Application of M1 protein related substance in preparation of medicine for treating diseases caused by avian influenza virus

Abstract

The invention provides application of M1 protein related substances in preparation of medicines for treating diseases caused by avian influenza viruses. The application is any one of the following applications: application of Y1 in preparation of medicines for preventing and / or treating diseases caused by avian influenza viruses or avian influenza virus infection; the Y2 is applied to preparation of an avian influenza virus inhibitor; the invention discloses application of Y3 in inhibition of avian influenza virus replication. After a mouse is immunized by the M1 recombinant protein, immune factors related to T cells are increased, secretion of proinflammatory factors is reduced, preparation is made for protecting a body from virus attack, and the M1 recombinant protein is also an expression for laterally improving the immunity of a host. The M1 protein has a good immune effect as an immunogen. The invention has application value in treatment of avian influenza virus and similar viruses.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of M1 protein-related substances in the preparation of drugs for diseases caused by avian influenza virus. Background Technology

[0002] Avian influenza virus (AIV) belongs to the genus Influenza A of the family Orthomyxoviridae. It has seasonal epidemic characteristics. Based on the differences in hemagglutinin protein (HA) and neuraminidase (NA) antigens on the surface of the viral particle envelope, it can be divided into 16 HA subtypes and 9 NA subtypes (N1-N9), which can combine in pairs to form a variety of subtype viruses. The H9N2 subtype AIV is a low pathogenic virus with a wide range of hosts, high isolation rate, and persistent harm, making it the most prevalent avian influenza virus in poultry farming [1-2]. During its spread, the H9N2 subtype AIV has undergone widespread mutation [3-4], and has also undergone gene recombination with H7N9, H10N8, or H5N1 zoonotic influenza subtypes, resulting in new genotypes. This has promoted the emergence of AIV recombinant virus pandemics and threatened global public health security [5-6].

[0003] Vaccination is a crucial means of controlling avian influenza, but its effectiveness is limited by viral genetic mutations and subtypes. Inactivated vaccines are the most widely used for avian influenza control, but they have drawbacks. Firstly, the inactivation of the virus may not be complete, leaving the virus potentially harmful. Secondly, inactivated vaccines rely on chicken embryos for production, and production is limited during severe outbreaks due to a shortage of chicken embryos. Furthermore, the long vaccine production cycle generates a large amount of waste chicken embryos, polluting the environment. Therefore, developing novel, safe, and highly effective avian influenza candidate vaccines is of paramount importance.

[0004] References:

[0005] [1] Cong Yanlong, Zhong Ying, Sun Yixue, et al. Research progress on the epidemiology of H9N2 subtype avian influenza virus and its vaccine [J]. Chinese Journal of Veterinary Medicine, 2017, 37(02):386-392.

[0006] [2]Wang J, Cao Z, Guo X, et al. Cytokine expression in three chicken hostsystems infected with H9N2 influenza viruses with different pathogenicities[J]. Avian Pathol. 2016; 45(6):630-639.

[0007] [3]Gao W, Zu Z, Liu J, et al. Prevailing I292V PB2 mutation in avianinfluenza H9N2 virus increases viral polymerase function and attenuates IFN-βinduction in human cell[J]sJ Gen Virol.2019Sep;100(9):1273-1281.

[0008] [4]Sun 14;12(5):541.doi:10.3390 / v12050541.PMID:32423002; PMCID:PMC7290818.

[0009] [5]Pu J, Wang S, Yin Y, et al. Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus[J]. Proc Natl Acad Sci US A. 2015 Jan 13;112(2):548-53.

[0010] [6]Li Summary of the Invention

[0011] The technical problem to be solved by the present invention is how to prepare drugs for preventing and / or treating diseases caused by avian influenza virus or avian influenza virus infection, and / or how to prepare avian influenza virus inhibitors.

[0012] To address the above technical problems, this invention provides the following applications of substances that enhance the activity of M1 protein and / or increase the expression level of the M1 protein gene and / or increase the content of M1 protein:

[0013] The use of Y1 in the preparation of drugs for the prevention and / or treatment of diseases caused by avian influenza virus or avian influenza virus infection;

[0014] Application of Y2 in the preparation of avian influenza virus inhibitors;

[0015] Application of Y3 in inhibiting avian influenza virus replication;

[0016] The M1 protein is a protein that is A1), A2), or A3 as follows:

[0017] A1) The amino acid sequence is the protein consisting of positions 3-254 of SEQ ID No. 2 in the sequence listing;

[0018] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1) that has more than 90% identity with the protein shown in A1) and has the same activity.

