Application of mGluR2 gene

Abstract

The present invention discloses the application of siRNA targeting the mGluR2 gene, monoclonal antibodies against the extracellular domain of the protein encoded by the mGluR2 gene, truncated peptides containing the extracellular domain of the protein encoded by the mGluR2 gene, or recombinant vectors containing the gRNA gene sequence targeting the mGluR2 gene and the CAS9 protein gene sequence in the preparation of preparations for inhibiting influenza virus, swine acute diarrhea syndrome coronavirus, porcine epidemic diarrhea virus, Japanese encephalitis virus, and porcine reproductive and respiratory syndrome virus. The present invention also discloses the application of a genetic engineering operation method for knocking out the mGluR2 gene in the preparation of a new mouse strain with enhanced influenza resistance. The aforementioned applications have prospects for promotion.

Description

Technical Field

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

[0002] Influenza A virus is a single-stranded negative-sense RNA virus, and its genome consists of eight segmented RNA segments. Influenza A virus is divided into different subtypes according to the different antigenicity of its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The influenza viruses existing in nature pose a great threat to the health of humans and animals. Since 1918, H1N1, H2N2, and H3N2 subtype influenza viruses have caused four human influenza pandemics, during which more than 50 million people died from influenza infection; in addition, currently, H1N1 and H3N2 viruses are still widely spread globally and cause annual seasonal epidemics. Some H5 and H7 subtypes of influenza virus show high pathogenicity in poultry and have caused more than 2,000 human infection cases with relatively high morbidity and mortality. Since 2020, different H5 viruses have caused thousands of outbreaks in Europe, Africa, Asia, and North America, resulting in the death 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 in turn leads to antigenic variation, and the continuous reduction of the protective effect of vaccines due to virus antigenic variation requires timely updating of vaccine seed viruses. Therefore, developing effective antiviral drugs and breeding disease-resistant animals will be important strategies for combating influenza viruses.

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

[0004] Influenza virus infection is triggered by the attachment of the viral HA protein to sialic acid receptors on the surface of the host cell membrane. However, the binding of influenza virus to sialylated cell membrane surface proteins does not always result in virus internalization, indicating 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, such as clathrin-mediated endocytosis (CME), caveolin-mediated endocytosis, and macropinocytosis, most virus particles enter cells through CME. CME is the main pathway for the endocytosis of cell membrane surface receptors and their bound ligands. Epidermal growth factor, transferrin, and low-density lipoprotein 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 de novo. However, the corresponding surface receptor that can transmit the virus binding signal and initiate CME as a ligand remains unknown.

[0005] Metabotropic glutamate receptor 2 (mGluR2), as a member of the glutamate receptor family, is an important neurotransmitter receptor mainly present in mammalian nerve cells. This receptor is a member of the G protein-coupled receptor C family, and there has been no report on the association between mGluR2 and influenza virus infection. Summary of the Invention

[0006] To solve the problems existing in the prior art, a first aspect of the present invention provides the use of a biological material 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: An inhibitor of mGluR2 gene transcription;

[0009] M2: An inhibitor of mGluR2 gene transcripts;

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

[0011] M4: An 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 deletion mutation of gene coding sequence fragments, deletion mutation of gene coding sequence bases, insertion mutation of gene coding sequence fragments, insertion mutation of gene coding sequence bases, point mutation of gene coding sequence, substitution mutation of gene coding sequence fragments, gene regulatory region mutation, or post-transcriptional regulation of genes to inactivate or weaken the mGluR2 gene in animals or animal cells 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 inducible expression promoter;

[0015] M6: Genetic engineering vector

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

[0017] M7: Host cell

[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, it is selected from any one or a combination of the following S1, S2, S3, S4, S5, S6, and S7;

[0032] S1: The inhibitor of the mGluR2 gene transcript is 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 influenza virus type H1N1, influenza virus type H5N6, influenza virus type H7N9, and influenza virus type H9N2;

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

[0039] The GenBank accession numbers of the protein sequences encoded by the mGluR2 gene are 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, it is selected from any one or a combination of the following A1, A2, A3, A4, and A5;

[0041] A1: The double-stranded sequences of the siRNA targeting the mGluR2 gene are as 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 - 567 of the protein sequence encoded by the mGluR2 gene; or

[0043] At least one antibody in the polyclonal antibody against the extracellular domain of the protein encoded by the mGluR2 gene recognizes a protein epitope that falls within positions 19 - 567 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 by the 19th to 567th positions 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 by the 19th to 567th positions of the protein sequence encoded by the mGluR2 gene and a tag peptide for protein separation and purification 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 mammalian influenza includes human influenza, swine influenza, and murine influenza.

[0048] The second aspect of the present invention provides an application of a genetic engineering operation method in the preparation of a new animal strain with improved disease resistance in animals;

[0049] The animals include: mice, pigs, chickens, pheasants, turkeys, ducks, geese, quails;

[0050] The diseases include:

[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 is: through genetic manipulation, making the animal a strain with inactivated or weakened mGluR2 gene.

