Application of mGluR2 gene

Abstract

The invention discloses application of siRNA (small interfering Ribonucleic Acid) of a targeted mGluR2 gene, a monoclonal antibody for resisting an extracellular domain of a protein coded by the mGluR2 gene, a truncated peptide containing the extracellular domain of the protein coded by the mGluR2 gene or a recombinant vector containing a gRNA (guide Ribonucleic Acid) gene sequence and a CAS9 protein gene sequence of the targeted mGluR2 gene in preparation of a medicine for inhibiting influenza virus, swine acute diarrhea syndrome coronavirus, swine acute diarrhea syndrome coronavirus, swine acute diarrhea syndrome coronavirus, swine acute diarrhea syndrome coronavirus, swine acute diarrhea syndrome coronavirus, swine acute diarrhea syndrome coronavirus, swine acute diarrhea syndrome coronavirus, swine acute diarrhea syndrome coronavirus, swine acute The invention also relates to application of the compound in preparations for treating porcine epidemic diarrhea virus, Japanese encephalitis virus and porcine reproductive and respiratory syndrome virus. The invention also discloses an application of the gene engineering operation method for knocking out the mGluR2 gene in preparation of a new mouse strain for improving influenza resistance. The application has a popularization prospect.

Description

Technical Field

[0001] The invention belongs to the medical field and relates to an application of mGluR2 gene. Background Art

[0002] Influenza A virus is a single-stranded negative-sense RNA virus whose genome consists of eight segmented RNA segments. Influenza A virus is divided into different subtypes based on the different antigenicity of its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Influenza viruses existing in nature pose a huge threat to human and animal health. Since 1918, influenza viruses of subtypes H1N1, H2N2, and H3N2 have caused four human influenza pandemics, during which more than 50 million people died from influenza infection; in addition, H1N1 and H3N2 viruses are still widely circulated worldwide and cause annual seasonal epidemics. Some H5 and H7 subtypes of influenza viruses show high pathogenicity in poultry and have caused more than 2,000 cases of human infection with high morbidity and mortality. Since 2020, different H5 viruses have caused thousands of outbreaks in Europe, Africa, Asia, and North America, resulting in the deaths of hundreds of millions of poultry, wild birds, and wild mammals. Vaccination is the main strategy for controlling influenza virus pandemics, but influenza viruses are prone to mutation, which leads to antigenic variation. Virus antigenic variation continuously reduces the protective effect of vaccines, and vaccine seed viruses need to be updated in a timely manner. Therefore, developing effective antiviral drugs and breeding disease-resistant animals will be important strategies to combat influenza viruses.

[0003] Three classes of influenza virus antiviral drugs have been licensed, including M2 channel inhibitors (e.g., amantadine), neuraminidase inhibitors (e.g., oseltamivir and zanamivir), and viral RNA polymerase inhibitors (e.g., favipiravir). However, amantadine is no longer used clinically because most influenza virus strains are resistant to amantadine. Some influenza A virus strains that are highly resistant to oseltamivir and oseltamivir-resistant are also increasingly being discovered. Two mutations in the PB2 and PA genes that are strongly resistant to favipiravir have been found in laboratory settings, suggesting that favipiravir-resistant strains may emerge. Antiviral drugs targeting viral proteins are easily ineffective due to the presence of resistant mutants, and host cell proteins do not mutate under drug-mediated selection pressure, which may be an ideal strategy for developing antiviral drugs based on host cell proteins.

[0004] Influenza virus infection is initiated by the attachment of the viral HA protein to sialic acid receptors on the host cell membrane surface, but the binding of influenza virus to sialylated cell membrane surface proteins does not always lead to viral internalization, suggesting that influenza virus attachment and endocytosis may be mediated by different host proteins or cell receptors. Although influenza virus enters host cells through different pathways for internalization, such as clathrin-mediated endocytosis (CME), caveolin-mediated endocytosis, and macropinocytosis, most viral particles enter cells through CME. CME is the main pathway for endocytosis of cell membrane surface receptors and their bound ligands, including epidermal growth factor, transferrin, and low-density lipoprotein, which all enter cells through CME. The initiation of CME is not a spontaneous process, and ligand-surface receptor interactions can initiate CME. Previous studies have clearly shown that influenza virus can initiate CME from scratch. However, the corresponding surface receptors of influenza virus as ligands that can transmit virus binding signals and thus initiate CME remain unknown.

[0005] Metabotropic glutamate receptor 2 (mGluR2), as a member of the glutamate receptor family, is an important neurotransmitter receptor that mainly exists in mammalian nerve cells. This receptor is a member of the G protein-coupled receptor C family. There have been no reports on the association between mGluR2 and influenza virus infection. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides, in a first aspect, the use of a biomaterial in the preparation of a preparation for inhibiting virus proliferation, preventing diseases, treating diseases, alleviating diseases or controlling diseases;

[0007] The biological material is selected from any one or a combination of the following M1, M2, M3, M4, M5, M6 and M7;

[0008] M1: inhibitor of mGluR2 gene transcription;

[0009] M2: inhibitor of mGluR2 gene transcript;

[0010] M3: inhibitor of the protein encoded by the mGluR2 gene;

[0011] M4: antagonist of the protein encoded by the mGluR2 gene;

[0012] M5: Gene expression cassette

[0013] The gene product in the gene expression cassette can mediate gene coding sequence fragment deletion mutation, gene coding sequence base deletion mutation, gene coding sequence fragment insertion mutation, gene coding sequence base insertion mutation, gene coding sequence point mutation, gene coding sequence fragment replacement mutation, gene regulatory region mutation, or gene transcription is regulated to inactivate or weaken the mGluR2 gene in an animal or animal cell containing the gene expression cassette or the product of the gene expression cassette;

[0014] The promoter of the gene expression cassette is a constitutive expression promoter, a tissue-specific expression promoter or an artificially induced expression promoter;

[0015] M6: Genetic Engineering Vector

[0016] The genetic engineering vector contains the gene expression cassette described in M5;

[0017] M7: Host cells

[0018] The host cell contains the genetic engineering expression vector described in M6;

[0019] The virus is selected from:

[0020] Influenza virus;

[0021] Porcine acute diarrhea syndrome coronavirus;

[0022] Porcine epidemic diarrhea virus;

[0023] Japanese encephalitis virus;

[0024] Porcine reproductive and respiratory syndrome virus;

[0025] The disease is selected from:

[0026] influenza;

[0027] Porcine acute diarrhea syndrome coronavirus disease;

[0028] Porcine epidemic diarrhea;

[0029] Japanese encephalitis;

[0030] Porcine reproductive and respiratory syndrome.

[0031] In some embodiments, any one or a combination thereof is selected from the following S1, S2, S3, S4, S5, S6 and S7;

[0032] S1: The inhibitor of the mGluR2 gene transcript is a siRNA or antisense RNA targeting the mGluR2 gene;

[0033] S2: The inhibitor of the protein encoded by the mGluR2 gene is a monoclonal antibody or a polyclonal antibody against the extracellular domain of the protein encoded by the mGluR2 gene;

[0034] S3: The antagonist of the protein encoded by the mGluR2 gene is a truncated peptide or a fusion peptide containing the extracellular domain of the protein encoded by the mGluR2 gene;

[0035] S4: The gene expression cassette contains a gRNA gene sequence targeting the mGluR2 gene and a CAS9 protein gene sequence;

[0036] S5: The influenza is mammalian influenza or avian influenza;

[0037] S6: The influenza virus is selected from the group consisting of H1N1 influenza virus, H5N6 influenza virus, H7N9 influenza virus and H9N2 influenza virus;

[0038] S7: The protein sequence encoded by the mGluR2 gene is shown in SEQ ID NO.1 or SEQ ID NO.16; or

[0039] The GenBank number of the protein sequence encoded by the mGluR2 gene is selected from NM_000839.4, NP_001153825.1, XP_020924514.1, XP_027323532.1, XP_035193215.1, XP_032050197.1, XP_019475446.1, XP_010716663.1 and XP_015730108.1.

[0040] In some embodiments, any one or a combination thereof is selected from the following A1, A2, A3, A4 and A5;

[0041] A1: The double-stranded sequences of the siRNA targeting the mGluR2 gene are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively;

[0042] A2: the protein epitope recognized by the monoclonal antibody against the extracellular domain of the protein encoded by the mGluR2 gene falls within positions 19 to 567 of the protein sequence encoded by the mGluR2 gene; or

[0043] The protein epitope recognized by at least one of the polyclonal antibodies against the extracellular domain of the protein encoded by the mGluR2 gene falls within the 19th to 567th positions of the protein sequence encoded by the mGluR2 gene;

[0044] A3: The truncated peptide containing the extracellular domain of the protein encoded by the mGluR2 gene is the protein shown at positions 19 to 567 of the protein sequence encoded by the mGluR2 gene; or

[0045] The fusion peptide containing the extracellular domain of the protein encoded by the mGluR2 gene is the protein shown at positions 19 to 567 of the protein sequence encoded by the mGluR2 gene and a label peptide used for separating and purifying the protein and / or for western blot detection;

[0046] A4: The target sequence of the gRNA gene targeting the mGluR2 gene is SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.20;

[0047] A5: The dairy animal influenza is selected from human influenza, swine influenza, and mouse influenza.

[0048] The second aspect of the present invention provides an application of a genetic engineering method in preparing a new animal strain that improves the animal's ability to resist disease;

[0049] The animal is selected from the group consisting of: mice, pigs, chickens, pheasants, turkeys, ducks, geese, and quails;

[0050] The disease is selected from:

[0051] influenza;

[0052] Porcine acute diarrhea syndrome coronavirus disease;

[0053] Porcine epidemic diarrhea;

[0054] Japanese encephalitis;

[0055] Porcine reproductive and respiratory syndrome;

[0056] The method comprises: using genetic manipulation to make the animals into a strain in which the mGluR2 gene is inactivated or weakened.