[0019] A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0020] In particular, positions 3-254 of SEQ ID No. 2 in the sequence listing consist of 252 amino acid residues.

[0021] In the above applications, the protein can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0022] In the above applications, the protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0023] In the above applications, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can be obtained.

[0024] In the above applications, the 90% or more of identity can be at least 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0025] In the above applications, the substance is a biomaterial related to the M1 protein, and is any one of the following:

[0026] B1) Recombinant M1 protein, wherein the recombinant M1 protein is any one of the following: B11) a protein whose amino acid sequence is positions 3-254 of SEQ ID No. 2; B12) a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein of B11) and having the same function as the protein of B11).

[0027] B2) A nucleic acid molecule encoding the M1 protein or a nucleic acid molecule encoding the recombinant M1 protein;

[0028] B3) An expression cassette containing the nucleic acid molecule described in B2);

[0029] B4) A recombinant vector containing the nucleic acid molecule described in B2), or a recombinant vector containing the expression cassette described in B3);

[0030] B5) Recombinant microorganisms containing the nucleic acid molecules described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4);

[0031] B6) A transgenic animal cell line containing the nucleic acid molecule described in B2), or a transgenic animal cell line containing the expression cassette described in B3), or a transgenic animal cell line containing the recombinant vector described in B4).

[0032] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0033] In the above applications, the nucleic acid molecule encoding the M1 protein is the nucleic acid molecule whose coding sequence is SEQ ID No. 1, positions 7-765; the nucleic acid molecule encoding the recombinant M1 protein is the nucleic acid molecule whose coding sequence is SEQ ID No. 1.

[0034] In the above application, the expression cassette (M1 gene expression cassette) containing the nucleic acid molecule described in B2) refers to a nucleic acid molecule capable of expressing M1 in host cells. This nucleic acid molecule may include not only a promoter to initiate M1 gene transcription but also a terminator to terminate M1 transcription. Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0035] A recombinant expression vector containing the M1 gene expression cassette can be constructed using existing prokaryotic expression vectors. The prokaryotic expression vector can be pET-32a, etc.

[0036] In the above applications, the diseases caused by the avian influenza virus may be avian influenza, etc.

[0037] This invention also protects products that are avian influenza virus inhibitors or drugs for the prevention and / or treatment of diseases caused by avian influenza virus or avian influenza virus infection, wherein the products contain substances that enhance the activity of M1 protein and / or increase the expression level of the M1 protein gene and / or increase the content of M1 protein; wherein the M1 protein is a protein of the following A1), A2), or A3):

[0038] A1) The amino acid sequence is the protein consisting of positions 3-254 of SEQ ID No. 2 in the sequence listing;

[0039] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1) that has more than 80% identity with the protein shown in A1) and has the same activity.

[0040] A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0041] In the above-mentioned products, the substance that enhances the activity of M1 protein and / or increases the expression level of the M1 protein gene and / or increases the content of M1 protein is a biomaterial related to M1 protein, and is any one of the following:

[0042] B1) Recombinant M1 protein, wherein the recombinant M1 protein is any one of the following: B11) a protein whose amino acid sequence is positions 3-254 of SEQ ID No. 2; B12) a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein of B11) and having the same function as the protein of B11).

[0043] B2) A nucleic acid molecule encoding the M1 protein or a nucleic acid molecule encoding the recombinant M1 protein;

[0044] B3) An expression cassette containing the nucleic acid molecule described in B2);

[0045] B4) A recombinant vector containing the nucleic acid molecule described in B2), or a recombinant vector containing the expression cassette described in B3);

[0046] B5) Recombinant microorganisms containing the nucleic acid molecules described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4);

[0047] B6) A transgenic animal cell line containing the nucleic acid molecule described in B2), or a transgenic animal cell line containing the expression cassette described in B3), or a transgenic animal cell line containing the recombinant vector described in B4).

[0048] In the above products, the nucleic acid molecule encoding the M1 protein is the nucleic acid molecule whose coding sequence is SEQ ID No. 1, positions 7-765; the nucleic acid molecule encoding the recombinant M1 protein is the nucleic acid molecule whose coding sequence is SEQ ID No. 1.

