H9N2 influenza virus M1 protein mutant and application thereof

Abstract

The invention discloses an H9N2 influenza virus M1 protein mutant and application thereof, and belongs to the technical field of biology. The H9N2 influenza virus M1 protein mutant is a protein obtained by mutating histidine H at the 222 position of an amino acid sequence of H9N2 influenza virus M1 protein into glutamine Q and keeping other amino acid residues unchanged, and the amino acid sequence of the H9N2 influenza virus M1 protein mutant is as shown in SEQ ID NO. 1. After a mouse is infected with the M1-H2222Q point mutation recombinant virus obtained by a reverse genetics technology, the weight loss of the mouse is obvious, the replication ability of the virus in the lung of the mouse is enhanced, and the pathological change is obvious. The M1 protein mutant disclosed by the invention can be used for rapidly obtaining the mouse adaptive H9N2 influenza virus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an H9N2 influenza virus M1 protein mutant and application thereof. BACKGROUND

[0002] In 1966, H9N2 influenza virus was first detected in domestic poultry in the United States, and in the following decades, migratory birds spread it to the Eurasian continent, the Middle East and Africa, and gradually became the dominant subtype in poultry and wild birds. H9N2 also repeatedly spread to mammals, including humans. Studies have shown that H9N2 influenza virus can act as a "donor" to provide internal genes for some highly pathogenic avian influenza viruses (such as H5N1, H5N6, H7N9 and H10N8).

[0003] Mice are ideal animal models for studying the pathogenic mechanism and host range determinants of influenza viruses. Some influenza virus strains cannot naturally infect and replicate in many mouse strains, which adds difficulty to the development of small animal infection models for vaccine and therapeutic research of influenza viruses. Although mouse-adapted mutants can be generated by continuous lung-lung passage, this process is time-consuming, and it usually takes 10 or more repeated continuous lung passages to obtain a mouse-adapted strain, and sometimes more than 30 passages are required to obtain a mouse lung-adapted strain. Wild-type H9N2 influenza virus usually only causes mild infection in mice, and the clinical symptoms are not obvious. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings and deficiencies existing in the prior art, and to provide an H9N2 influenza virus M1 protein mutant and application thereof. The M1 protein mutant is an M1-H2222Q mutant, which is a point mutation recombinant virus obtained by reverse genetics technology. After infecting mice, the mice have obvious weight loss, the virus replication ability in the lungs of mice is enhanced, and the pathological changes are obvious. The M1 protein mutant of the present application can be used to quickly obtain a mouse-adapted H9N2 influenza virus.

[0005] The purpose of the present application is achieved by the following technical solutions The present application provides an M1 protein mutant, which is a protein obtained by mutating the histidine H at position 222 of the amino acid sequence of the influenza virus M1 protein to glutamine Q, and other amino acid residues remain unchanged.

[0006] In the above-mentioned M1 protein mutant, the influenza virus M1 protein is derived from H9N2 influenza virus. The amino acid sequence of the M1 protein mutant is preferably as shown in SEQ ID NO. 1.

[0007] The present application also provides a gene encoding the above-mentioned M1 protein mutant, and the nucleotide is preferably as shown in SEQ ID NO. 2.

[0008] The present application also provides a biological material related to the above-mentioned M1 protein mutant, which comprises any one of the following (1) to (4): (1) a nucleic acid molecule encoding the M1 protein mutant; (2) an expression cassette containing the nucleic acid molecule of (1); (3) a recombinant vector containing the nucleic acid molecule of (1), or a recombinant vector containing the expression cassette of (2); (4) a host cell containing the nucleic acid molecule of (1), or a host cell containing the expression cassette of (2), or a host cell containing the recombinant vector of (3).

[0009] The present application also provides the use of the above-mentioned M1 protein mutant, or the above-mentioned gene, or the above-mentioned biological material in increasing the replication ability of influenza virus.

[0010] The present application also provides the use of the above-mentioned M1 protein mutant, or the above-mentioned gene, or the above-mentioned biological material in preparing a recombinant influenza virus. The recombinant influenza virus is an influenza virus with enhanced replication ability, or the recombinant influenza virus is an influenza virus mouse lung-adapted strain.

[0011] The present application also provides a recombinant influenza virus containing a gene encoding the above-mentioned M1 protein mutant. The expressed M1 protein of the recombinant influenza virus is the above-mentioned M1 protein mutant, i.e. the recombinant influenza virus mutates the amino acid at position 222 of the wild-type influenza virus M1 protein to glutamine Q compared with the wild-type influenza virus.