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

[0058] B1: A strain with inactivated or weakened mGluR2 gene in the animal through deletion mutation of gene coding sequence fragments, deletion mutation of gene coding sequence bases, insertion mutation of gene coding sequence fragments, insertion mutation of gene coding sequence bases, point mutation of gene coding sequence, substitution mutation of gene coding sequence fragments, gene regulatory region mutation, or post-transcriptional regulation of genes;

[0059] B2: The protein sequence encoded by the mGluR2 gene is as shown in SEQ ID NO.1 or SEQ ID NO.16, or the GenBank numbers of the protein sequences encoded by the mGluR2 gene are 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, make the animal a homozygous strain with inactivated or weakened mGluR2 gene.

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

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

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

[0064] P2: Perform in vitro transcription on the recombinant plasmid, microinject the in vitro transcribed Cas9 mRNA and sgRNA into mouse fertilized eggs, and transplant the mouse fertilized eggs into the uterus of a mouse to obtain 4 groups of F0 generation mice;

[0065] P3: Through the identification of the mGluR2 gene sequence of the F0 generation mice, screen and obtain the positive F0 generation mice with knocked-out mGluR2 gene.

[0066] In some embodiments, the positive F0 generation mice with knocked-out mGluR2 gene are self-crossed to obtain positive homozygous mice with knocked-out mGluR2 gene.

[0067] In some embodiments, the target sequences of the sgRNA gene are 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 Shows the influence of siRNA targeting mGluR2 on the ability of influenza virus to infect.

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

[0070] Figure 3 Shows that mGluR2 promotes the internalization of influenza virus by directly interacting with HA protein.

[0071] Figure 4 Shows the results of the internalization assay of mGluR2 in influenza virus-infected cells.

[0072] Figure 5 Shows that mGluR2 is involved in clathrin-mediated endocytosis of influenza virus.

[0073] Figure 6 Shows that mGluR2 affects the internalization of other subtypes of influenza virus.

[0074] Figure 7 Shows the change in the resistance of mGluR2 gene knockout mice to different influenza viruses.

[0075] Figure 8 Shows that mGluR2 affects the titer of influenza virus infecting DF1 cells.

[0076] Figure 9 Shows that mGluR2 affects the infection of porcine acute diarrhea syndrome coronavirus, porcine epidemic diarrhea virus, Japanese encephalitis virus and porcine reproductive and respiratory syndrome virus. Detailed implementation mode

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

[0078] The materials and instruments not described in the present invention are conventional materials and instruments in the art, the 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 usage instructions provided by the software provider, and the kits used in the present invention are operated by conventional methods with reference to the kit instruction manual.

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

[0080] Viruses and cell lines

[0081] Viruses:

[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, influenza A virus subtype H1N1 [A / WSN / 1933 (H1N1) strain]: an influenza virus isolated from a human patient, with the full genome GISAID number EPI_ISL_69745.

[0084] Influenza A virus subtype H5N6: A / duck / Guangdong / S1330 / 2016 (H5N6), with the GISAID number EPI1921655.

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

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

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

[0088] Porcine epidemic diarrhea virus: LNCT2 strain, with the GenBank numbers KT323980.1 respectively.

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

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

[0091] Porcine reproductive and respiratory syndrome virus HP-PRRSV HuN4 strain, with the GenBank number EF635006.1.

[0092] Cell line

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

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

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

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

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

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

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

[0100] IPI-2I cells: Purchased from the 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 plasmids

[0103] The ORF of human mGluR2 cDNA (ORF1, GenBank accession number of the coding sequence is NM_000839.4) was constructed into the pCAGGS plasmid (supplier HonorGene, catalog number HG-VPA0057), with a Flag tag at the N-terminus, named pmGluR2.

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

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

[0106] MGSLLALLALLLLWGAVAEGPAKKVLTLEGDLVLGGLFPVHQKGGPAEDCGPVNEHRGIQRLEAMLFALDRINRDPHLLPGVRLGAHILDSCSKDTHALEQALDFVRASLSRGADGSRHICPDGSYATHGDAPTAITGVIGGSYSDVSIQVANLLRLFQIPQISYASTSAKLSDKSRYDYFARTVPPDFFQAKAMAEILRFFNWTYVSTVASEGDYGETGIEAFELEARARNICVATSEKVGRAMSRAAFEGVVRALLQKPSARVAVLFTRSEDARELLAASQRLNASFTWVASDGWGALESVVAGSEGAAEGAITIELASYPISDFASYFQSLDPWNNSRNPWFREFWEQRFRCSFRQRDCAAHSLRAVPFEQESKIMFVVNAVYAMAHALHNMHRALCPNTTRLCDAMRPVNGRRLYKDFVLNVKFDAPFRPADTHNEVRFDRFGDGIGRYNIFTYLRAGSGRYRYQKVGYWAEGLTLDTSLIPWASPSAGPLPASRCSEPCLQNEVKSVQPGEVCCWLCIPCQPYEYRLDEFTCADCGLGYWPNASLTGCFELPQEYIRWGDAWAVGPVTIACLGALATLFVLGVFVRHNATPVVKASGRELCYILLGGVFLCYCMTFIFIAKPSTAVCTLRRLGLGTAFSVCYSALLTKTNRIARIFGGAREGAQRPRFISPASQVAICLALISGQLLIVVAWLVVEAPGTGKETAPERREVVTLRCNHRDASMLGSLAYNVLLIALCTLYAFKTRKCPENFNEAKFIGFTMYTTCIIWLAFLPIFYVTSSDYRVQTTTMCVSVSLSGSVVLGCLFAPKLHIILFQPQKNVVSHRAPTSRFGSAAARASSSLGQGSGSQFVPTVCNGREVVDSTTSSL