[0049] The products described above may be substances that enhance the activity of M1 protein and / or increase the expression level of the M1 protein gene and / or increase the content of M1 protein, and may also contain carriers or excipients.

[0050] The present invention also provides a method for preparing the recombinant M1 protein, comprising: expressing the encoding gene of the recombinant M1 protein in an organism to obtain the recombinant M1 protein; wherein the organism is a microorganism, a plant or a non-human animal.

[0051] In the above method, the organism is a microorganism, and the expression includes introducing the coding gene of the recombinant M1 protein into a recipient microorganism to obtain a recombinant microorganism expressing the coding gene of the recombinant M1 protein, culturing the recombinant microorganism, and expressing the fusion protein.

[0052] In the above method, the coding gene is a nucleic acid molecule whose coding sequence is SEQ ID No. 1.

[0053] The present invention also protects substances that enhance the activity of M1 protein and / or increase the expression level of the M1 protein gene and / or increase the content of M1 protein.

[0054] This invention, by immunizing mice with recombinant M1 protein, elevates T-cell-related immune factors and reduces the secretion of pro-inflammatory factors, preparing the body to protect against viral attack and indirectly enhancing the host's immunity. M1 protein, as an immunogen, exhibits good immune effects. This invention has application value in the treatment of avian influenza virus infection and similar viruses. Attached Figure Description

[0055] Figure 1 The image shows the electrophoresis results of the M1 gene PCR amplification products in Example 1 of this invention, where lane M is the Trans 2K DNA Marker; lanes 1 to 3 are all M1 gene amplification products.

[0056] Figure 2 The results of double enzyme digestion identification of the recombinant vector pET-32a-M1 in Example 1 of this invention are shown. Lane M is the Trans 8K DNA Marker; lanes 1-2 are both double enzyme digestion products of pET-32a-M1.

[0057] Figure 3 The results of SDS-PAGE electrophoresis and solubility analysis of recombinant protein M1 in Example 1 of this invention are shown. Wherein, M is the protein marker; 1 is the pET-32a empty vector; 2 is the total protein; 3 is the soluble protein; and 4 is the insoluble protein.

[0058] Figure 4 The results are Western blot verification results of the recombinant M1 protein in Example 1 of this invention. Wherein, M is the protein marker; 1 is the empty pET-32a vector; and 2 is the pET-32a-M1 vector.

[0059] Figure 5 The level of serum IgG antibodies in M1-immunized mice in Example 1 of this invention.

[0060] Figure 6 The results of multi-cytokine metabolism in mouse spleen cell supernatant immunized with M1 recombinant protein in Example 1 of this invention are shown. * indicates significant difference, and ** indicates extremely significant difference.

[0061] Preservation Instructions

[0062] Bacterial strain name: Avian influenza virus

[0063] Strain ID: A / chicken / Guangxi / CWM / 2019(H9N2)

[0064] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0065] Collection institution abbreviation: CGMCC

[0066] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0067] Deposit date: June 20, 2022

[0068] CGMCC Registration Number: 45210 Detailed Implementation

[0069] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are conventional biochemical reagents and are commercially available.

[0071] In the following examples, the H9N2 subtype avian influenza virus strain A / chicken / Guangxi / CWM / 2019(H9N2) was deposited at the China General Microbiological Culture Collection Center on June 20, 2022, with the collection center registration number CGMCC No. 45210.

[0072] In the examples described below, BALB / c mice were purchased from the Guangdong Animal Center and housed in individually ventilated cages.

[0073] In the following examples, the Gene JET RNA Purification Kit is a product of Gene JET Life Sciences, Inc.

[0074] In the following examples, the complete Freund's adjuvant was a product of Sigma.

[0075] In the following examples, the vectors pMD18-T and pET-32a, the ligase T4 DNA, the restriction endonucleases NotⅠ and EcoRI, and the competent cells DH5α and BL21 were all products of Takara Bio Engineering (Dalian) Co., Ltd.

[0076] In the following examples, the 10-well protein electrophoresis precast gel (15%) and the DAB enhancement reagent were both products of Beijing Solarbio Science & Technology Co., Ltd.

[0077] In the following examples, the His-labeled mouse monoclonal antibody and the HRP-labeled goat anti-mouse IgG (H+L) were both products of Wuhan Sanying Technology Co., Ltd.