[0012] The substance for mutating the amino acid at position 222 of the influenza virus M protein to glutamine Q is specifically a primer for mutating the plasmid of the influenza virus M protein to a plasmid carrying the influenza virus M protein mutant; The above-mentioned primer is specifically as follows in the examples: mH222Q-F: GGGACTCAaCCTAACTCCAGTACTGGTCTAAAGGA (SEQ ID NO. 3), mH222Q-R: GAGTTAGGtTGAGTCCCGACTGTTCTCATAGC (SEQ ID NO. 4).

[0013] The present application also provides the use of the above-mentioned recombinant influenza virus in constructing an influenza virus mouse infection model.

[0014] The present application also provides a method for constructing an influenza virus mouse infection model, which comprises the step of infecting a mouse with the above-mentioned recombinant influenza virus.

[0015] Further, the mode of infection is nasal drop infection.

[0016] Further, the nasal drop infection dose is: 1×10 5 PFU-1×10 7 / 50µL / mouse.

[0017] The present application has the following advantages and beneficial effects relative to the prior art: the M1-H2222Q mutation improves the replication ability of the H9N2 influenza virus in mice, so that the H9N2 influenza virus can effectively amplify in mice without the traditional mouse lung adaptation process, and can be used as a tool strain for establishing a mouse infection model of the H9N2 influenza virus. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The M1-H222Q virus mutation site was identified. The sequencing results were compared with the nucleotide sequence of the M gene using SnapGene software, and the 666th base was mutated from C to A, i.e., the 222nd amino acid of the M1 protein was mutated from histidine (CAC) to glutamine (CAA).

[0019] Figure 2 The growth curve of the virus on A549 cells. Statistical analysis was performed using GraphPad Prism software, and the analysis method was selected as Two-way ANOVA, and all experiments were repeated three times. P <0.05, P <0.01, P <0.001, P <0.0001. WT represents the wild-type virus.

[0020] Figure 3 The effect of the M1-H222Q mutation on the pathogenicity of mice. (A) The change in the body weight of mice after infection with the recombinant virus. (B) The change in the viral load in the lungs of mice after infection with the recombinant virus. The statistical method was mutiple t-test, P <0.05, P <0.01, P <0.001, P <0.0001. (C) Lung histopathology of mice infected with wild-type and M1-H222Q mutant viruses at 3 d.p.i. The pictures are 10-fold magnified, and the bar in the figure represents 100 μm. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present application will now be described in detail, with reference to the figures. The detailed description is merely intended to teach a few examples of the present application and is not intended to put limits on the scope of the application. Rather, the scope of the present application is only limited by the appended claims.

[0022] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise stated, each intervening value of the parameter is also specifically included within the scope of the present application. The intervening values of the parameter are combined with a stated value of the parameter in range form. These are only examples of the various values of the parameter, which are encompassed by the present application. Other examples of the disclosed and claimed inventive aspects will be apparent to those of ordinary skill in the art from consideration of the specification and practice of the application disclosed herein.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited.

[0024] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Additional implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. For example, the methods and materials described herein can be used in a variety of applications including, but not limited to, the applications described herein. The specification and examples given herein are exemplary only. It is therefore contemplated to cover any and all modifications, variations, combinations or equivalents that fall within the scope of the present application. It is intended that the scope of the present application shall include all changes and modifications that come within the meaning and range of equivalents of the claims.

[0025] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0026] The viruses, cells, strains and plasmid vectors used in the present application are as follows: Virus: The influenza virus strain used is A / Chicken / Hunan / 8.27YYGK3W3-OC / 2018 (H9N2), referred to as 3W3 (EPI_ISL_699478), which was isolated by the applicant from a poultry market (Bi Y et al. Dominant subtype switch in avian influenza viruses during 2016-2019 in China. Nat Commun. 2020 Nov 20;11(1):5909. doi: 10.1038 / s41467-020-19671-3. PMID: 33219213; PMCID: PMC7679419.) and stored at -80°C.

[0027] Cells: MDCK cells, A549 cells, 293T cells.

[0028] Strains: Escherichia coli (E. coli) DH5a competent cells. Escherichia coli

[0029] Plasmid vector: 3W3 influenza virus genome bidirectional transcription vector PHW2000, including PB2, PB1, PA, HA, NP, NA, M, NS eight gene fragments, respectively referred to as PHW-PB2, PHW-PB1, PHW-PA, PHW-HA, PHW-NP, PHW-NA, PHW-M, PHW-NS, the construction method is referred to in the reference E Hoffmann et al. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A. 2000 May 23;97(11):6108-13. doi: 10.1073 / pnas.100133697. PMID: 10801978; PMCID: PMC18566.

[0030] Example 1: Rescue of recombinant virus (1) Construction of point mutation plasmid Point mutation was performed on the PHW-M plasmid to mutate the histidine at position 222 of the M1 protein to glutamine. The point mutation primers are mH222Q-F and mH222Q-R.