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

[0108] MKAKLLVLLYAFVATDADTICIGYHANNSTDTVDTIFEKNVAVTHSVNLLEDRHNGKLCKLKGIAPLQLGKCNITGWLLGNPECDSLLPARSWSYIVETPNSENGACYPGDFIDYEELREQLSSVSSLERFEIFPKESSWPNHTFNGVTVSCSHRGKSSFYRNLLWLTKKGDSYPKLTNSYVNNKGKEVLVLWGVHHPSSSDEQQSLYSNGNAYVSVASSNYNRRFTPEIAARPKVKDQHGRMNYYWTLLEPGDTIIFEATGNLIAPWYAFALSRGFESGIITSNASMHECNTKCQTPQGSINSNLPFQNIHPVTIGECPKYVRSTKLRMVTGLRNIPSIQYRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNNLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDLNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI

[0109] Example 2: Experiment on the effect of mGluR2 on influenza virus infection

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

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

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

[0113] During the experiment, first add 40 μL / well of Opti-MEM medium into a 1.5 mL RNase-free centrifuge tube. Then add 1 pmol of siRNA into the Opti-MEM, and pipette 5 times with a RNase-free pipette tip to mix well, taking care not to let the liquid stick to the pipette tip wall and ensuring no air bubbles. Then add 0.4 μL of RNAiMAX, and pipette 5 times with a RNase-free pipette tip to mix well. Let it stand at room temperature for 30 min. During this process, digest and count the cells, and dilute the cells to 8000 cells per 60 μL of cell suspension. Add the siRNA, Opti-MEM, and RNAiMAX mixture prepared in the centrifuge tube into a 96-well plate, and add the diluted A549 cells, 60 μL of the diluted cell suspension per well. Then transfer the plate to an incubator at 37 °C for culture. The experiment uses Scramble siRNA as a negative control (siControl).

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

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

[0116] CGAUUGGACGAAUUCACUUUU.

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

[0118] AAGUGAAUUCGUCCAAUCGGU.

[0119] siControl is: a random non-targeting RNA sequence, purchased from Thermofisher, catalog number 4390843.

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

[0121] Step (1): 72 hours after transfection, total RNA of A549 cells treated with simGluR2 and siControl in the first part of the wells was extracted and reverse-transcribed. The operation for extracting total cellular RNA was as follows: Add 1 mL / well of Trizol lysis solution to the cells, let it stand at room temperature for 5 min, then collect the Trizol lysis solution into a 1.5 mL centrifuge tube. Add 200 μL of chloroform to the tube, cover the centrifuge tube cap and shake vigorously for 20 s, then let it stand at room temperature for 10 min to wait for solid-liquid phase separation. Carefully aspirate 460 μL of the supernatant (liquid phase) and add it to a new centrifuge tube, then add 230 μL of absolute ethanol, mix well with a pipette tip, and transfer all of it into the centrifuge tube column of an RNA extraction kit (Supplier: Tiangen, Catalog number: DP419). Centrifuge at 12000 g at 4 °C for 1 min. Add 500 μL of protein removal solution RD (add absolute ethanol to the newly opened protein removal solution RD in advance) to the centrifuge tube column, centrifuge at 12000 g at 4 °C for 1 min, and discard the waste liquid in the lower centrifuge tube. Add 500 μL of washing solution RW (add absolute ethanol to the newly opened washing solution RW in advance) to the centrifuge tube column, centrifuge at 12000 g at 4 °C for 1 min, and discard the waste liquid in the lower centrifuge tube. Add 500 μL of washing solution RW (add absolute ethanol to the newly opened washing solution RW in advance) to the centrifuge tube column, centrifuge at 12000 g at 4 °C for 1 min, and discard the waste liquid in the lower centrifuge tube. After centrifuging at 12000 g at 4 °C for 3 min, insert the centrifuge tube column into a new centrifuge tube, and add 40 μL of RNase-free water to the center of the centrifuge tube column. Let it stand at room temperature for 5 min, then centrifuge at 8000 g at 4 °C for 3 min, discard the centrifuge tube column, and keep the centrifuge tube. Detect the concentration of the RNA extracted in the above process, label it on the centrifuge tube wall, and place it on ice waiting for reverse transcription.