[0078] In the following examples, the His-tagged protein purification kit (soluble protein) was a product of Beijing Kangwei Century Co., Ltd.

[0079] In the following examples, the main instruments and their models and manufacturers are as follows: the NanoDrop2000 micro-nucleic acid analyzer is a product of Thermo Fisher Scientific; the Gel Doc™ XR+ gel imaging analyzer is a product of Bio-Rad Scientific, USA; the clean bench (SG404) is a product of Beijing Donglian Haer Instrument Co., Ltd.; the shaking incubator (MQT-60R) is a product of Shanghai Minquan Instrument Co., Ltd.; and the inverted fluorescence microscope (ECLIPSETi2-U) is a product of Nikon Corporation, Japan.

[0080] Unless otherwise specified, all quantitative experiments in the following examples were performed in triplicate, and the results were averaged. GraphPad Prism8 software was used for graphing. Student t-tests were performed on the two groups of data. P < 0.05 was considered statistically significant and marked with *, and P < 0.01 was considered highly statistically significant and marked with **.

[0081] Example 1

[0082] The M1 protein, located inside the viral particle and being the most abundant, connects to the viral envelope and plays a crucial role in maintaining the integrity of the viral particle. It exhibits high conservation across different AIV subtypes and participates in important activities throughout the viral life cycle. Studies have found that the M1 protein participates in avian influenza virus replication and transcription, transports substances from the host cell nucleus and cytoplasm, and participates in the transport of viral ribonucleoproteins during the later stages of replication. The M1 protein is highly conserved across different AIV subtypes, making it an ideal broad-spectrum target protein for universal vaccine research. This embodiment uses pET-32a-M1 to induce recombinant M1 protein expression, providing antigen for immunizing mice. The immunogenicity was explored by measuring neutralizing antibodies and multiple cytokines in mouse spleen cell culture supernatant, providing a reference basis for influenza vaccine development.

[0083] 1. Construction and identification of recombinant expression vector pET-32a-M1

[0084] 1.1 Primer synthesis and coding region sequence amplification

[0085] The amino acid sequence of the M1 protein of the H9N2 subtype avian influenza virus strain (A / chicken / Guangxi / CWM / 2019(H9N2)) is shown in positions 3-254 of SEQ ID No. 2, and the CDS sequence of the M1 gene is shown in positions 7-765 of SEQ ID No. 1. Specific primers were designed using Prime 5.0 software, and restriction enzyme sites were added for PCR amplification. The primer sequences are as follows:

[0086] Upstream primer sequence (5′-3′): CCG GAATTC ATGAGTCTTCTAACCGAGGTCGAAA

[0087] (The underlined area indicates the EcoRI enzyme recognition site);

[0088] Downstream primer sequence (5′-3′) (The wavy line indicates the NotI enzyme recognition site.)

[0089] Primer synthesis was performed by Guangzhou Ruibo Company.

[0090] 1.2 Viral RNA extraction and M1 gene coding region amplification

[0091] Following the instructions of the Gene JET RNA Purification Kit, viral RNA was extracted from the H9N2 subtype avian influenza virus strain (A / chicken / Guangxi / CWM / 2019(H9N2)) and then reversed into cDNA using Takara reverse transcription reagent.

[0092] Using the synthesized cDNA as a template, the M1 gene was amplified. The total reaction volume was 50 μL: 25 μL of 2×TransTaq-TPCR SuperMix, 1 μL each of forward and reverse primers (primer concentration 10 μmol / L), 4 μL of cDNA template, and nuclease-free water to make up to 50 μL.

[0093] Reaction procedure: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 56℃ annealing for 1 min, 72℃ extension for 1 min, 34 cycles; 72℃ final extension for 5 min. PCR products were separated by 1.5% agarose gel electrophoresis.

[0094] The results showed that the M1 gene PCR amplification yielded a band of approximately 759 bp, consistent with the expected size. (See attached image) Figure 1 The target band is extracted and recovered from the gel, yielding the M1 gene fragment.

[0095] The M1 gene fragment was ligated into the pMD18-T vector, transformed into competent DH5α cells, identified by double enzyme digestion, and sent to BGI for sequencing. The vector with completely accurate alignment was named T-M1 and stored at -20℃ for later use.