[0031] mH222Q-F: GGGACTCAaCCTAACTCCAGTACTGGTCTAAAGGA (SEQ ID NO. 3); ​mH222Q-R: GAGTTAGGtTGAGTCCCGACTGTTCTCATAGC (SEQ ID NO. 4).

[0032] The point mutation plasmid was amplified by PCR with PHW-M plasmid as a template according to the following PCR system:

[0033] The reaction conditions were as follows: (1) 98°C for 2 min; (2) 98°C for 10 s, (3) 61°C for 30 s, (4) 68°C for 5 min 30 s, (5) GO to (2) for 35 cycles; (6) 68°C for 5 min, 8°C hold.

[0034] After the reaction, 2 µL DpnI digestion enzyme was used to remove the template plasmid, and after 37°C digestion overnight, agarose gel electrophoresis was performed and the gel was recovered. 100 ng of the recovered product was used to connect Exnase II homologous recombination enzyme at 37°C for 30 min, and then transformed into E. coli DH5α competent cells. After 16 h, three single colonies were picked and shaken, and sent to Genescript (Wuhan) Biotechnology Co., Ltd. for Sanger sequencing bacterial liquid identification. The positive colonies with correct sequencing were cultured and expanded, and the plasmid was extracted by using a plasmid extraction kit, and the PHW-M1-H222Q plasmid was obtained for subsequent experiments.

[0035] The amino acid sequence of the M1-H222Q mutant (SEQ ID NO. 1): MSLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNADLEALMEWIKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYKKLKREMTFHGAKEVALSYSTGALASCMGLIYNRMGTVTAEGALGLVCATCEQIADAQHRSHRQMATTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEVASQARQMVQAMRTVGTQPNSSTGLKDDLIENLQAYQNRMGVQLQRFK .

[0036] The nucleotide sequence encoding the M1-H222Q mutant (SEQ ID NO. 2): atgagccttctaaccgaggtcgaaacgtacgttctctctatcattccatcaggccccctcaaagccgagatcgcgcagagacttgaggatgtttttgcagggaagaacgcagatctcgaggctctcatggagtggataaagacaagaccaatcctgtcacctctgactaaggggattttagggtttgtgtttacgctcaccgtgcccagtgagcgaggactgcagcgtagacggtttgtccaaaacgccctaaatgggaatggagacccaaacaacatggacaaggcagttaaattatacaagaaactgaagagggagatgacatttcatggagcaaaggaagttgcactcagttactcaactggtgcgcttgccagctgcatgggtctcatatacaacaggatggggacagtaactgcagaaggggctcttggactggtatgtgctacttgtgagcagattgctgacgcacaacatcggtcccacagacagatggcaactactaccaacccactaattaggcatgagaatagaatggtactagccagtactacggctaaggctatggagcagatggctggatcaagtgagcaggcagcggaagccatggaagtcgcaagccaggctaggcaaatggtgcaggctatgagaacagtcgggactcaacctaactccagtactggtctaaaggatgatcttattgaaaatttgcaggcttaccagaaccggatgggagtgcaactgcagcggttcaagtga.

[0037] (2) Virus Rescue The 293T cells were inoculated in 6-well plates in advance, so that the density was 80-90% when used, and transfected according to the lipo8000 transfection reagent instruction. 500 ng of each of the 8 fragment plasmids of the virus was transfected (among them, PHW-M plasmid and PHW-M1-H222Q plasmid were used respectively to rescue wild type and M1-H222Q site mutant virus). 24 h after transfection, the culture medium was discarded, washed twice with PBS, then 2 mL of virus maintenance liquid (DMEM + 0.3% BSA + 1% P.S. + 1 mM HEPES) was added to each well, and TPCK-treated trypsin was added to a final concentration of 0.5 μg / mL. After 48 h of continuous culture, the cell supernatant was collected, centrifuged at 12000 g at 4°C for 5 min to remove cell debris, and the supernatant was the rescued virus.

[0038] The cell supernatant obtained in the previous step was used to inoculate 2 10-day-old SPF chicken embryos, 200 μL per embryo. After 72 h, the allantoic fluid of the embryos was collected, the precipitate was removed at 12000 rpm at 4°C, and the supernatant was the rescued virus. The virus liquid was aliquoted and stored at -80°C for standby.

[0039] (3) Virus sequence verification After the virus was rescued by reverse genetics, the viral RNA was extracted by a kit and reversed to cDNA, PCR amplification was performed with detection primers (detection primer sequences: M1check-F: ATGAGCCTTCTAACCGAGGTCG (SEQ ID NO. 5); M1check-R: GTAAGCCTGCAAATTTTCAATAAGAT (SEQ ID NO. 6)), and the amplification product was sent to GenScript Biotech Co., Ltd. for sequencing identification. The results are shown in Figure 1 The sequencing results were aligned with the M gene nucleotide sequence using SnapGene software. The 666th base was mutated from C to A, i.e., the 222nd amino acid of the M1 protein was mutated from histidine (CAC) to glutamine (CAA), and no mutation occurred at other sites.

