequence is shown as SEQ ID NO: 1 is shown.

2. An anti-influenza virus mask comprises an outer filter layer, an anti-virus layer and an inner filter layer, wherein the inner filter layer is a layer in contact with the skin, and the anti-influenza virus mask is characterized in that: the antiviral active peptide is infiltrated on the antiviral layer; wherein the amino acid sequence of the antiviral active peptide is shown as SEQ ID NO: 1 is shown.