[0057] In some embodiments, any one or a combination thereof is selected from B1, B2 and B3;

[0058] B1: A strain in which the mGluR2 gene of the animal is inactivated or weakened by gene coding sequence fragment deletion mutation, gene coding sequence base deletion mutation, gene coding sequence fragment insertion mutation, gene coding sequence base insertion mutation, gene coding sequence point mutation, gene coding sequence fragment replacement mutation, gene regulatory region mutation or gene transcriptional regulation;

[0059] B2: the protein sequence encoded by the mGluR2 gene is shown in SEQ ID NO.1 or SEQ ID NO.16, or the GenBank number of the protein sequence encoded by the mGluR2 gene is selected from NM_000839.4, NP_001153825.1, XP_020924514.1, XP_027323532.1, XP_035193215.1, XP_032050197.1, XP_019475446.1, XP_010716663.1 and XP_015730108.1;

[0060] B3: Through genetic manipulation, the animals are made into homozygous strains in which the mGluR2 gene is inactivated or weakened.

[0061] In some embodiments, the genetic engineering manipulation method is to use the Crispr-CAS9 method to make the animal a strain with inactivated or weakened mGluR2 gene.

[0062] In some embodiments, the genetic engineering method comprises the following steps:

[0063] P1: Designing a sgRNA target sequence targeting the mGluR2 gene, inserting a DNA sequence corresponding to the sgRNA target sequence into a plasmid to obtain a recombinant plasmid containing a CAS enzyme gene expression cassette and a sgRNA gene expression cassette;

[0064] P2: in vitro transcription of the recombinant plasmid was performed, the in vitro transcribed Cas9 mRNA and sgRNA were microinjected into mouse fertilized eggs, and the mouse fertilized eggs were transplanted into the mouse uterus to obtain 4 groups of F0 mice;

[0065] P3: The mGluR2 gene sequence of F0 mice was identified and the mGluR2 gene knockout positive F0 mice were screened.

[0066] In some embodiments, the mGluR2 gene knockout-positive F0 mice are self-bred to obtain mGluR2 gene knockout-positive homozygous mice.

[0067] In some embodiments, the target sequence of the sgRNA gene is SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.20. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 The effect of siRNA targeting mGluR2 on the infectivity of influenza virus is shown.

[0069] Figure 2Shown is the effect of siRNA targeting mGluR2 on the adsorption and internalization of influenza virus.

[0070] Figure 3 It is shown that mGluR2 promotes influenza virus internalization by directly interacting with HA protein.

[0071] Figure 4 Shown are the results of an internalization assay of mGluR2 in influenza virus infected cells.

[0072] Figure 5 mGluR2 is shown to be involved in clathrin-mediated endocytosis of influenza virus.

[0073] Figure 6 mGluR2 was shown to affect the internalization of other subtypes of influenza virus.

[0074] Figure 7 Shown is the altered resistance of mGluR2 knockout mice to different influenza viruses.

[0075] Figure 8 Shown is the effect of mGluR2 on the titer of influenza virus infection in DF1 cells.

[0076] Fig. 9 It was shown that mGluR2 affects infection with porcine acute diarrhea syndrome coronavirus, porcine epidemic diarrhea virus, Japanese encephalitis virus, and porcine reproductive and respiratory syndrome virus. DETAILED DESCRIPTION

[0077] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0078] Materials and instruments not described in the present invention are conventional materials and instruments in the art, operation details not described in the present invention are conventional operations in the art, the software used in the present invention is operated by conventional methods with reference to the instructions of the software provider, and the kit used in the present invention is operated by conventional methods with reference to the kit instruction manual.

[0079] The nucleic acid sequences shown in the present invention are all written from left to right in the direction of 5' to 3', and the proteins are written from left to right in the direction of N-terminus to C-terminus.

[0080] Viruses and cell lines

[0081] Virus:

[0082] The H1N1, H5N6, H7N9 and H9N2 influenza virus strains used in the present invention are all preserved by the National Avian Influenza Reference Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0083] Specifically, H1N1 influenza virus [A / WSN / 1933 (H1N1) strain]: influenza virus isolated from human patients, the full genome GISAID number is EPI_ISL_69745.

[0084] H5N6 influenza virus: A / duck / Guangdong / S1330 / 2016(H5N6), GISAID number is EPI1921655.

[0085] H7N9 influenza virus: A / chicken / Guangdong / SD008 / 2017(H7N9)-PB2 / 627K, reference: PMID:29151586.

[0086] Influenza virus (H9N2): A / chicken / Liaoning / SD007 / 2017(H9N2).

[0087] Swine acute diarrhea syndrome coronavirus: SADS-CoV / GDWT-P7 strain, GenBank number MK994934.1.

[0088] Porcine epidemic diarrhea virus: LNCT2 strain, GenBank number is KT323980.1.

[0089] Adenovirus type 5: purchased from Stratagene, catalog number 240010.

[0090] Japanese encephalitis virus SA14 strain, GenBank number is M55506.1.

[0091] The HP-PRRSV HuN4 strain has a GenBank number of EF635006.1.

[0092] Cell lines

[0093] A549 cells were purchased from the American Type Culture Collection, catalog number CCL-185.

[0094] MDCK cells: purchased from American Type Culture Collection, catalog number PTA-6500.

[0095] HEK293 cells were purchased from the American Type Culture Collection, catalog number CRL-1573.

[0096] N2a cells: purchased from American Type Culture Collection, catalog number CCL-131.

[0097] Vero-E6 cells: purchased from American Type Culture Collection, catalog number CRL-1586.

[0098] PK-15 cells: purchased from American Type Culture Collection, catalog number CCL-33.

[0099] DF1 cells: purchased from American Type Culture Collection, catalog number CRL-3586.

[0100] IPI-2I cells were purchased from China Center for Type Culture Collection.

[0101] MARC145 cells: A derivative line of MA104 cells derived from the embryonic kidney tissue of African green monkeys, preserved by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0102] Example 1: Construction of plasmid

[0103] The ORF of human mGluR2 cDNA (ORF1, GenBank No. NM_000839.4 for the coding sequence) was constructed into pCAGGS plasmid (supplier HonorGene, catalog No. HG-VPA0057) and carried a Flag tag at the N-terminus, and named pmGluR2.

[0104] The ORF (ORF2, the GenBank number of the coding sequence is J02176.1) of the HA gene derived from the human influenza virus H1N1.

[0105] The amino acid sequence of ORF1 translation is (SEQ ID NO.1):

[0106] MGSLLALLALLLLWGAVAEGPAKKVLTLEGDLVLGGLFPVHQKGGPAEDCGPVNEHRGIQRLEAMLFALDRINRDPHLLPGVRLGAHILDSCSKDTHALEQALDFVRASLSRGADGSRHICPDGSYATHGDAPTAITGVIGGSYSDVSIQVANLLRLFQIPQISYASTSAKLSDKSRYDYFARTVPPDFFQAKAMAEILRFFNWTYVSTVASEGDYGETGIEAFELEARARNICVATSEKVGRAMSRAAFEGVVRALLQKPSARVAVLFTRSEDARELLAASQRLNASFTWVASDGWGALESVVAGSEGAAEGAITIELASYPISDFASYFQSLDPWNNSRNPWFREFWEQRFRCSFRQRDCAAHSLRAVPFEQESKIMFVVNAVYAMAHALHNMHRALCPNTTRLCDAMRPVNGRRLYKDFVLNVKFDAPFRPADTHNEVRFDRFGDGIGRYNIFTYLRAGSGRYRYQKVGYWAEGLTLDTSLIPWASPSAGPLPASRCSEPCLQNEVKSVQPGEVCCWLCIPCQPYEYRLDEFTCADCGLGYWPNASLTGCFELPQEYIRWGDAWAVGPVTIACLGALATLFVLGVFVRHNATPVVKASGRELCYILLGGVFLCYCMTFIFIAKPSTAVCTLRRLGLGTAFSVCYSALLTKTNRIARIFGGAREGAQRPRFISPASQVAICLALISGQLLIVVAWLVVEAPGTGKETAPERREVVTLRCNHRDASMLGSLAYNVLLIALCTLYAFKTRKCPENFNEAKFIGFTMYTTCIIWLAFLPIFYVTSSDYRVQTTTMCVSVSLSGSVVLGCLFAPKLHIILFQPQKNVVSHRAPTSRFGSAAARASSSLGQGSGSQFVPTVCNGREVVDSTTSSL

[0107] The amino acid sequence translated from ORF2 is (SEQ ID NO.2):

[0108] MKAKLLVLLYAFVATDADTICIGYHANNSTDTVDTIFEKNVAVTHSVNLLEDRHNGKLCKLKGIAPLQLGKCNITGWLLGNPECDSLLPARSWSYIVETPNSENGACYPGDFIDYEELREQLSSVSSLERFEIFPKESSWP NHTFNGVTVSCSHRGKSSFYRNLLWLTKKGDSYPKLTNSYVNNKGKEVLVLWGVHHPSSSDEQQSLYSNGNAYVSVASSNYNRRFTPEIAARPKVKDQHGRMNYYWTLLEPGDTIIFEATGNLIAPWYAFALSRGFESGII TSNASMHECNTKCQTPQGSINSNLPFQNIHPVTIGECPKYVRSTKLRMVTGLRNIPSIQYRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNNLEKRMENL NKKVDDGFLDIWTYNAELLVLLENERTLDFHDLNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI

[0109] Example 2: mGluR2 affects influenza virus infection experiment

[0110] The present invention firstly performs an RNA interference experiment to detect whether mGluR2 is involved in the infection process of influenza virus.

[0111] (I) RNAi experiment in A549 cells in 96-well plates

[0112] The transfection system for each well of a 96-well plate is 1 pmol siRNA (simGluR2, for targeted silencing of the mGluR2 gene), 0.4 μL RNAiMAX transfection reagent (supplier Invitrogen, catalog number 13778150), 40 μL Opti-MEM medium (supplier Invitrogen, catalog number 31985070) and 60 μL A549 cell suspension (containing a total of 8000 cells, the solvent is water), and the final concentration of siRNA is 10 nM. A549 cells are routinely cultured in F-12K medium containing 10% (v / v) fetal bovine serum (FBS), and cell counts are performed during passage.

[0113] In the experiment, 40 μL / well of Opti-MEM medium was first added to a 1.5 mL RNase-free centrifuge tube, and then 1 pmol of siRNA was added to Opti-MEM. The mixture was mixed by blowing 5 times with an RNase-free pipette tip. Be careful not to let the liquid hang on the wall of the pipette tip and there should be no bubbles. Then 0.4 μL RNAiMAX was added and mixed by blowing 5 times with an RNase-free pipette tip. The mixture was left at room temperature for 30 minutes. During this process, the cells were digested and counted, and the cells were diluted to 8,000 cells per 60 μL cell suspension. The prepared siRNA, Opti-MEM and RNAiMAX mixture in the centrifuge tube was added to a 96-well plate, and the diluted A549 cells were added. 60 μL of the diluted cell suspension was added to each well, and then the plate was transferred to a 37°C incubator for culture. Scramble siRNA was used as a negative control (siControl) in the experiment.