[0096] 1.3 Construction of expression vectors

[0097] Following the EcoRI and NotI restriction enzyme digestion methods, the T-M1 vector obtained in step 1.2 was double-digested in a 50 μL digestion system at 37 °C for 4 h. The digestion products were then separated by 1.0% agarose gel electrophoresis to recover the target fragment.

[0098] Following the EcoRI and NotI restriction enzyme digestion methods, the pET-32a(+) vector (Takara Bio Engineering (Dalian) Co., Ltd.) was double-digested with 50 μL of enzyme digestion system at 37 °C for 4 h. The digestion products were separated by 1.0% agarose gel, and the linearized pET-32a(+) vector was recovered.

[0099] The linearized pET-32a(+) vector and the target fragment were ligated overnight at 16°C in a 1:3 ratio. The ligation product was then transformed into competent BL21 cells, plated, and cultured overnight. Single colonies were selected for double enzyme digestion and sequencing verification.

[0100] The results of the double enzyme verification are shown below. Figure 2 Two bands were obtained after double enzyme digestion of the recombinant expression vector. Analysis of the band size showed that it was consistent with the size of the target gene fragment (759bp) and the vector fragment (5900bp), indicating that the target fragment of the M1 gene was successfully inserted into the pET-32a(+) vector.

[0101] Sequencing results showed that the target fragment was successfully inserted into the pET-32a(+) vector. Specifically, the fragment between the EcoRI and NotI recognition sites of the restriction endonucleases in the pET-32a(+) vector (including the small fragment containing both EcoRI and NotI recognition sites) was replaced by the M1 gene fragment (nucleotide sequence shown in SEQ ID No. 1 of the sequence listing), while keeping the other sequences of the pET-32a(+) vector unchanged, resulting in a recombinant expression vector for the M1 protein. The correctly sequenced vector was named pET-32a-M1.

[0102] The pET-32a-M1 expresses the M1 recombinant protein (recombinant influenza M1 protein) whose amino acid sequence is SEQ ID No. 2 (containing the amino acid sequence encoding the protein of the M1 gene with the insertion of pET-32a(+) and the amino acid sequence encoded by the nucleotide sequence fused to pET-32a(+)). pET-32a-M1 contains a His-tagged M1 recombinant protein gene, the nucleotide sequence (CDS) of which is SEQ ID No. 1, encoding the M1 recombinant protein shown in SEQ ID No. 2. In SEQ ID No. 1, positions 1-2 are the sequence from the start codon to the EcoRI recognition site on pET-32a(+), and positions 3-254 are the M1 protein gene sequence.

[0103] 2. Target protein induction and solubility analysis

[0104] The correctly sequenced positive colonies pET-32a-M1 were expanded and inoculated at a ratio of 1% into 50 mL LB broth. The culture was carried out at 37°C with a shaker at 200 rpm until the OD value reached 0.6–0.8. IPTG was then added to a final concentration of 1.0 mmol / L, and the culture was continued at 37°C for 6 h. The cells were collected by centrifugation at 4000 rpm for 5 min, washed twice with PBS, and resuspended with appropriate amounts of RIPA bacterial lysis buffer and PMSF. The mixture was then placed on ice for lysis for 30 min, followed by sonication until clear. The total protein was collected, and after centrifugation, the supernatant (soluble protein) and the precipitate (insoluble protein) were subjected to SDS-PAGE electrophoresis. Using the pET-32a(+) empty vector as a control, the cells were stained with Coomassie brilliant blue, destained with water, and the protein forms were analyzed.

[0105] See results Figure 3 The M1 recombinant protein is well expressed in E. coli, mainly in the supernatant, and is a soluble protein. The fusion protein is approximately 49 kDa (including the tag protein).

[0106] 3. Western blot identification of recombinant proteins

[0107] After separation by SDS-PAGE, the proteins were transferred to a PVDF membrane using a "sandwich" method via a transfer apparatus. The membrane was blocked with 5% skim milk at room temperature for 4 hours, followed by incubation at 4°C overnight with His-labeled mouse monoclonal antibody (1:5000). The primary antibody was discarded, and the membrane was washed four times with PBST. Then, HRP-labeled goat anti-mouse IgG (H+L) secondary antibody (1:2000) was added, and the membrane was incubated at room temperature for 1 hour. After washing four times with PBST, DAB-enhanced reagent was added for light-protected development for 1–5 minutes, followed by imaging using a BIO-RAD imaging system. pET-32a(+) empty vector control was used.