[0122] The specific operation of reverse transcription was as follows: The reaction system was 2 μg of RNA, 1 μL of Primer Mix, 1 μL of RT Enzyme Mix, 4 μL of 5×RT Buffer, and ddH2O was added to 20 μL. Put the prepared system into a PCR instrument, and the reverse transcription program was: 37 °C for 15 min, 98 °C for 5 min. After completion, store the reverse-transcribed cDNA at 4 °C or -20 °C.

[0123] (3) Fluorescent quantitative qPCR and data analysis

[0124] The fluorescence quantitative qPCR experiment was operated as follows: The cDNA obtained from A549 cells treated with simGluR2 and siControl above was equally divided into two parts by volume, with each part being 10 μL. The qPCR system for the first part of cDNA was: 50 μL ChamQUniversal SYBR qPCR Master Mix, 2 μL of the internal reference gene GAPDH Forward primer (10 μM), 2 μL of the internal reference gene GAPDH Reverse primer (10 μM), 10 μL of cDNA, and 36 μL of ddH2O. The total volume was 100 μL. After mixing the system, it was aliquoted into the first 4 wells of an eight-strip tube, 20 μL per tube. The qPCR system for the second part of cDNA was: 50 μL ChamQUniversal SYBR qPCR Master Mix, 2 μL of the primer for the gene to be detected, mGluR2 Forward primer (10 μM), mGluR2 Reverse primer (10 μM), 10 μL of cDNA, and 36 μL of ddH2O. The total volume was 100 μL. After mixing the system, it was aliquoted into the last 4 wells of an eight-strip tube, 20 μL per tube, that is, the left 4 of a row of eight-strip tubes were amplified with the fluorescence quantitative primer of the internal reference gene, and the right 4 were amplified with the fluorescence quantitative primer of the gene to be detected.

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

[0126] 5’-GCACAGGCAAGGAGACAGC-3’

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

[0128] 5’-GAGGCAGCCAAGCACCAC-3’

[0129] In all the experiments of the present invention, both the siControl group and the simGluR2 group were repeated 4 wells within the group, that is, 4 aliquots of 20 μl cDNA of the siControl group and 4 aliquots of 20 μl cDNA of the simGluR2 group were obtained by extracting RNA and reverse transcription, for a total of 8 cDNA samples. Each cDNA sample was further repeated 4 wells each with the internal reference gene primer and the gene primer to be detected during the fluorescence quantitative qPCR process to ensure the accuracy of the experiment. Therefore, a large reaction system was prepared in advance according to different primers. If there are 8 samples, the above 100 μl reaction system was scaled up 9 times proportionally and then aliquoted into centrifuge tubes; if there are 12 samples, the above 100 μl reaction system was scaled up 13 times proportionally and then aliquoted into centrifuge tubes, 90 μl per tube. Subsequently, 10 μl of cDNA was added and gently pipetted to mix evenly without generating bubbles. After adjusting the pipette range to 20 μl, it was aliquoted into an eight-strip tube. The eight-strip tube with the added samples was placed into the fluorescence quantitative qPCR instrument, and the program was set as follows: (1) Pre-denaturation: 95 °C for 30 s, 1 cycle; (2) Cycling 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 showed the cycle number (Cycle threshold, CT) of the samples, and △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; the 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 results of the expression level of mGluR2 mRNA in A549 cells treated with simGluR2 and siControl are shown in Figure 1 Figure A. It can be seen that compared with the siControl transfected cells, the expression of mGluR2 mRNA in the simGluR2 transfected cells was significantly reduced 48 hours after transfection ( Figure 1 Figure 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 for studying the function of mGluR2.

[0133] (4) Virus inhibition experiment

[0134] For the A549 cells treated with simGluR2 and siControl in the second part of the wells in step (1), after 48 hours of transfection, add the pCAGGS plasmid at a dosage of 0.5 μg / well. The transfection reagent is ExFect Transfection Reagent (Supplier: Vazyme, Product Number: T101-02). After 24 hours of transfection, infect the A549 cells in the wells with H1N1 virus (MOI = 0.01). After incubation for 24 hours, detect the TCID of H1N1 virus under the four operations 50 .