[0114] simGluR2: purchased from Thermo, catalog number s6195.

[0115] The positive chain sequence of simGluR2 is (SEQ ID NO.3):

[0116] CGAUUGGACGAAUUCACUUUU.

[0117] The antisense sequence of simGluR2 is (SEQ ID NO.4):

[0118] AAGUGAAUUCGUCCAAUCGGU.

[0119] siControl is a random non-targeted RNA sequence purchased from Thermofisher with the catalog number 4390843.

[0120] (II) Total RNA extraction and reverse transcription

[0121] 72 hours after transfection in step (a), the total RNA of A549 cells treated with simGluR2 and siControl in the first part of the wells was extracted and reverse transcribed. The total RNA extraction of cells was performed as follows: 1 mL / well of Trizol lysis solution was added to the cells, and after standing at room temperature for 5 minutes, the Trizol lysis solution was collected in a 1.5 mL centrifuge tube, 200 μL of chloroform was added to the tube, and the centrifuge tube was covered with a cap and shaken vigorously for 20 seconds. The tube was stood at room temperature for 10 minutes to wait for solid-liquid phase separation, and 460 μL of the supernatant (liquid phase) was carefully drawn into a new centrifuge tube, and then 230 μL of anhydrous ethanol was added, and the mixture was mixed by blowing with a pipette tip, and all were transferred into the centrifuge column of the RNA extraction kit (supplier: Tiangen, item number: DP419), and centrifuged at 12000g, 4°C for 1 minute. Add 500 μL of deproteinized solution RD (add anhydrous ethanol to the newly opened deproteinized solution RD in advance) to the centrifuge column, centrifuge at 12000g, 4℃ for 1min, and discard the waste liquid in the centrifuge tube below. Add 500 μL of rinse solution RW (add anhydrous ethanol to the newly opened deproteinized solution RW in advance) to the centrifuge column, centrifuge at 12000g, 4℃ for 1min, and discard the waste liquid in the centrifuge tube below. Add 500 μL of rinse solution RW (add anhydrous ethanol to the newly opened deproteinized solution RW in advance) to the centrifuge column, centrifuge at 12000g, 4℃ for 1min, and discard the waste liquid in the centrifuge tube below. Centrifuge at 12000g, 4℃ for 3min, insert the centrifuge column into a new centrifuge tube, and add 40 μL of RNase-free water to the center of the centrifuge column. After standing at room temperature for 5min, centrifuge at 8000g, 4℃ for 3min, discard the centrifuge column, and keep the centrifuge tube. The RNA extracted in the above process was tested for concentration, marked on the wall of the centrifuge tube, and placed on ice waiting for reverse transcription.

[0122] The specific operation of reverse transcription is as follows: the reaction system is 2 μg RNA, 1 μL Primer Mix, 1 μL RT Enzyme Mix, 4 μL 5× RT Buffer, ddH 2 Add 20 μL of 1% HO to the reaction mixture. Place the prepared system in a PCR instrument and set the reverse transcription procedure to: 37°C for 15 min, 98°C for 5 min. After the reverse transcription, store the cDNA at 4°C or -20°C.

[0123] (III) Fluorescence quantitative qPCR and data analysis

[0124] The fluorescence quantitative qPCR experiment was performed as follows: the cDNA obtained from the A549 cells treated with simGluR2 and siControl was divided into two equal volumes, each with 10 μL. The qPCR system for the first cDNA was 50 μL ChamQUniversal SYBR qPCR Master Mix, 2 μL internal reference gene GAPDH Forward primer (10 μM), 2 μL internal reference gene GAPDH Reverse primer (10 μM), 10 μL cDNA, 36 μL ddH 2 O. The total volume is 100 μL. Mix the system and dispense it into the first 4 wells of the eight-tube strip, 20 μL per tube. The qPCR system for the second cDNA is: 50 μL of CamQUniversal SYBR qPCR Master Mix, 2 μL of the primers of the mGluR2 gene to be tested, mGluR2 Forward primer (10 μM), mGluR2 Reverse primer (10 μM), 10 μL of cDNA, 36 μL of ddH 2 O. The total volume is 100 μL. Mix the system and dispense it into the last 4 wells of the eight-tube concatenation, 20 μL per tube, that is, the 4 on the left of a row of eight-tube concatenations are amplified with the internal reference gene fluorescence quantitative primers, and the 4 on the right are amplified with the fluorescence quantitative primers of the gene to be tested.

[0125] The mGluR2 Forward primer sequence is (SEQ ID NO.5):

[0126] 5'-GCACAGGCAAGGAGACAGC-3'

[0127] The mGluR2 reverse primer sequence is (SEQ ID NO.6):

[0128] 5'-GAGGCAGCCAAGCACCAC-3'

[0129] In all experiments of the present invention, 4 wells will be repeated in the siControl group and the simGluR2 group, that is, 4 cDNAs of 20 μl each for the siControl group and 4 cDNAs of 20 μl each for the simGluR2 group, for a total of 8 cDNAs. Each cDNA will be repeated in 4 wells with the internal reference gene primers and the test gene primers during the fluorescence quantitative qPCR process to ensure the accuracy of the experiment. Therefore, when conducting the experiment, the large system will be configured in advance according to the different primers. If there are 8 samples, the above 100 μl system will be expanded 9 times in a proportional manner and then divided into centrifuge tubes; if there are 12 samples, the above 100 μl system will be expanded 13 times in a proportional manner and then divided into centrifuge tubes, 90 μl per tube, and then 10 μl of cDNA will be added and gently blown with the gun tip to mix, and no bubbles will be generated. The pipette range is adjusted to 20 μl and then divided into eight tubes. Place the eight-tube strips with samples into a fluorescent quantitative qPCR instrument and set the following program: (1) pre-denaturation: 95°C for 30 s, 1 cycle; (2) cycle reaction: 95°C for 10 s, 60°C for 30 s (signal collection), 40 cycles; (3) melting curve: 95°C for 15 s, 60°C for 60 s, 95°C for 15 s (signal collection), 1 cycle.

[0130] The data analysis process is as follows. The fluorescence quantitative results show the cycle number (Cycle threshold, CT) of the sample, △CT sample =CT sample -CT GAPDH ; △CT sample-siControl Average value = (△CT sample-siControl 1 +△CT sample-siControl 2 +△CT sample-siControl 3 +△CT sample-siControl 4 ) / 4; relative mRNA or viral RNA level of the sample = 2 -△CTsample-siGene / 2 -△CTsample-siControl Average value*100%.

[0131] Expression level of mGluR2 mRNA: The expression level of mGluR2 mRNA in A549 cells treated with simGluR2 and siControl is shown in Figure 1 A, It can be seen that the expression of mGluR2 mRNA in simGluR2-transfected cells was significantly decreased 48 hours after transfection compared with siControl-transfected cells ( Figure 1 A).

[0132] It can be seen that the simGluR2 used in this example can effectively reduce the expression level of mGluR2 mRNA and can be effectively used to study the function of mGluR2.

[0133] (IV) Virus inhibition experiment

[0134] For the A549 cells treated with simGluR2 and siControl in the second part of the wells in step (I), 48 hours after transfection, pCAGGS plasmid was added at an amount of 0.5 μg / well, and the transfection reagent was ExFect Transfection Reagent (supplier: Vazyme, catalog number: T101-02). 24 hours after transfection, the A549 cells in the wells were infected with H1N1 virus (MOI = 0.01), and the TCID of H1N1 virus under the four operations was detected after incubation for 24 hours. 50 .

[0135] TCID 50 Detection steps: MDCK cells were cultured with DMEM medium containing 5% (v / v) FBS. The MDCK cell stock solution was diluted 6 times one day in advance and then transferred to a 96-well plate. The next day, when the MDCK cells in the cell plate grew to 80% abundance, the medium was discarded and replaced with DMEM medium without serum. Take another 96-well plate and add Opti-MEM medium containing TPCK trypsin (0.5μg / mL) as a virus dilution, adding 180μL to each well. During titration, use a spray gun to take 20μL of the cell supernatant from the two treatments (simGluR2 treatment with pCAGGS plasmid, siControl treatment with pCAGGS plasmid) and add 10 1 Discard the pipette tip and replace it with a new one. Use a 10-fold virus dilution to dilute. 1 Add 20 μL of 10-fold virus dilution 2 Dilute the cell supernatant to 10 7 Discard the serum-free DMEM in the cell plate, replace the pipette tip, and 7 times to 10 1Transfer the virus dilution to the cell plate in turn, adding 100 μL to each well. Transfer the cell plate to a 37°C incubator and culture for 1 hour, then take it out, wash the cells with serum-free DMEM medium and discard the DMEM, add fresh Opti-MEM medium containing TPCK trypsin (0.5 μg / mL) again, and culture it in a 37°C incubator. After 48 hours, use a row gun to transfer the cell supernatant to the blood coagulation plate in turn, 25 μL TPCK trypsin per well (the gun tip needs to be replaced for each row), and then add 25 μL phosphate buffer (PBS, pH = 7.2-7.4) and 25 μL PBS containing 1% (v / v) chicken red blood cells. After standing for 15 minutes, determine the results. The occurrence of blood coagulation is recorded as a positive well, and the blood sedimentation flow is recorded as a negative well. Calculate the positive rate of the recorded results and use the Reed-Muench formula to calculate the equal ratio to determine the sample TCID 50 result.

[0136] Titer statistics see Figure 1 B, The results showed that the viral titer in mGluR2-silenced cells was significantly lower than that in control cells ( Figure 1 B), indicating that mGluR2 is involved in the infection process of influenza virus.

[0137] Figure 1 In the figure, A shows that the expression level of mGluR2 mRNA in cells with simGluR2 interference was significantly reduced; B shows that knocking down mGluR2 expression significantly inhibited viral infection. *p<0.05, **p<0.01. Figure 1 A and Figure 1 B shows that simGluR2 can reduce the expression of mGluR2 and the titer of influenza virus H1N1.