[0040] Example 2: Virus growth curve determination A549 cells were plated in a 24-well plate at a density of 2 x 10 5 The cells were plated in a 24-well plate at a density of 2 x 10 Figure 2As shown, the replication efficiency of the M1-H222Q virus at the four time points was significantly higher than that of the wild-type (WT) virus, indicating that the M1-H222Q point mutation improved the virus's replication ability in mammalian cell lines.

[0041] Example 3: Effect of virus pathogenicity on mice The virus was diluted to 1 × 10 5 PFU / 50µL. Eight BALB / c mice (4-6 weeks old) were anesthetized by intraperitoneal injection of 2.5% Avertin at 150µL / 10g. Each mouse was intranasally infected with 50µL of diluted wild-type or M1-H222Q mutant virus. A separate group of mice received PBS intranasally as a control. Three days after infection, lungs from three mice were homogenized in a pre-prepared extraction tube containing 400µL of 0.1% BSA-PBS solution. The homogenate was centrifuged at 12,000g for 15 minutes at 4°C, and the supernatant was collected and used for plaque assays using MDCK cells to determine lung virus titers. A small amount of lung tissue was fixed in a centrifuge tube with 5mL of tissue fixative for tissue sectioning and HE staining. Five mice per group were left for body weight monitoring. The mice were weighed and their status recorded daily at the same time until 14 days after infection.

[0042] like Figure 3 As shown, the virus titer in the lungs of mice infected with the M1-H222Q mutant virus was significantly higher than that of mice infected with the wild-type virus. Weight monitoring results also showed that mice infected with the wild-type virus had fully recovered their weight on day 7, while the weight of mice infected with the M1-H222Q mutant virus had dropped to 83.1% of the original weight. Statistical analysis data also showed that from day 4 to day 14, there was a significant difference in the weight of mice in the mutant virus group and the wild-type virus group ( P). Mice infected with the mutant virus showed obvious clinical symptoms after day 4: piloerection and weight loss, slow movement, and crouching in the corner, and the symptoms gradually recovered at the later stage of infection, while mice infected with the wild-type virus did not show obvious clinical symptoms after infection. Lung histopathology also showed that mice infected with M1-H222Q mutant virus showed thickening of alveolar walls, partial obstruction, and inflammatory cell infiltration. These results showed that the M1 protein H222Q mutation enhanced the replication ability of the virus in mice, and the M1-H222Q mutant virus could effectively replicate in the lungs of mice without mouse lung adaptation, causing weight loss in mice and obvious clinical symptoms (similar to human influenza virus infection). Therefore, the above results show that the M1 protein H222Q mutation increases the adaptability of the virus in mice, so that the mutant virus obtained can directly infect mice without the traditional mouse lung adaptation method and cause obvious clinical symptoms, indicating the feasibility of using the mutant virus as a tool virus to construct a mouse infection model of H9N2 influenza virus.

[0043] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application as defined by the claims.

Claims

1. A mutant of the H9N2 influenza virus M1 protein, characterized in that: The protein is obtained by mutating the 222nd histidine H in the amino acid sequence of the H9N2 influenza virus M1 protein to glutamine Q, while the other amino acid residues remain unchanged.

2. The M1 protein mutant according to claim 1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

3. A gene encoding the M1 protein mutant according to claim 1 or 2.

4. A biomaterial, characterized in that: Any of the following (1) to (4): (1) A nucleic acid molecule encoding the M1 protein mutant according to claim 1 or 2; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule described in (1), or a recombinant vector containing the expression cassette described in (2); (4) A host cell containing the nucleic acid molecule described in (1), or a host cell containing the expression cassette described in (2), or a host cell containing the recombinant vector described in (3).

5. Use of the M1 protein mutant according to claim 1 or 2, the gene according to claim 3, or the biomaterial according to claim 4 for increasing the replication ability of influenza virus.

6. Use of the M1 protein mutant according to claim 1 or 2, the gene according to claim 3, or the biological material according to claim 4 in preparing a recombinant influenza virus.

7. The use according to claim 6, characterized in that: The recombinant influenza virus is an influenza virus with enhanced replication ability, or the recombinant influenza virus is an influenza virus mouse lung adapted strain.

8. A recombinant influenza virus, characterized in that: Contains a gene encoding the M1 protein mutant according to claim 1 or 2.

9. Use of the recombinant influenza virus according to claim 8 in constructing an influenza virus infection mouse model.

10. A method for constructing an influenza virus infection model in mice, characterized by: The method comprises the step of infecting mice with the recombinant influenza virus according to claim 8.

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