[0135] TCID 50 Detection steps: Culture MDCK cells in DMEM medium containing 5% (v / v) FBS. Dilute the original MDCK cell solution 6-fold one day in advance and transfer it to a 96-well plate. The next day, when the MDCK cells in the cell plate grow to 80% confluence, discard the medium and replace it with serum-free DMEM medium. Take another 96-well plate and add Opti-MEM medium containing TPCK trypsin (0.5 μg / mL) as the virus serial dilution solution, 180 μL per well. During titration, use a multichannel pipette to take 20 μL of the cell supernatant from the wells of the two treatments (adding the pCAGGS plasmid to the simGluR2 treatment and adding the pCAGGS plasmid to the siControl treatment) and add it to the 10 1 -fold virus dilution solution for pipetting and dilution. Discard the pipette tip and replace it with a new one. Use a multichannel pipette to take 20 μL of the 10 1 -fold virus dilution solution and add it to the 10 2 -fold virus dilution solution for pipetting and dilution until the cell supernatant is diluted to 10 7 -fold. Discard the serum-free DMEM in the cell plate, change the pipette tip, and from 10 7 -fold to 10 1Transfer the virus dilution to the cell plate in multiples, adding 100 μL to each well. Transfer the cell plate to an incubator at 37 °C and incubate for 1 h. 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 place it in an incubator at 37 °C. After 48 h, use a multi-channel pipette to transfer the cell supernatant to a hemagglutination plate, adding 25 μL of TPCK trypsin to each well (change the pipette tip for each row). Then add 25 μL of phosphate buffer (PBS, pH = 7.2 - 7.4) and 25 μL of PBS containing 1% (v / v) chicken red blood cells. After standing for 15 min, judge the results. Wells showing hemagglutination are recorded as positive wells, and those with blood sediment flowing down are recorded as negative wells. Calculate the positive rate of the recorded results and use the Reed-Muench formula to calculate the equal interval ratio and then determine the TCID of the sample 50 Results

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

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

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

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

[0140] To study whether mGluR2 is involved in the internalization process, the mGluR2-silenced cells (A549 cells treated with simGluR2) and their control cells (A549 cells treated with siControl) cultured by the same method as in step (i) of Example 2 for 72 hours were incubated with H1N1 virus (MOI = 5) at 4 °C for 1 hour, and then incubated 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. The 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 indicates that knockdown of mGluR2 significantly reduces the internalization of influenza virus.

[0141] After internalization treatment of A549 cells with mGluR2 silencing (A549 cells treated with simGluR2) and control cells (cells treated with siControl), 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, cat. no. 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, cat. no. 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 cell nuclei. Images were acquired by a 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 assays (see Figure 2 C and 2D). The above results indicate that mGluR2 is important for the internalization of influenza virus.

[0143] Figure 2The internalization of influenza virus regulated by mGluR2 is shown. A shows that knocking down the expression of mGluR2 does not affect the adsorption process of influenza virus; B shows that knocking down the expression of mGluR2 significantly reduces the internalization process of influenza virus; in the simGluR2 treatment group and the siControl treatment group of C, the upper figure is the merged fluorescence image, and the lower figure is the enlarged image of the square field of view in the upper figure. In the left figure of the enlarged image is the color of the secondary antibody reflecting the HA protein of H1N1 virus, and in the right figure of the enlarged image is the merged image of the color of the secondary antibody reflecting the HA protein of H1N1 virus and the DAPI color. It can be seen that after simGluR2 treatment, the virus particles on the cell surface are significantly increased, indicating that the confocal-based imaging experiment confirms that knocking down the expression of mGluR2 reduces the internalization of H1N1 virus, nucleus (blue), virus HA (green); Figure D shows the statistical data of the green fluorescence intensity of the two groups, ***p<0.001, ****p<0.0001.

[0144] It can be seen that after knocking down mGluR2 by simGluR2, the internalization of influenza virus is extremely significantly reduced, and mGluR2 mediates the internalization of influenza virus.

[0145] Example 4: Experiment on mGluR2 promoting the internalization of influenza virus 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. To figure out whether mGluR2 directly interacts with the HA of influenza virus, the present invention conducted a pull-down experiment. First, the present invention constructed recombinant HA (HA-His) by replacing the transmembrane region of the HA protein of H1N1 with a six-histidine (6*His) tag and a fold domain (the natural trimerization domain of the T4 phage fibritin) (the construction principle is shown in Figure 3 A).

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

[0149] MKAKLLVLLYAFVATDADTICIGYHANNSTDTVDTILEKNVAVTHSVNLLEDSHNGKLCKLKGIAPLQLGKCNITGWLLGNPECDSLLPARSWSYIVETPNSENGACYPGDLIDYEELREQLSSVSSLERFEIFPKESSWPNHTFNGVTVSCSHRGKSSFYRNLLWLTKKGDSYPKLTNSYVNNKGKEVLVLWGVHHPSSSDEQQSLYSNGNAYVSVASSNYNRRFTPEIAARPKVRDQHGRMNYYWTLLEPGDTIIFEATGNLIAPWYAFALSRGFESGIITSNASMHECNTKCQTPQGAINSNLPFQNIHPVTIGECPKYVRSTKLRMVTGLRNIPSIQYRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNNLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDLNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKIDGVKLESMGVYQ

[0150] The amino acid sequence of the folded subdomain 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 Pymol software ( Figure 3 B). Then, the purified eukaryotic-expressed HA-His was mixed with the lysate of HEK293 cells transfected with mGluR2-Flag (plasmid pmGluR2 in Example 1) for pull-down experiments.