[0138] Example 3: Experiment on mGluR2 regulation of influenza virus adsorption and internalization

[0139] The present invention then studies whether mGluR2 affects the invasion process of influenza virus. The process of virus invasion of host cells mainly includes adsorption and internalization. The present invention first studies whether mGluR2 affects the adsorption of influenza virus. The mGluR2-silenced A549 cells (simGluR2-treated A549 cells) and control cells (siControl-treated A549 cells) cultured for 72 hours in the same manner as in step (a) of Example 2 were incubated with H1N1 virus (MOI=5) on ice for 1 hour, and then immediately washed with cold neutral PBS (pH=7.2) to remove unbound viruses. The vRNA levels of attached viruses were quantified by qPCR. For statistical results, see Figure 2 A, the present invention found that the virus binding to mGluR2 silenced cells was comparable to that of control cells (the difference was not significant) ( Figure 2 A), indicating that mGluR2 has little effect on the adsorption process of influenza virus.

[0140] In order to investigate whether mGluR2 is involved in the internalization process, mGluR2-silenced cells (simGluR2-treated A549 cells) and control cells (siControl-treated A549 cells) cultured for 72 hours in the same manner as in step (i) of Example 2 were incubated with H1N1 virus (MOI = 5) at 4°C for 1 hour and then at 37°C for 2 hours. The virus on the cell surface was removed by washing the cells with cold acidic PBS (pH = 1.3), and then the vRNA of the virus in the cells was tested by qPCR. Statistical results are shown in Figure 2 B, the present invention found that the vRNA level of mGluR2 silenced cells was significantly lower than that of control cells ( Figure 2 B). This shows that knockdown of mGluR2 significantly reduced influenza virus internalization.

[0141] After internalization treatment of mGluR2-silenced A549 cells (simGluR2-treated A549 cells) and control cells (siControl-treated cells), the cells were fixed with 4% paraformaldehyde (PFA) aqueous solution for 30 minutes, blocked with 5% BSA in PBS for 1 hour, and then incubated with the primary antibody (rabbit anti-influenza virus HA protein monoclonal antibody, purchased from SinoBiological, catalog number 11692-T62) for 2 hours. The cells were washed 3 times with PBS (pH = 7.2) and incubated with the secondary antibody (AlexaFluor 488-labeled goat anti-rabbit IgG, purchased from Invitrogen, catalog number A11034) for 1 hour. After washing three times, the cells were incubated with DAPI (4', 6-diamidino-2-phenylindole) for 15 minutes to stain the nuclei. Images were collected by confocal laser scanning microscope 880 (Carl Zeiss, Germany). The fluorescence intensity of each cell was calculated by the software of the confocal laser scanning microscope 880.

[0142] The present invention further confirmed this result by microscopy-based assay (see Figure 2 C and 2D). The above results indicate that mGluR2 is important for the internalization of influenza virus.

[0143] Figure 2The figure shows that mGluR2 regulates the internalization of influenza virus. A shows that knocking down mGluR2 expression does not affect the adsorption process of influenza virus; B shows that knocking down mGluR2 expression significantly reduces the internalization process of influenza virus; in the simGluR2 treatment group and siControl treatment group in C, the upper figure is a merged fluorescence image, and the lower figure is an enlarged image of the box field of the upper figure. The left image in the enlarged image reflects the secondary antibody color of the HA protein of the H1N1 virus, and the right image in the enlarged image reflects the secondary antibody color of the HA protein of the H1N1 virus and the DAPI color. It can be seen that after simGluR2 treatment, the virus particles on the cell surface increased significantly, indicating that the confocal imaging experiment confirmed that knocking down mGluR2 expression reduced the internalization of H1N1 virus, cell nucleus (blue), virus HA (green); Figure D shows the green fluorescence intensity statistics of the two groups, ***p<0.001, ****p<0.0001.

[0144] These results indicate that the internalization of influenza virus was significantly reduced after knocking down mGluR2 by simGluR2, indicating that mGluR2 mediates the internalization of influenza virus.

[0145] Example 4: Experiment in which mGluR2 promotes influenza virus internalization by directly interacting with HA protein

[0146] (1) Vector construction

[0147] The surface protein HA of influenza virus is responsible for its internalization. mGluR2 is involved in the internalization of influenza virus. In order to find out whether mGluR2 directly interacts with the HA of influenza virus, the present invention conducted a pull-down experiment. First, the present invention constructed a recombinant HA (HA-His) by replacing the transmembrane region of the HA protein of H1N1 with a hexahistidine (6*His) tag and a foldon domain (the natural trimerization domain of T4 phage fiber protein) (the construction principle is shown in FIG. Figure 3 A).

[0148] The amino acid sequence from the HA portion of HA-His is (SEQ ID NO.7):

[0149] MKAKLLVLLYAFVATDADTICIGYHANNSTDTVDTILEKNVAVTHSVNLLEDSHNGKLCKLKGIAPLQLGKCNITGWLLGNPECDSLLPARSWSYIVETPNSENGACYPGDLIDYEELREQLSSVSSLERFE IFPKESSWPNHTFNGVTVSCSHRGKSSFYRNLLWLTKKGDSYPKLTNSYVNNKGKEVLVLWGVHHPSSSDEQQSLYSNGNAYVSVASSNYNRRFTPEIAARPKVRDQHGRMNYYWTLLEPGDTIIFEATGNL IAPWYAFALSRGFESGIITSNASMHECNTKCQTPQGAINSNLPFQNIHPVTIGECPKYVRSTKLRMVTGLRNIPSIQYRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVN SVIEKMNTQFTAVGKEFNNLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDLNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKIDGVKLESMGVYQ

[0150] The amino acid sequence of the foldon domain is (SEQ ID NO.8):

[0151] GYIPEAPRDGQAYVRKDGEWVLLSTFL

[0152] The structure of the HA-His trimer was predicted by the software Alphafold 2 and visualized by the software Pymol ( Figure 3 B) The purified eukaryotically expressed HA-His was then mixed with HEK293 cell lysates transfected with mGluR2-Flag (plasmid pmGluR2 in Example 1) for a pull-down experiment.

[0153] (2) Pull-down experiment

[0154] (i) Experimental group

[0155] The steps of the pull-down experiment are as follows: 250 μL of HEK293 cell lysate transfected with plasmid (pmGluR2-Flag) was mixed with 10 μL of agarose beads coupled with anti-Flag antibody (Flag agarose beads), and the mixture was inverted at 4°C for 2 hours. 1 mL of PBS containing 1% NP-40 (ethylphenyl polyethylene glycol) was added, and the mixture was centrifuged at 3,000×g for 5 minutes at 4°C, and the washing was repeated twice. After the agarose beads were resuspended with 100 μL PBS, 10 μg of purified soluble protein (HA-His) was added, and the mixture was inverted at 4°C for 6 hours. The beads were washed 5 times with PBS containing 1% NP-40, and finally the agarose beads were resuspended with 70 μL PBS and 4× protein loading buffer, and the beads were placed in a boiling water bath for 15 minutes. The mixture containing the material shed from the agarose beads (eluate) was obtained as sample 1, and then the Western blot experiment was performed.

[0156] (ii) Control 1

[0157] Take 250 μL of HEK293 cell lysate without plasmid transfection and mix it with 10 μL of agarose beads coupled with anti-Flag antibody (Flag agarose beads), flip at 4°C for 2h. Add 1mL of PBS containing 1% NP-40 (ethylphenyl polyethylene glycol), centrifuge at 3,000×g for 5min at 4°C, and repeat the washing twice. After resuspending the agarose beads with 100 μL PBS, add 10 μg of purified soluble protein (HA-His) and mix, and place in a boiling water bath for 15min to obtain a mixture (eluate) containing the material shed from the agarose beads as sample 2, and then perform Western blot experiments.

[0158] (3) Western blot experiment

[0159] WB1: For HA-His, the primary antibody was rabbit anti-histidine tag antibody (purchased from Sigma-Aldrich, catalog number 492635533), and the secondary antibody was HPR-labeled sheep anti-rabbit IgG antibody (purchased from Invitrogen, catalog number 31460).

[0160] WB2: For mGluR2, the primary antibody was rabbit anti-Flag tag monoclonal antibody (purchased from Genscript, catalog number A00170), and the secondary antibody was HPR-labeled goat anti-rabbit IgG antibody (purchased from Invitrogen, catalog number 31460).

[0161] Conventional WB procedures were used and developed using ECL luminescent substrate.

[0162] and Figure 3C corresponds to the first and second gels (counted from top to bottom, the same below), the protein molecular weight marker is added to the first lane, the Flag agarose bead eluate (sample 1) after pull-down of the experimental group is added to the second lane, and the third lane is sample 2. The first gel was treated with anti-Flag antibody and then developed (WB2) to detect mGluR2, and the second gel was treated with anti-histidine tag antibody and then developed (WB1) to detect HA-His. It can be seen that the pull-down experiment was successful, and pmGluR2-Flag and HA-His were present in the Flag agarose bead eluate after pull-down.

[0163] Note: pmGluR2-Flag exists in the form of a mixture of dimers and monomers.

[0164] In the third gel, the protein molecular weight marker was added to the first lane, the HEK293 cell lysate transfected with pmGluR2-Flag and 10 μg purified soluble protein (HA-His) mixture was added to the second lane, and the HEK293 cell lysate that was not transfected and not incubated with Flag agarose beads and 10 μg purified soluble protein (HA-His) mixture was added to the third lane. After treatment with anti-Flag antibody, the expression level of mGluR2 after transfection was detected by development (WB2). It can be seen that mGluR2 was contained in the lysate before incubation with Flag agarose beads.

[0165] In the fourth gel, the protein molecular weight marker was added to the first lane, the HEK293 cell lysate transfected with pmGluR2-Flag and 10 μg purified soluble protein (HA-His) mixture was added to the second lane, and the HEK293 cell lysate not transfected and not incubated with Flag agarose beads and 10 μg purified soluble protein (HA-His) mixture was added to the third lane. After treatment with anti-histidine tag antibody, development (WB1) was performed to detect the purification effect of HA-His. As can be seen from the fourth gel, the method of the present invention can effectively detect HA-His.

[0166] The present invention found that HA-His was successfully pulled down by mGluR2 ( Figure 3 C), Demonstration that mGluR2 directly interacts with HA of influenza virus.