[0108] Western blot results showed that the recombinant M1 protein and the His-labeled mouse monoclonal antibody exhibited a specific band at 49 kDa, while the control empty vector did not show a corresponding band at this location, indicating that the recombinant influenza M1 protein had good reactivity. Figure 4 .

[0109] 4. Immunization of mice with M1 recombinant protein and detection of serum IgG antibody levels in mice by ELISA

[0110] After purifying the M1 protein solution, it was dialyzed (the supernatant from step 2 was purified using a His-tagged protein purification kit) and mixed with complete Freund's adjuvant. The mixture was then fully emulsified to achieve an M1 protein concentration of 100 μg / mL, resulting in a mixture of M1 and complete Freund's adjuvant.

[0111] The M1 protein solution was replaced with PBS and mixed with complete Freund's adjuvant. The volume of PBS used was the same as that of the M1 protein solution to obtain a mixture of PBS and complete Freund's adjuvant.

[0112] Ten 8-week-old BALB / c mice were randomly divided into two groups:

[0113] Treatment group: Subcutaneous injection of a mixture of M1 and complete Freund's adjuvant, 1.0 mL / animal, 14 days later, followed by a second booster immunization.

[0114] Control group: Subcutaneous injection of a mixture of PBS and Freund's adjuvant, 1.0 mL / animal, 14 days later, for the second immunization.

[0115] Fourteen days after booster immunization, blood was collected from the orbital cavity to separate serum. The serum was incubated at 37°C for one hour, then incubated at 4°C overnight, centrifuged at 3000 r / min for 10 min at 4°C, collected from mice, aliquoted and labeled, and stored at -20°C.

[0116] The ELISA method is as follows:

[0117] (1) The supernatant from step 2 was purified using a His-tagged protein purification kit to obtain purified M1 protein, which was diluted to 10 μg / mL with ELISA coating buffer and aliquoted into 96-well ELISA plates, 100 μL / well, and incubated overnight at 4°C.

[0118] (2) Wash the plate with PBST, 200 μL / well, 3 times, and pat dry. Block with 1% bovine serum albumin (BSA), 100 μL / well, and incubate at 37°C for 1 h.

[0119] (3) Wash the plates as above. Dilute mouse serum with PBST from 1:1,000, 1:2,000, 1:3,000, 1:4,000, 1:5,000, 1:6,000, 1:7,000, 1:8,000, 1:9,000, 1:10,000, 1:20,000, 1:30,000, 1:40,000, 1:50,000, 1:60,000, 1:70,000, 1:80,000, 1:90,000 to 1:100,000. Each concentration was repeated in 3 wells. A control was set up using negative mouse serum. The blank control was incubated with PBST at 37°C for 1 hour.

[0120] (4) Wash the plate 3 times, dilute the secondary antibody (goat anti-mouse IgG) with PBST 1:4000, and incubate at 37℃ for 1h.

[0121] (5) Wash the plate 3 times, add 100 μL of colorimetric reagent per well, incubate at room temperature in the dark for 10 min, and then add 50 μL of stop solution per well.

[0122] (6) OD measurement using an enzyme-linked immunosorbent assay (ELISA) reader 450 The maximum dilution with a positive serum OD value (P) / negative serum OD value (N) > 2 is the mouse serum IgG antibody titer.

[0123] The results of the ELISA test are shown below. Figure 5 The results showed that the serum of immunized mice produced a certain amount of IgG antibodies with a titer of 1:30000, indicating that M1 recombinant protein immunization of mice produced specific IgG binding antibodies.

[0124] 5. Luminex assay for multiple cytokines in mouse spleen cell culture supernatant

[0125] (1) The mice in step 4 were sacrificed on the 14th day after booster immunization, soaked in 75% alcohol for 5 minutes, the abdominal skin of the mice was cut open, and the spleen was removed with tweezers.

[0126] (2) Place the spleen in a 100μm cell sieve, add 2mL of DMEM culture medium, and grind the spleen thoroughly with a 10mL syringe tip.

[0127] (3) Add 3 mL of DMEM to clean the syringe and cell sieve, and transfer all the filtrate to a 15 mL centrifuge tube.

[0128] (4) Centrifuge at 1500 r / min for 5 min, discard the supernatant, and wash the spleen cells once with DMEM.