[0153] (2) Pull-down experiments

[0154] (i) Experimental group

[0155] The steps of the pull-down experiment are as follows: Mix 250 μL of the lysate of HEK293 cells transfected with plasmid (pmGluR2-Flag) with 10 μL of agarose beads conjugated with anti-Flag antibody (Flag agarose beads), and invert at 4 °C for 2 h. Add 1 mL of PBS containing 1% NP-40 (ethyl phenyl polyethylene glycol), centrifuge at 3,000×g for 5 min at 4 °C, and repeat the washing 2 times. After resuspending the agarose beads with 100 μL of PBS, then add 10 μg of purified soluble protein (HA-His), and invert at 4 °C for 6 h. Wash 5 times with PBS containing 1% NP-40, and finally resuspend the agarose beads with 70 μL of PBS and 4× protein loading buffer, and incubate in a boiling water bath for 15 min. The mixture containing the substances detached from the agarose beads (eluate) is used as Sample 1, and then Western blot experiment is carried out.

[0156] (ii) Control 1

[0157] Mix 250 μL of the lysate of HEK293 cells not transfected with plasmid with 10 μL of agarose beads conjugated with anti-Flag antibody (Flag agarose beads), and invert at 4 °C for 2 h. Add 1 mL of PBS containing 1% NP-40 (ethyl phenyl polyethylene glycol), centrifuge at 3,000×g for 5 min at 4 °C, and repeat the washing 2 times. After resuspending the agarose beads with 100 μL of PBS, then add 10 μg of purified soluble protein (HA-His) and mix, and incubate in a boiling water bath for 15 min. The mixture containing the substances detached from the agarose beads (eluate) is used as Sample 2, and then Western blot experiment is carried out.

[0158] (3) Western blot experiment

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

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

[0161] Adopt the conventional WB operation and develop the image with ECL luminescent substrate.

[0162] And Figure 3Corresponding to C, in the first gel and the second gel (counting from top to bottom, the same below), in the first lane, a protein molecular weight Marker was added; in the second lane, the eluate of the Flag agarose beads after pull-down of the experimental group (sample 1) was added; in the third lane, it was sample 2. The first gel was developed after being treated with an anti-Flag antibody (WB2) to detect mGluR2, and the second gel was developed after being treated with an anti-histidine tag antibody (WB1) to detect HA-His. Thus, it can be seen that the pull-down experiment was successful, and both pmGluR2-Flag and HA-His were present in the eluate of the Flag agarose beads after pull-down.

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

[0164] In the third gel, in the first lane, a protein molecular weight Marker was added; in the second lane, a mixture of the lysate of HEK293 cells transfected with pmGluR2-Flag that was not incubated with Flag agarose beads and 10 μg of purified soluble protein (HA-His) was added; in the third lane, a mixture of the lysate of HEK293 cells that was not transfected and not incubated with Flag agarose beads and 10 μg of purified soluble protein (HA-His) was added. It was developed after being treated with an anti-Flag antibody (WB2) to detect the expression level of mGluR2 after transfection. Thus, it can be seen that the lysate contained mGluR2 before incubation with Flag agarose beads.

[0165] In the fourth gel, in the first lane, a protein molecular weight Marker was added; in the second lane, a mixture of the lysate of HEK293 cells transfected with pmGluR2-Flag that was not incubated with Flag agarose beads and 10 μg of purified soluble protein (HA-His) was added; in the third lane, a mixture of the lysate of HEK293 cells that was not transfected and not incubated with Flag agarose beads and 10 μg of purified soluble protein (HA-His) was added. It was developed after being treated with an anti-histidine tag antibody (WB1) to detect the purification effect of HA-His. It can be seen from the fourth gel that 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), proving that mGluR2 directly interacts with the 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. After blocking with PBS containing 5% BSA for 1 hour, the HA of 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 nuclei were stained with DAPI. After washing, photographs were taken through a confocal laser scanning microscope 880. One of the typical photographs is shown in Figure 3 D.

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

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

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

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

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

[0174] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK

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

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

[0177] (iv) The murine IgG2a antibody, isotype control, with no specifically targeted molecule, was 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) respectively. Cell viability tests were performed according to the instructions of the manufacturer of the CellTiter-Glo kit (Promega, G9242). The present invention found that cell viability was basically not affected by the antibody ( Figure 3 E), from which it can be seen that these four materials have no obvious toxicity to A549 cells.