[0167] (4) Immunofluorescence assay

[0168] A549 cells were infected with H1N1 virus at a dose of MOI = 50. After 1 hour of infection, the cells were fixed with 4% PFA aqueous solution for 30 minutes, blocked with PBS containing 5% BSA for 1 hour, and HA obtained from H1N1 virus was fluorescently labeled with rabbit anti-HA antibody IgG (purchased from SinoBiological, catalog number 11692-T62) and Alexa Fluor488-conjugated goat anti-rabbit IgG (purchased from Invitrogen, catalog number A11034). mGluR2 was labeled with mouse anti-mGluR2 antibody IgG (purchased from SantaCruz Biotechnology, catalog number sc271654) and Alexa Fluor 568-conjugated goat anti-mouse IgG (purchased from abcam, catalog number ab175473). The cell nucleus was stained with DAPI. After washing, the cells were photographed by focusing laser scanning microscope 880. One of the typical photos is shown in Figure 3 D.

[0169] Immunofluorescence assay further confirmed that mGluR2 and H1N1 virus particles were clearly co-localized in A549 cells ( Figure 3 D).

[0170] (5) Antibody blocking and soluble protein neutralization experiments

[0171] To investigate whether the interaction between mGluR2 and HA is important for influenza virus infection, the present invention performed antibody blocking experiments and mGluR2 soluble protein neutralization.

[0172] The following four biological materials are used in this step:

[0173] (i) GST tag, protein sequence is as follows (SEQ ID NO.9):

[0174] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRI AYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK

[0175] (ii) The fusion protein of the extracellular domain of mGluR2 and the GST tag (named: mGluR2-GST) was expressed and purified in prokaryotes and called a soluble protein. The protein sequence of the extracellular domain was from position 19 to position 567 of ORF1 shown in Example 1.

[0176] (iii) mAb-mGluR2, an antibody targeting the extracellular domain of mGluR2, was purchased from Santa Cruz Biotechnology, catalog number sc271654.

[0177] (iv) Mouse IgG2a antibody, isotype control, no specific targeting molecule, purchased from Southern Biotech, catalog number 0103-01.

[0178] A549 cells were incubated with GST tag (concentration 800 μg / mL), mGluR2-GST (concentration 800 μg / mL), mAB-mGluR2 (concentration 40 μg / mL), and IgG2a (concentration 40 μg / mL). Cell viability was tested according to the manufacturer's instructions of the CellTiter-Glo kit (Promega, G9242). The present invention found that cell viability was not substantially affected by the antibodies ( Figure 3 E), it can be seen that the four materials have no obvious toxicity to A549 cells.

[0179] A549 cells were grown on 96-well plates for 16 hours in opti-MEM medium. The cells were first placed on ice for 15 minutes, then the supernatant in the wells was discarded, and 40μg / ml IgG2a, 10μg / ml mAB-mGluR2, 20μg / ml mAB-mGluR2, 30μg / ml mAB-mGluR2, and 40μg / ml mAB-mGluR2 (antibody diluent was opti-MEM) were added to the A549 cells, 100μl / well, and the cells were placed on ice for 1 hour. The supernatant was discarded, and influenza virus solution H1N1 (MOI=0.001) diluted with 40 μg / ml IgG2a, 10 μg / ml mAB-mGluR2, 20 μg / ml mAB-mGluR2, 30 μg / ml mAB-mGluR2, and 40 μg / ml mAB-mGluR2 (the antibody diluent was opti-MEM) was added to the A549 cell wells, 100 μl / well, and the cells were placed on ice for another hour. After 1 hour, the supernatant was discarded, and the cells were washed three times with PBS. 40 μg / ml IgG2a, 10 μg / ml mAB-mGluR2, 20 μg / ml mAB-mGluR2, 30 μg / ml mAB-mGluR2, and 40 μg / ml mAB-mGluR2 (the antibody diluent was opti-MEM) were added to the wells of the five treatments, 100 μl / well. After incubation for 48 hours, the virus titer (TCID 50 ). For titer statistics, see Figure 3 F, it can be seen that compared with the mouse IgG2a antibody, the mGluR2 antibody mAb-mGluR2 effectively inhibited the H1N1 virus infection in A549 cells in a dose-dependent manner. It can be seen that mAb-mGluR2 can shield the receptor mGluR2 on the cell surface, thereby hindering the infection of the virus, while IgG2a, as the isotype control of mAb-mGluR2, randomly and non-specifically binds to the cell membrane protein, so it cannot significantly hinder the infection of the virus.

[0180] The present invention conducted a soluble protein blocking experiment. A549 cells were grown on a 96-well plate for 16 hours, and the culture medium was DMEM containing 10% fetal bovine serum. The supernatant was discarded and replaced with opti-MEM. 800 μg / ml GST and different concentrations (400 μg / ml, 600 μg / ml, 800 μg / ml) of mGluR2-GST purified protein (soluble protein) in the solvent of opti-MEM were respectively mixed with influenza virus H1N1 (10 4 TCID 50 / ml) and mix thoroughly, incubate at 4°C with rotation for 24 hours. Take 100 μL of the four protein virus mixture and inoculate it into the aforementioned 96-well cell culture plate. After infection at 37°C for 1 hour, discard the supernatant and add Opti-MEM culture medium containing different concentrations of purified proteins (800 μg / ml GST, 400 μg / ml mGluR2-GST, 600 μg / ml mGluR2-GST, 800 μg / ml mGluR2-GST) in turn, 100 μl / well. 24 hours after infection, according to the cell CPE, the Reed-Muench formula was used to determine the titer of the virus in the cell supernatant (TCID 50 ), and the relative infection rate was calculated based on the titer. The results of GST were taken as 100% as the reference, and the relative infection rates of other treatments were calculated. The results are shown in Figure 3 G, mGluR2-GST inhibited H1N1 virus infection in A549 cells in a dose-dependent manner. These results indicate that mGluR2-GST competes with mGluR2 on the cell membrane of A549 cells for binding to H1N1, thereby reducing H1N1 infection of cells.

[0181] Figure 3 The figure shows that mGluR2 promotes influenza virus internalization by directly interacting with HA protein. A is a schematic diagram of the wild-type H1N1 HA and 6×His-tagged HA (HA-His) domains; B is the HA-His visualization structure prediction result made by Alphafold2 and Pymol; C is the result of the pull-down experiment to prove the direct interaction between mGluR2 and HA; D is the co-localization of mGluR2 and HA confirmed by confocal imaging. Cell nucleus (blue), mGluR2 (red) and viral HA (green); E shows that mGluR2-GST or mGluR2 antibody treatment does not affect the viability of A549 cells; F shows that mGluR2 antibody treatment of cells significantly reduced the replication of H1N1 virus in A549 cells; G shows that mGluR2-GST treatment significantly reduced the replication of H1N1 virus in A549 cells. *p<0.05.

[0182] Example 5: Internalization assay of mGluR2 in influenza virus infected cells

[0183] According to the results of Example 3, mGluR2 is involved in the internalization of influenza virus. In order to find out whether mGluR2 is internalized together with the virus, the abundance of mGluR2 on the cell membrane before and after H1N1 virus infection was quantitatively determined by microscopy.

[0184] The experiment was divided into three groups. A549 cells were cultured with opti-MEM. The A549 cells in the first group were not treated and incubated at 37°C for 2 hours. The A549 cells in the second group were incubated at 4°C for 1 hour, and then influenza virus H1N1 (MOI = 50) was added and incubated at 4°C for 1 hour, and then the unadsorbed virus particles were washed off with PBS. The third group was incubated at 37°C for 2 hours based on the treatment of the second group. The three groups of cells were fixed with 4% paraformaldehyde (PFA) aqueous solution for 30 minutes, and then immunofluorescence experiments were performed without permeabilization to mark mGluR2 on the cell membrane. The primary antibody was mouse anti-mGluR2 antibody IgG (purchased from Santa Cruz Biotechnology, catalog number sc271654), and the secondary antibody was Alexa Fluor 488-conjugated goat anti-mouse IgG (purchased from abcam, catalog number ab150113). The nucleus was stained with DAPI, and the fluorescence intensity of each cell was calculated by the software of the confocal laser scanning microscope 880.

[0185] Typical photos see Figure 4 , it can be seen that when the H1N1 virus is internalized, the fluorescence intensity of mGluR2 on the cell membrane is significantly lower than that of uninfected control cells or virus-bound cells ( Figure 4 A and 4B ), indicating that mGluR2 is internalized along with influenza virus.

[0186] Figure 4 The results of mGluR2 internalization assay in influenza virus infected cells are shown. The left, middle and right images in A are typical photos obtained from the first, second and third groups, respectively. B is the statistics of fluorescence intensity on the cell membrane in A. A and B show that mGluR2 is internalized into A549 cells together with H1N1 virus. Cell nucleus (blue), mGluR2 (green). Scale bar, 5 μm. ns, no significant difference, ***p<0.001.

[0187] This shows that mGluR2 mediates virus internalization and then enters the cell membrane, where it is internalized together with the influenza virus.

[0188] Example 6: mGluR2 is involved in clathrin-mediated influenza virus endocytosis

[0189] Influenza viruses use different infection pathways to enter cells, and the known pathways include CME (clathrin-mediated endocytosis), caveolin-mediated endocytosis, and macropinocytosis. Key molecules involved in these three pathways have been identified: clathrin light chain (CLTC) is a key molecule for CME, caveolin 1 (CAV1) is a key molecule for caveolin-mediated endocytosis, and Rac family small GTPase 1 (RAC1) is a key molecule for macropinocytosis. In order to study the pathways involved in influenza virus infection, the present invention first performed a microscopy-based assay to evaluate the changes in the abundance of mGluR2 on the membranes of A549 cells with CLTC, CAV1 or RAC1 silenced and their control A549 cells after H1N1 virus infection.

[0190] For transferrin, cholera toxin B, and dextran endocytosis assays, A549 cells were transfected with siRNA-CLTC (siRNA targeting CLTC), siRNA-CAV1 (siRNA targeting CAV1), and siRNA-RAC1 (siRNA targeting RAC1) for 72 hours, respectively, with a transfection dose of 1 pmol. The cells were then washed twice with PBS (pH = 7.2) and replaced with Opti-MEM for 2 hours at 37°C. The cells were then washed with PBS and incubated with 5 μg / mL Alexa Fluor 488-labeled transferrin (supplier Invitrogen, catalog number T13342), 5 μg / mL Alexa Fluor 594-labeled cholera toxin B (Invitrogen, C34777), and 100 μg / mL Alexa Fluor 647-labeled dextran (Invitrogen, D22914) for 30 minutes. The cells were then washed twice with ice-cold PBS and then incubated twice for 2 minutes in a buffer (50 mM glycine, 100 mM NaCl, pH = 3.0). After removing the residual labeled protein on the membrane, the cells were fixed for confocal laser scanning microscopy analysis. The fluorescence intensity of each cell was calculated by the software of the confocal laser scanning microscope 880.