[0129] (5) Centrifuge for 5 min, discard the supernatant, add 5 mL of red blood cell lysis buffer to resuspend the cells, place at room temperature for 5 min, add culture medium to stop lysis, centrifuge and discard the supernatant.

[0130] (6) Resuspend spleen cells in 5 mL of culture medium, measure cell density, and seed into 6-well plates. Add 2 mL of HA protein (10 μg / mL) as a stimulant to each well. Include a control well without stimulant in each group. Incubate cells at 37°C with 5% CO2 for 72 h. Collect cell supernatant and send it to Shanghai Laids Biotechnology Co., Ltd. for cytokine detection.

[0131] See results Figure 6 The results showed that the levels of cytokines IL-2 (108.17 pg / mL), IL-4 (11.05 pg / mL), and IL-6 (6617.96 pg / mL) in the spleen cell supernatant of M1-immunized mice were significantly higher than those in the control group (9.91, 3.08, and 4011.25 pg / mL, respectively) (p<0.01). Compared with the control group, the secretion of IL-1β, IL-18, and IFN-γ in the spleen cell culture supernatant of the M1-immunized group was significantly lower (p<0.01), while the secretion of IL-5, IL-12p70, and GM-CMF was significantly lower (p<0.05), and the secretion of IL-13 and TNF-α showed no significant difference (p>0.05). This indicates that the level of T cell-related immune factors increases and the secretion of pro-inflammatory factors decreases after immunization, preparing the body to protect against viral attack.

[0132] Avian influenza viruses are prevalent worldwide, threatening the healthy development of the livestock industry. The H9N2 subtype of influenza virus has a wide host range and a high prevalence rate, and during transmission, it recombines with other subtypes of influenza virus to produce new subtypes. The M1 protein is the matrix protein of type A influenza virus and is highly conserved across all subtypes of type A influenza virus.

[0133] Compared to traditional seasonal influenza vaccines, recombinant antigen proteins offer several advantages as immunogens for next-generation influenza vaccines. Current seasonal influenza vaccines are typically produced using eggs. This production method requires a long preparation time, and for human influenza vaccines, patients with egg allergies cannot receive this type of vaccine. Furthermore, egg supplies can be very limited during influenza pandemics. Recombinant antigen proteins are obtained through genetic engineering techniques. Genes with multiple antigenic sites are cloned into vectors, transferred into bacteria or cells, and induced to express, yielding a large quantity of a single antigen. This technology is mature, and the resulting antigens are highly safe for humans and animals. In this embodiment, the H9 subtype M1 protein is expressed in prokaryotes. The recombinant protein exists in a soluble form, creating favorable conditions for purification.

[0134] Subunit vaccines induce humoral and cellular immune responses in the host after immunization, and a single vaccination can provide longer-lasting immune protection. In this example, mice were immunized twice with M1 recombinant protein. Two weeks later, specific IgG antibodies were produced in the serum, with an antibody titer of 1:30,000, indicating that M1 recombinant protein, as a genetically engineered subunit vaccine, elicited a good immune response in mice. T-cell-mediated cellular immunity is a current hot topic in vaccine research and a research direction for universal influenza vaccines. Influenza vaccines induce T-cell immune responses, which can produce broad-spectrum protective effects. After immunizing mice with M1 recombinant protein, mouse spleen cells were isolated and stimulated with M1 recombinant protein. The results showed that the levels of IL-2, IL-4, and IL-6 in the supernatant of mouse spleen cells were significantly increased compared with the control group (p<0.01). The secretion of IL-1β, IL-18, and IFN-γ was significantly reduced (p<0.01). IL-1β and IL-18 belong to the IL-1 family and are pro-inflammatory cytokines. In the early stages of viral infection, IL-1β and IL-18 play a protective role by promoting CD8+ T cell activity and inducing antibody secretion. However, excessive production can disrupt this beneficial outcome. IFN-γ's main function is antiviral, but excessive production also leads to adverse consequences. Studies have found that IFN-γ plays an important role in acute lung injury caused by H1N1 virus infection. The secretion of IL-5, IL-12p70, and GM-CMF was significantly reduced (p<0.05), while the reduction in IL-13 and TNF-α secretion was not significant (p>0.05). TNF-α is also a typical pro-inflammatory cytokine, and studies have shown that TNF-α exacerbates the severity of influenza damage during H5N1 AIV infection. Inflammatory factors can provide protection during viral infection, but excessive accumulation can lead to inflammatory damage and acute death in the host. This indicates that immunization of mice with recombinant M1 protein reduces the accumulation of inflammatory factors, thereby mitigating the damage caused by viral infection and indirectly enhancing the host's immunity. The M1 protein, as an immunogen, exhibits good immune effects.