[0179] A549 cells were grown on a 96-well plate for 16 h in opti-MEM. First, the cells were placed on ice and allowed to stand for 15 min, then the supernatant in the wells was discarded. 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 (antibody diluent was opti-MEM) were added to A549 cells, respectively, at 100 μl / well, and the cells were placed on ice for 1 hour. The supernatant was discarded, and the wells treated as described above were sequentially added with influenza virus 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 (antibody diluent was opti-MEM), at 100 μl / well, and the cells were kept on ice for another 1 hour. After 1 hour, the supernatant was discarded, and the cells were washed 3 times with PBS. The wells treated as described above were sequentially added 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 (antibody diluent was opti-MEM), at 100 μl / well. After incubation for 48 hours, the virus titer (TCID 50 ) in the cell supernatant was measured using the same method as in Example 2. For the titer statistical results, see Figure 3 Figure F. It can be seen that compared with the murine IgG2a antibody, the mGluR2 antibody mAb-mGluR2 effectively inhibits H1N1 virus infection in A549 cells in a dose-dependent manner. Thus, it can be seen that mAb-mGluR2 can block the receptor mGluR2 on the cell surface, thereby hindering virus infection, while IgG2a, as an isotype control of mAb-mGluR2, randomly binds to cell membrane proteins non-specifically, so it cannot significantly hinder virus infection.

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

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

[0182] Example 5: Determination of the internalization of mGluR2 in influenza virus-infected cells

[0183] According to the results of Example 3, it can be seen that mGluR2 is involved in the internalization of influenza virus. To figure out whether mGluR2 is internalized together with the virus, the present invention quantitatively measured the abundance of mGluR2 on the cell membrane before and after H1N1 virus infection by microscopic determination.

[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, then influenza virus H1N1 (MOI = 50) was added, and incubation was continued at 4 °C for 1 hour. Then, the unadsorbed virus particles were washed off with PBS. The third group was incubated at 37 °C for 2 hours on the basis of the treatment of the second group. The cells in the three groups were fixed with 4% paraformaldehyde (PFA) aqueous solution for 30 minutes, and then immunofluorescence experiments were carried out under the condition of non-permeabilized membrane 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 nuclei were stained with DAPI, and the fluorescence intensity of each cell was calculated by the software of the confocal laser scanning microscope 880.

[0185] See the typical photos in Figure 4 , thus it can be seen that when the H1N1 virus internalizes, 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 internalizes with the influenza virus.

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

[0187] Thus it can be seen that after mGluR2 mediates virus internalization, it enters the interior of the cell membrane and internalizes with the influenza virus.

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

[0189] Influenza viruses enter cells through different infection pathways, 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. To study the pathway by which mGluR2 is involved in influenza virus infection, the present invention first conducted a microscopy-based assay to evaluate the change in the abundance of mGluR2 on the cell membrane of A549 cells with CLTC, CAV1, or RAC1 silencing after H1N1 virus infection and their control A549 cells.

[0190] For the 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) in sequence for 72 hours at a transfection dose of 1 pmol. Then the cells were washed twice with PBS (pH = 7.2) and replaced with Opti-MEM at 37°C for 2 hours. Then the cells were 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) in sequence for 30 minutes. Subsequently, the cells were washed twice with ice-cold PBS and then incubated twice in buffer (50 mM glycine, 100 mM NaCl, pH = 3.0) for 2 minutes. After removing the residual labeled proteins 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 sense strand sequence of siRNA-CLTC is (SEQ ID NO.10):

[0192] CAUUAUGACCGGGCUCAUAUU

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

[0194] UAUGAGCCCGGUCAUAAUGUU

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

[0196] CCCUAAACACCUCAACGAUUU

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

[0198] AUCGUUGAGGUGUUUAGGGUU

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

[0200] GCAAACAGAUGUGUUCUUAUU

[0201] The antisense strand sequence 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 confirmed 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 was able to reduce the intracellular uptake of transferrin, cholera toxin B, and dextran, respectively, indicating that the model of the present invention was established, i.e., treatment of A549 cells with siRNA was able to attenuate 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 in sequence. The transfection dose was 1 pmol, the culture medium was Opti-MEM, and 100 μl / well. Then the cells were washed twice with PBS (pH = 7.2) and replaced with Opti-MEM at 37°C for 2 hours. The cells treated with the aforementioned four treatments were incubated with H1N1 virus (MOI = 5) at 4°C for 1 hour, and then incubated at 37°C for 2 hours. Then the cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) aqueous solution for 30 minutes. Immunofluorescence experiments were performed to label mGluR2 on the cell membrane without permeabilization. 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 nuclei were stained with DAPI, and the fluorescence intensity of each cell was calculated by the software of the confocal laser scanning microscope 880. The photograph results are shown in Figure 5 C, and the fluorescence intensity of mGluR2 on the cell membrane is shown in Figure 5 D.

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

[0206] Figure 5 It shows the situation of mGluR2 participating in clathrin-mediated influenza virus endocytosis. 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 can significantly reduce the internalization of mGluR2 in H1N1 virus-infected A549 cells. Cell nuclei (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 together with influenza virus through the CME pathway.