[0191] The positive strand sequence of siRNA-CLTC is (SEQ ID NO.10):

[0192] CAUUAUGACCGGGCUCAUAUU

[0193] The sequence of the antisense strand of siRNA-CLTC is (SEQ ID NO.11):

[0194] UAUGAGCCCGGUCAUAAUGUU

[0195] The positive strand sequence of siRNA-CAV1 is (SEQ ID NO.12):

[0196] CCCUAAACACCUCAACGAUUU

[0197] The sequence of the antisense strand of siRNA-CAV1 is (SEQ ID NO.13):

[0198] AUCGUUGAGGUGUUUAGGGUU

[0199] The positive strand sequence of siRNA-RAC1 is (SEQ ID NO.14):

[0200] GCAAACAGAUGUGUUCUUAUU

[0201] The sequence of the antisense strand of siRNA-RAC1 is (SEQ ID NO.15):

[0202] UAAGAACACAUCUGUUUGCUU

[0203] Transferrin, cholera toxin B, and dextran were used as positive controls for previously reported CME, caveolin-mediated endocytosis, and macropinocytosis, respectively. The present invention demonstrated that the internalization of transferrin, cholera toxin B, and dextran was significantly reduced in CLTC, CAV1, and RAC1 silenced cells, respectively, compared to control cells ( Figure 5 A and 5B). Treatment of A549 cells with siRNA can reduce the intracellular uptake of transferrin, cholera toxin B and dextran, respectively, indicating that the model of the present invention is established, that is, treatment of A549 cells with siRNA can weaken the effects of CME, caveolin-mediated endocytosis and macropinocytosis, respectively.

[0204] A549 cells were transfected with the aforementioned siRNA-CLTC, siRNA-CAV1, siRNA-RAC1, and siControl (control) used in Example 2 for 72 hours, respectively, with a transfection dose of 1 pmol and a culture medium of Opti-MEM, 100 μl / well. The cells were then washed twice with PBS (pH=7.2) and replaced with Opti-MEM for 2 hours at 37°C. The cells treated with the aforementioned four methods were incubated with H1N1 virus (MOI=5) at 4°C for 1 hour, and then incubated at 37°C for 2 hours. The cells were then washed with PBS and fixed with a 4% paraformaldehyde (PFA) aqueous solution for 30 minutes. Immunofluorescence experiments were performed without permeabilization to label mGluR2 on the cell membrane. The primary antibody was mouse anti-mGluR2 antibody IgG (purchased from Santa Cruz Biotechnology, catalog number sc271654), and the secondary antibody was Alexa Fluor 488-conjugated goat anti-mouse IgG (purchased from abcam, catalog number ab150113). The cell nucleus was stained with DAPI, and the fluorescence intensity of each cell was calculated by the software of the confocal laser scanning microscope 880. The photographic results are shown in Figure 5 C, mGluR2 fluorescence intensity on the cell membrane see Figure 5 D.

[0205] It can be seen that the fluorescence intensity of mGluR2 on the membrane of CLTC-silenced cells was significantly higher than that of control cells, while the fluorescence intensity of mGluR2 on the membrane of CAV1 or RAC1-silenced cells was comparable to that of control cells ( Figure 5 C and 5D), indicating that after CLTC silencing, more mGluR2 was retained on the cell membrane surface, and less mGluR2 was internalized into the cell along with the virus particles, indicating that the CME pathway is involved in mGluR2-mediated influenza virus internalization.

[0206] Figure 5 The involvement of mGluR2 in clathrin-mediated endocytosis of influenza virus is shown. A and B show the internalization of transferrin, cholera toxin B, or dextran in CLTC-, CAV1-, and RAC1-silenced A549 cells. C and D show that knocking down the expression of CLTC significantly reduced the internalization of mGluR2 in A549 cells infected with H1N1 virus. Nucleus (blue), mGluR2 (green). Scale bar, 5 μm. ns, no significant difference, *p<0.05, **p<0.01, ****p<0.0001. It can be seen that mGluR2 is internalized with influenza virus through the CME pathway.

[0207] Example 7: mGluR2 affects the internalization of other influenza virus subtypes

[0208] The above studies show that mGluR2 promotes CME of H1N1 virus. It is not clear whether other subtypes of influenza viruses also internalize cells through this mechanism. Therefore, the present invention conducted a series of cytological experiments on H5N6 virus A / duck / Guangdong / S1330 / 2016 (H5N6) (GenBank No. EPI1921655, hereinafter referred to as H5N6 virus) and H7N9 virus A / chicken / Guangdong / SD008 / 2017 (H7N9)-PB2 / 627K (reference: PMID: 29151586, hereinafter referred to as H7N9). Using the same method and operating parameters as Example 3, only the influenza virus strains used were different. The present invention first compared the replication of H5N6 and H7N9 viruses in differently treated cells. The present invention found that the viral titers of the two viruses in A549 cells with mGluR2 silenced were significantly lower than those in control cells ( Figure 6 A). The levels of internalized H5N6 and H7N9 virus vRNA in mGluR2 knockdown cells were significantly lower than those in control cells ( Figure 6 B). Using the same method and operating parameters as in Example 4, except for the different influenza virus strains, it was shown that mGluR2 is important for the internalization of both viruses in A549 cells, which was further confirmed by microscopy-based assays ( Figure 6 C, D). In addition, the present invention found that antibodies against the extracellular domain of mGluR2 effectively blocked the infection of the two viruses ( Figure 6 E), and mGluR2-GST inhibited the infection of both viruses in a dose-dependent manner ( Figure 6 F), Figure 6 The 0 in F represents the mGluR2-GST dosage of 0, i.e., 800ug / ml of GST protein. In summary, these results suggest that mGluR2-related CME may be the main mechanism of influenza virus internalization.

[0209] Figure 6 The figure shows the effect of mGluR2 on the internalization of other influenza virus subtypes; A shows that the replication of H5N6 and H7N9 in A549 cells with mGluR2 silencing is significantly reduced; B shows that the internalization of H5N6 and H7N9 in A549 cells with mGluR2 silencing is significantly reduced; C and D show that knocking down the expression of mGluR2 significantly reduces the internalization of H5N6 and H7N9 into A549 cells; E shows that mGluR2 antibody treatment of cells significantly reduces the replication of H5N6 and H7N9 in A549 cells; F shows that mGluR2-GST treatment significantly reduces the replication of H5N6 and H7N9 in A549 cells. *p<0.05, **p<0.01, ****p<0.0001.

[0210] This shows that mGluR2 can also mediate the internalization of H5N6 and H7N9.

[0211] Example 8: Experiment on the changes in resistance of mGluR2 knockout mice to different influenza virus infections

[0212] 1. Preparation of Knockout Mice

[0213] Extensive cell studies have shown that the mGluR2 gene plays a direct role in the internalization of influenza virus. To further investigate whether the mGluR2 gene affects influenza virus infection in individuals, the authors evaluated the effects of different influenza viruses in wild-type mice (C57BL / 6J mice) and mGluR2 knockout homozygotes (mGluR2 - / - ) Replication titers and lethality in mice.

[0214] Homozygous mGluR2 knockout - / - ) The method for preparing mice is as follows.

[0215] Parental mouse strain: C57BL / 6J mice.

[0216] The amino acid sequence encoded by the parental mouse mGluR2 gene (target gene) is shown in GenBank No. NP_001153825.1, wherein the mGluR2 gene genomic sequence Gene ID No. is 108068.

[0217] The amino acid sequence of the mGluR2 gene translation is (SEQ ID NO.16):

[0218] MESLLRFLALLLLRGAVAEGPAKKVLTLEGDLVLGGLFPVHQKGGPAEECGPVNEHRGIQRLEAMLFALDRINRDPHLLPGVRLGAHILDSCSKDTHALEQALDFVRASLSRGADGSRHICPDGSYATLSDAPTAITGVIGGSYSDVSIQVANLLRLFQIPQISYASTSAKLSDKSRYDYFARTVPPDFFQAKAMAEILRFFNWTYVSTVASEGDYGETGIEAFELEARARNICVATSEKVGRAMSRAAFEGVVRALLQKPSARVAVLFTRSEDARELLAATQRLNASFTWVASDGWGALESVVAGSERAAEGAITIELASYPISDFASYFQNLDPWNNSRNPWFREFWEERFRCSFRQRDCAAHSLRAVPFEQESKIMFVVNAVYAMAHALHNMHRALCPNTTRLCDAMRPVNGRRLYKDFVLNVKFDAPFRPADTDDEVRFDRFGDGIGRYNIFTYLRAGNGRYRYQKVGYWAEGLTLDTSIIPWASPSAGTLPASRCSEPCLQNEVKSVQPGEVCCWLCIPCQPYEYRLDEFTCADCGLGYWPNASLTGCFELPQEYIRWGDAWAVGPVTIACLGALATLFVLGVFVRHNATPVVKASGRELCYILLGGVFLCYCMTFIFIAKPSTAVCTLRRLGLGTAFSVCYSALLTKTNRIARIFGGAREGAQRPRFISPASQVAICLALISGQLLIVAAWLVVEAPGIGKETAPERREVVTLRCNHRDASMLGSLAYNVLLIALCTLYAFKTRKCPENFNEAKFIGFTMYTTCIIWLAFLPIFYVTSSDYRVQTTTMCVSVSLSGSVVLGCLFAPKLHIILFQPQKNVVSHRAPTSRFGSAAPRASANLGQGSGSQLVPTVCNGREVVDSTTSSL

[0219] (I) Vector construction

[0220] According to the sequence characteristics of the genomic fragment where the mGluR2 gene is located and the mouse genome sequence characteristics, the following four sgRNA target sequences shown in Table 1 were designed, and the DNA fragments corresponding to the sgRNA target sequences were synthesized respectively, and the DNA fragments were cloned into the Crispr-CAS9 vector plasmid to form 4 recombinant plasmids.