[0135] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

[0136] sequence list

[0137] SEQ ID No. 1 (underlined indicates EcoRI enzyme recognition site, wavy line indicates NotI enzyme recognition site):

[0138]

[0139]

[0140] SEQ ID No. 2:

[0141] EFMSLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNADLEALMEWIKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYKKLKREMTFHGAKEVALSYSTGALA SCMGLIYNRMGTVTAEGALGLVCATCEQIADAQHRSHRQMATTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEVASQARQMVQAMRTVGTHPNSSTGLKDDLIENLQAYQNRMGVQLQRFK.

Claims

1. The application of substances that enhance the activity of M1 protein and / or increase the expression level of the M1 protein gene and / or increase the content of M1 protein, wherein the application is any one of the following: The use of Y1 in the preparation of drugs for the prevention and / or treatment of diseases caused by avian influenza virus or avian influenza virus infection; Application of Y2 in the preparation of avian influenza virus inhibitors; Application of Y3 in inhibiting avian influenza virus replication; The M1 protein is a protein that is A1), A2), or A3 as follows: A1) The amino acid sequence is the protein located at positions 3-254 of SEQ ID No. 2 in the sequence listing; A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1) that has more than 80% identity with the protein shown in A1) and has the same activity. A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

2. The application according to claim 1, characterized in that: The substance is any one of the following: B1) Recombinant M1 protein, wherein the recombinant M1 protein is any one of the following: B11) a protein whose amino acid sequence is positions 3-254 of SEQ ID No. 2; B12) a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein of B11) and having the same function as the protein of B11). B2) A nucleic acid molecule encoding the M1 protein or a nucleic acid molecule encoding the recombinant M1 protein; B3) An expression cassette containing the nucleic acid molecule described in B2); B4) A recombinant vector containing the nucleic acid molecule described in B2), or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the nucleic acid molecules described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic animal cell line containing the nucleic acid molecule described in B2), or a transgenic animal cell line containing the expression cassette described in B3), or a transgenic animal cell line containing the recombinant vector described in B4).

3. The application according to claim 2, characterized in that: The nucleic acid molecule encoding the M1 protein is the nucleic acid molecule whose coding sequence is SEQ ID No. 1, positions 7-765; the nucleic acid molecule encoding the recombinant M1 protein is the nucleic acid molecule whose coding sequence is SEQ ID No.

1.

4. A product, wherein the product is an avian influenza virus inhibitor or a drug for the prevention and / or treatment of diseases caused by avian influenza virus or avian influenza virus infection, characterized in that: The product contains substances that enhance the activity of M1 protein and / or increase the expression level of the M1 protein gene and / or increase the content of M1 protein; the M1 protein is a protein of the following type A1), A2), or A3): A1) The amino acid sequence is the protein located at positions 3-254 of SEQ ID No. 2 in the sequence listing; A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1) that has more than 80% identity with the protein shown in A1) and has the same activity. A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

5. The product according to claim 4, characterized in that: The substance is the substance described in claim 2.

6. The product according to claim 5, characterized in that: The nucleic acid molecule encoding the M1 protein is the nucleic acid molecule whose coding sequence is positions 7-765 of SEQ ID No. 1; the nucleic acid molecule encoding the recombinant M1 protein has the coding sequence of SEQ ID No.

1.

7. A method for preparing the recombinant M1 protein of claim 2, comprising: The recombinant M1 protein is obtained by expressing the gene encoding the recombinant M1 protein in an organism, wherein the organism is a microorganism, plant, or non-human animal.

8. The method according to claim 7, characterized in that: The organism is a microorganism, and the expression includes introducing the coding gene of the recombinant M1 protein into a recipient microorganism to obtain a recombinant microorganism expressing the coding gene of the recombinant M1 protein, culturing the recombinant microorganism, and expressing the fusion protein.

9. The method according to claim 8, characterized in that: The coding gene is a nucleic acid molecule whose coding sequence is SEQ ID No.

1.

10. The substance as described in claim 2.

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