[0221] Table 1. List of sgRNA target sequences

[0222] sgRNA name sgRNA target sequence (5'→3') PAM S1 (SEQ ID NO. 17) CCTCTTACTCCGTGGCATAT AGG S2 (SEQ ID NO.18) TGGGGATGAGAGCTAACACT GGG S3 (SEQ ID NO. 19) GCGACCAGATCCCCTAGGTC TGG S4 (SEQ ID NO. 20) TGTAGAGTTTAAGGCTCGCC TGG

[0223] 2. Microinjection and transplantation

[0224] The above four recombinant plasmids were transcribed in vitro, mice were superovulated, sperm of the same strain of mice were fertilized in vitro to obtain fertilized eggs, Cas9 mRNA and sgRNA transcribed in vitro were microinjected into the fertilized eggs, and the fertilized eggs were transplanted into the mouse uterus to obtain four groups of F0 mice;

[0225] (III) Identification and breeding of F0 generation mice

[0226] Each of the above four groups of F0 mice was operated separately, and the mice born by microinjection were subjected to PCR and sequencing identification, and the mGluR2 gene knockout positive F0 mice (heterozygotes) were screened. The above mGluR2 gene knockout F0 female mice were selected for self-fertilization to obtain F1 mice. The F1 mice included mGluR2 gene knockout homozygotes, heterozygotes and wild types.

[0227] (IV) mGluR2 gene knockout homozygote

[0228] F1 mice were screened by PCR and sequencing to obtain mGluR2 gene double knockout positive F1 mice and obtain mGluR2 gene knockout homozygous (mGluR2 - / - ) mice. Two pairs of primers were used for PCR. The upstream primer and downstream primer 1 were used to amplify the band, and the upstream primer and downstream primer 2 were used to amplify the band. The mice without the band were mGluR2 gene knockout homozygotes (mGluR2 - / - ) mice. The PCR products were sequenced and further confirmed to be homozygous for complete knockout of the mGluR2 gene.

[0229] Upstream primer (SEQ ID NO.21): CGTGGCCTGATATCTCTACCGT

[0230] Downstream primer 1 (SEQ ID NO.22): ACCGTCTCCTAGAAGAGTGGACA

[0231] Downstream primer 2 (SEQ ID NO.23): TGTCCACAGTGTGGTGCTGAAT

[0232] Knockout mice with various knockout mutant mGluR2 gene sequences prepared for the above-mentioned four sgRNA target sequences were kept for use in subsequent experiments.

[0233] 2. Test of antiviral ability of transgenic mice

[0234] Each group consisted of 16 wild-type and 16 mGluR2 - / - The mice were intranasally inoculated with a lethal dose of influenza virus and each group was divided into three groups. In the first group, three wild-type mice and three mGluR2 - / - The mice were euthanized on day 3 and their organs were collected for virus titration. - / - Mice were euthanized on day 5 and their organs were collected for virus titration. The remaining 10 wild-type mice and 10 mGluR2 - / - The body weight changes and survival rates of mice were observed for a total of two weeks.

[0235] Specifically, 6-week-old mGluR2 - / - Mice and wild-type mice (C57BL / 6J) were inoculated with 50 μL of H1N1 influenza virus (10 3.8 PFU), H5N6 influenza virus (10 4.5 EID 50 ) and H7N9 influenza virus (10 3.5 EID 50 ) virus, and checked mental state and survival twice a day for 14 days. The survival rate was calculated using GraphPad Prism software, and the statistical significance was analyzed by the software's built-in Log-rank (Mantel-Cox) test. On the 3rd and 5th days, the nasal concha, lungs, brain, spleen and kidneys of the first and second groups of mice were collected for virus titration. 1g of tissue was placed in 1ml of normal saline, and each tissue was prepared into a homogenate. The titration method was the same as step (iv) of Example 2, except that the homogenate was used as the sample to be tested.

[0236] For changes in the resistance of mGluR2 knockout mice to different influenza viruses, see Figure 7 Sixteen wild-type C57BL / 6J mice and mGluR2 - / - The results of intranasal inoculation of mice with H1N1 virus are shown in A, B, and C, the corresponding results of H5N6 virus are shown in D, E, and F, and the corresponding results of H7N9 virus are shown in G, H, and I. Three wild-type mice and three mGluR2- / - Mice were euthanized on days 3 and 5, respectively, and their organs were collected for virus titration in cells, see A, D, G for results. The weight changes (see B, E, H for results) and 2-week survival (see C, F, I for results) of the remaining 10 mice in each group were evaluated. The data in Figures A, D, and G are SD ± mean. Virus titers were statistically analyzed using Student's t test. Log-rank (Mantel-Cox) test was used to analyze wild-type and mGluR2 - / - Statistical differences in mouse survival rate. **p<0.01, ***p<0.001, ****p<0.0001.

[0237] Combining the results of the first and second groups, H1N1 virus was detected in the nasal turbinates, lungs, and kidneys of all three wild-type mice. Figure 7 A), but only at mGluR2 - / - H1N1 virus was detected in the nasal turbinates and lungs of mice, and the titer was significantly lower than that of wild-type mice ( Figure 7 A). Wild-type mice lost nearly 29% of their body weight and died on day 9 ( Figure 7 B, C) mGluR2 - / - The mice also lost nearly 29% of their body weight, and four died by day 9, but the other six survived the infection. Figure 7 B, C).

[0238] Combining the results of the first and second groups, we found that in all five organs tested in wild-type mice and except for mGluR2 - / - H5N6 virus was detected in four tested organs other than mouse brain, mGluR2 - / - The titers in the nasal turbinates, lungs, and spleens of mice were significantly lower than those in wild-type mice ( Figure 7 D). Wild-type mice lost nearly 23% of their body weight and died between days 7 and 12 ( Figure 7 E, F) mGluR2 - / - The mice lost about 12% of their body weight, and three died between days 9 and 11, while the other seven recovered and survived ( Figure 7 E, F).

[0239] Combining the results of the first and second panels, H7N9 virus was detected in four of the five tested organs, except for the spleen of wild-type mice, but only in the mGluR2 - / - H7N9 virus, mGluR2, were detected in the nasal turbinates and lungs of mice - / - The titers of mice were significantly lower than those of wild-type mice ( Figure 7 G). Wild-type mice lost nearly 19% of their body weight and died within 11 days of infection; mGluR2 - / -The mice lost about 8% of their body weight, and three died between days 8 and 10 after infection, while the other seven survived ( Figure 7 H, I). These animal studies suggest that mGluR2 plays an important role in influenza virus replication and lethality in vivo.

[0240] This shows that compared with wild-type mice, mGluR2 - / - After mice were infected with different influenza viruses, the types of susceptible target organs decreased, the virus titer of target organs that could be infected decreased, and the mortality rate was significantly reduced. - / - The mice were significantly more resistant to influenza virus than wild-type mice.

[0241] Example 9: mGluR2 affects influenza virus infection of avian cells

[0242] The present invention conducted RNA interference experiments on chicken cell line DF1 cells (supplier ATCC, catalog number CRL-3586) to detect whether mGluR2 is involved in influenza virus infection of avian cells. The simGluR2 targeting mGluR2 and the control siControl were the same as those in Example 2, and the RNAi experiment was performed using the same steps and methods as in Example 2.

[0243] After mGluR2 knockdown, H1N1, H5N6, H7N9 and H9N2 viruses were infected respectively, and the supernatant was collected and titrated 24 hours after infection. The results showed that the virus titer in mGluR2 silenced cells was significantly lower than that in control cells ( Figure 8 ), indicating that mGluR2 is involved in the infection process of influenza virus.

[0244] Figure 8 The figure shows the effect of mGluR2 on influenza virus infection of DF1 cells. Knockdown of mGluR2 expression significantly inhibited H1N1 (A), H5N6 (B), H7N9 (C) and H9N2 (D) virus infection of chicken-derived DF1 cells. *p<0.05, **p<0.01.

[0245] Example 10: mGluR2 affects infection of porcine acute diarrhea syndrome coronavirus and porcine epidemic diarrhea virus

[0246] In order to explore whether mGluR2 affects the infection of other important livestock and poultry viruses, the present invention selects porcine acute diarrhea syndrome coronavirus (SADS-CoV) SADS-CoV / GDWT-P7 strain (GenBank No. MK994934.1), porcine epidemic diarrhea virus (PEDV) LNCT strain (GenBank No. KT323980.1) and adenovirus type 5 (Adenovirus type 5) (purchased from Stratagene, catalog number 240010) for experiments. After mGluR2 was knocked down, SADS-CoV and PEDV were infected respectively, and then the supernatant was collected for titration. For SADS-CoV, Vero-E6 cells and IPI-2I cells were tested respectively, PEDV was tested with Vero-E6 cells, and Adenovirus type 5 was tested with A549 cells. The simGluR2 targeting mGluR2 is the same as the control siControl in Example 2, and the siCLTC is the same as Example 6. The RNAi experiment is carried out using the same steps and methods as Examples 2 and 6, respectively.

[0247] Fig. 9 The effect of mGluR2 on porcine acute diarrhea syndrome coronavirus and porcine epidemic diarrhea virus infection is shown. Knockdown of mGluR2 expression by simGluR2 significantly inhibited the infection of porcine acute diarrhea syndrome coronavirus (A) and porcine epidemic diarrhea virus (B), but did not affect the infection of adenovirus type 5 (C). ns, no significant difference, *p<0.05, **p<0.01.

[0248] The results showed that the viral titer in mGluR2-silenced cells was significantly lower than that in control cells ( Fig. 9 AB), indicating that mGluR2 is involved in the infection process of SADS-CoV and PEDV. It is worth noting that reducing the expression of mGluR2 did not affect the expression of adenovirus type 5 ( Fig. 9 C), silencing of mGluR2 did not affect the efficiency of adenovirus type 5 in infecting mGluR2-positive A549 cells, but silencing of CLTC affected the efficiency of adenovirus type 5 in infecting mGluR2-positive A549 cells, suggesting that mGluR2-mediated viral infection is virus species specific.

[0249] Example 11: Effect of mGluR2 on Japanese encephalitis virus infection experiment

[0250] 1. Materials

[0251] Cultured cells: N2a cells (supplier ATCC, catalog number CCL-131).

[0252] Virus strain: SA14, GenBank number: M55506.1.

[0253] mGluR2 antibody: Antibody targeting the extracellular domain of mGluR2 was purchased from Santa Cruz Biotechnology, catalog number sc271654.

[0254] Mouse IgG2a antibody: isotype control, no specific targeting molecule, purchased from Southern Biotech, catalog number 0103-01.

[0255] (II) Antibody blocking experiment

[0256] N2a cells were grown on a 96-well plate for 16 hours in opti-MEM. The cells were first placed on ice for 15 minutes, then the supernatant in the wells was discarded, 40 μg / ml IgG2a and 40 μg / ml mAB-mGluR2 (antibody diluent was opti-MEM) were added to the N2a cells, 100 μl / well, and the cells were placed on ice for 1 hour. The supernatant was discarded, and Japanese encephalitis virus (MOI = 0.1) diluted with 40 μg / ml IgG2a and 40 μg / ml mAB-mGluR2 (antibody diluent was opti-MEM) in the wells treated above was added to the N2a cell wells, 100 μl / well, and the cells were placed on ice for another 1 hour. After 1 hour, discard the supernatant, wash the cells 3 times with PBS, and add 40μg / ml IgG2a and 40μg / ml mAB-mGluR2 (antibody diluent is opti-MEM) to the above wells, 100μl / well. After 24 hours of incubation, measure the virus titer (PFU) in the cell supernatant. For titer statistics, see Fig. 9 D. It can be seen that compared with the mouse IgG2a antibody, the mGluR2 antibody mAb-mGluR2 effectively inhibits Japanese encephalitis virus infection in N2a cells. It can be seen that mAb-mGluR2 can shield the receptor mGluR2 on the cell surface, thereby hindering the infection of the virus, while IgG2a, as the isotype control of mAb-mGluR2, randomly and non-specifically binds to cell membrane proteins, so it cannot significantly hinder the infection of the virus.

[0257] It can be seen that the antibody targeting mGluR2 used in this example can effectively reduce the proliferation ability of Japanese encephalitis virus, and targeting mGluR2 is expected to be used to inhibit Japanese encephalitis virus so as to treat or alleviate Japanese encephalitis.

[0258] Example 12: Experiment on the effect of knockdown of mGluR2 on blue ear disease virus infection

[0259] 1. Materials

[0260] Cultured cells: MARC145.

[0261] Blue ear disease (porcine reproductive and respiratory syndrome) virus strain: HP-PRRSV HuN4, GenBank number EF635006.1.

[0262] mGluR2 antibody: Antibody targeting the extracellular domain of mGluR2 was purchased from Santa Cruz Biotechnology, catalog number sc271654.

[0263] Mouse IgG2a antibody: isotype control, no specific targeting molecule, purchased from Southern Biotech, catalog number 0103-01.

[0264] (II) Antibody blocking experiment

[0265] Except that the cells were MARC145 and the antibody concentration was 10 μg / ml, the remaining steps were the same as those in Example 11. The infection dose of blue ear disease virus was MOI = 0.05. 72 hours after infection, the virus titer (TCID 50 ). For titer statistics, see Fig. 9 E, The results show that compared with the mouse IgG2a antibody, the mGluR2 antibody mAb-mGluR2 effectively inhibits the blue ear disease virus infection in MARC145 cells. This shows that mAb-mGluR2 can shield the receptor mGluR2 on the cell surface, thereby hindering the infection of the virus.

[0266] It can be seen that the antibody targeting mGluR2 used in this example can effectively reduce the proliferation ability of the blue ear disease virus, and targeting mGluR2 is expected to be used to inhibit the blue ear disease virus in order to slow down the blue ear disease.

[0267] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. Use of a biomaterial in the preparation of a preparation for inhibiting virus proliferation, preventing, treating, slowing down or controlling a disease; The biological material is selected from any one or a combination of the following M1, M2, M3, M4, M5, M6 and M7; M1: inhibitor of mGluR2 gene transcription; M2: inhibitor of mGluR2 gene transcript; M3: inhibitor of the protein encoded by the mGluR2 gene; M4: antagonist of the protein encoded by the mGluR2 gene; M5: Gene expression cassette The gene product in the gene expression cassette can mediate gene coding sequence fragment deletion mutation, gene coding sequence base deletion mutation, gene coding sequence fragment insertion mutation, gene coding sequence base insertion mutation, gene coding sequence point mutation, gene coding sequence fragment replacement mutation, gene regulatory region mutation, or gene transcription is regulated to inactivate or weaken the mGluR2 gene in an animal or animal cell containing the gene expression cassette or the product of the gene expression cassette; The promoter of the gene expression cassette is a constitutive expression promoter, a tissue-specific expression promoter or an artificially induced expression promoter; M6: Genetic Engineering Vector The genetic engineering vector contains the gene expression cassette described in M5; M7: Host cells The host cell contains the genetic engineering expression vector described in M6; The virus is selected from: Influenza virus; Porcine acute diarrhea syndrome coronavirus; Porcine epidemic diarrhea virus; Japanese encephalitis virus; Porcine reproductive and respiratory syndrome virus; The disease is selected from: influenza; Porcine acute diarrhea syndrome coronavirus disease; Porcine epidemic diarrhea; Japanese encephalitis; Porcine reproductive and respiratory syndrome.

2. The use according to claim 1, characterized in that Any one or a combination thereof selected from the following S1, S2, S3, S4, S5, S6 and S7; S1: The inhibitor of the mGluR2 gene transcript is a siRNA or antisense RNA targeting the mGluR2 gene; S2: The inhibitor of the protein encoded by the mGluR2 gene is a monoclonal antibody or a polyclonal antibody against the extracellular domain of the protein encoded by the mGluR2 gene; S3: The antagonist of the protein encoded by the mGluR2 gene is a truncated peptide or a fusion peptide containing the extracellular domain of the protein encoded by the mGluR2 gene; S4: The gene expression cassette contains a gRNA gene sequence targeting the mGluR2 gene and a CAS9 protein gene sequence; S5: The influenza is mammalian influenza or avian influenza; S6: The influenza virus is selected from the group consisting of H1N1 influenza virus, H5N6 influenza virus, H7N9 influenza virus and H9N2 influenza virus; S7: The protein sequence encoded by the mGluR2 gene is shown in SEQ ID NO.1 or SEQ ID NO.16; or The GenBank number of the protein sequence encoded by the mGluR2 gene is selected from NM_000839.4, NP_001153825.1, XP_020924514.1, XP_027323532.1, XP_035193215.1, XP_032050197.1, XP_019475446.1, XP_010716663.1 and XP_015730108.

1.

3. The use according to claim 2, characterized in that Selected from any one or a combination of the following A1, A2, A3, A4 and A5; A1: The double-stranded sequences of the siRNA targeting the mGluR2 gene are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively; A2: the protein epitope recognized by the monoclonal antibody against the extracellular domain of the protein encoded by the mGluR2 gene falls within positions 19 to 567 of the protein sequence encoded by the mGluR2 gene; or The protein epitope recognized by at least one of the polyclonal antibodies against the extracellular domain of the protein encoded by the mGluR2 gene falls within the 19th to 567th positions of the protein sequence encoded by the mGluR2 gene; A3: The truncated peptide containing the extracellular domain of the protein encoded by the mGluR2 gene is the protein shown at positions 19 to 567 of the protein sequence encoded by the mGluR2 gene; or The fusion peptide containing the extracellular domain of the protein encoded by the mGluR2 gene is the protein shown at positions 19 to 567 of the protein sequence encoded by the mGluR2 gene and a label peptide used for separating and purifying the protein and / or for western blot detection; A4: The target sequence of the gRNA gene targeting the mGluR2 gene is SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.20; A5: The dairy animal influenza is selected from human influenza, swine influenza, and mouse influenza.

4. Application of a genetic engineering method in the preparation of new animal strains that enhance the animal's ability to resist disease; The animal is selected from the group consisting of: mice, pigs, chickens, pheasants, turkeys, ducks, geese, and quails; The disease is selected from: influenza; Porcine acute diarrhea syndrome coronavirus disease; Porcine epidemic diarrhea; Japanese encephalitis; Porcine reproductive and respiratory syndrome; The method comprises: using genetic manipulation to make the animals into a strain in which the mGluR2 gene is inactivated or weakened.

5. The method according to claim 4, characterized in that Any one or a combination thereof selected from B1, B2 and B3; B1: A strain in which the mGluR2 gene of the animal is inactivated or weakened by gene coding sequence fragment deletion mutation, gene coding sequence base deletion mutation, gene coding sequence fragment insertion mutation, gene coding sequence base insertion mutation, gene coding sequence point mutation, gene coding sequence fragment replacement mutation, gene regulatory region mutation or gene transcriptional regulation; B2: the protein sequence encoded by the mGluR2 gene is shown in SEQ ID NO.1 or SEQ ID NO.16, or the GenBank number of the protein sequence encoded by the mGluR2 gene is selected from NM_000839.4, NP_001153825.1, XP_020924514.1, XP_027323532.1, XP_035193215.1, XP_032050197.1, XP_019475446.1, XP_010716663.1 and XP_015730108.1; B3: Through genetic manipulation, the animals are made into homozygous strains in which the mGluR2 gene is inactivated or weakened.

6. The method according to claim 4 or 5, characterized in that The genetic engineering operation method is to use the Crispr-CAS9 method to make the animal a strain with inactivated or weakened mGluR2 gene.

7. The method according to any one of claims 4 to 6, characterized in that The genetic engineering operation method comprises the following steps: P1: Designing a sgRNA target sequence targeting the mGluR2 gene, inserting a DNA sequence corresponding to the sgRNA target sequence into a plasmid to obtain a recombinant plasmid containing a CAS enzyme gene expression cassette and a sgRNA gene expression cassette; P2: in vitro transcription of the recombinant plasmid was performed, the in vitro transcribed Cas9 mRNA and sgRNA were microinjected into mouse fertilized eggs, and the mouse fertilized eggs were transplanted into the mouse uterus to obtain 4 groups of F0 mice; P3: The mGluR2 gene sequence of F0 mice was identified and the mGluR2 gene knockout positive F0 mice were screened.

8. The method according to claim 7, characterized in that The mGluR2 gene knockout positive F0 generation mice were self-fertilized to obtain the mGluR2 gene knockout positive homozygous mice.

9. The use according to claim 7, characterized in that The target sequence of the sgRNA gene is SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.20.

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