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

[0208] The above research shows that mGluR2 promotes the CME of H1N1 virus. It is not yet clear whether other subtypes of influenza virus 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 accession number: 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 in Example 3, only the influenza virus strains used were different. The present invention first compared the replication of H5N6 and H7N9 viruses in cells treated differently. The present invention found that the virus titers of both viruses in mGluR2-silenced A549 cells were significantly lower than those in control cells ( Figure 6 A). The vRNA levels of internalized H5N6 and H7N9 viruses 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, only the influenza virus strains used were different, indicating 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 both viruses ( Figure 6 E), and mGluR2-GST inhibited the infection of both viruses in a dose-dependent manner ( Figure 6 F), Figure 6 0 in F represents that the dosage of mGluR2-GST is 0, that is, 800 μg / ml of GST protein. In summary, these results indicate that mGluR2-related CME may be the main mechanism for influenza virus internalization.

[0209] Figure 6 Shows the effect of mGluR2 on the internalization of other subtypes of influenza virus; A shows a significant decrease in the replication of H5N6 and H7N9 in mGluR2-silenced A549 cells; B shows a significant decrease in the internalization of H5N6 and H7N9 in mGluR2-silenced A549 cells; 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 treatment of cells with mGluR2 antibody significantly reduces the replication of H5N6 and H7N9 in A549 cells; F shows that treatment with mGluR2-GST significantly reduces the replication of H5N6 and H7N9 in A549 cells. *p<0.05, **p<0.01, ****p<0.0001.

[0210] Thus, it can be seen that mGluR2 can also mediate the internalization of H5N6 and H7N9.

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

[0212] I. Preparation of gene knockout mice

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

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

[0215] Variety of parental mice: C57BL / 6J mice.

[0216] The amino acid sequence encoded by the mGluR2 gene (target gene) of the parental mice is as shown in GenBank accession number NP_001153825.1, and the Gene ID number of the mGluR2 gene genomic sequence is 108068.

[0217] The amino acid sequence translated by the mGluR2 gene 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 sequence characteristics of the mouse genome, four sgRNA target sequences shown in Table 1 were designed. 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 kinds of 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] (II) Microinjection and transplantation

[0224] For the above-mentioned 4 kinds of recombinant plasmids, in vitro transcription was carried out respectively. Mice were superovulated, in vitro fertilization was carried out with sperm of the same inbred strain of mice to obtain fertilized eggs. The fertilized eggs were microinjected with in vitro transcribed Cas9 mRNA and sgRNA, and the fertilized eggs were transplanted into the uterus of mice; 4 groups of F0 generation mice were obtained;

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

[0226] For each of the above-mentioned 4 groups of F0 generation mice, operations were carried out respectively. The newly born mice after microinjection were identified by PCR and sequencing, and the positive F0 generation mice (heterozygotes) with mGluR2 gene knockout were screened. The above-mentioned F0 generation female mice with mGluR2 gene knockout were self-crossed to obtain F1 generation mice. The F1 generation mice included mGluR2 gene knockout homozygotes, heterozygotes and wild types.

[0227] (IV) mGluR2 gene knockout homozygotes

[0228] F1 generation mice were screened by PCR and sequencing to obtain F1 generation mice with double knockout of mGluR2 gene, and mGluR2 gene knockout homozygotes (mGluR2 - / - ) mice. PCR was carried out using two pairs of primers. Mice with a band amplified using the upstream primer and downstream primer 1 and no band amplified using the upstream primer and downstream primer 2 were mGluR2 gene knockout homozygotes (mGluR2 - / - ) mice. The PCR products were further verified by sequencing to further confirm the homozygotes with complete knockout of 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] For the gene knockout mice with multiple knockout mutant mGluR2 gene sequences prepared for the foregoing 4 sgRNA target sequences, they were reserved for subsequent experiments.

[0233] II. Test for antiviral ability of transgenic mice

[0234] In each group, 16 wild-type and 16 mGluR2 - / - mice were respectively inoculated intranasally with a lethal dose of influenza virus. Each group was divided into three subgroups. In the first subgroup, 3 wild-type mice and 3 mGluR2 - / - mice were respectively euthanized on the 3rd day, and their organs were collected for virus titration. In the second subgroup, 3 wild-type mice and 3 mGluR2 - / - mice were respectively euthanized on the 5th day, and their organs were collected for virus titration. Monitor the body weight changes and survival rates of the remaining 10 wild-type mice and 10 mGluR2 - / - mice in the third subgroup for a total of two weeks.

[0235] Specifically, 6-week-old mGluR2 - / - mice and wild-type mice (C57BL / 6J) were respectively inoculated intranasally with 50 μL of influenza virus of type H1N1 (10 3.8 PFU), influenza virus of type H5N6 (10 4.5 EID 50 ) and influenza virus of type H7N9 (10 3.5 EID 50 ). Check the mental state and survival condition twice a day for 14 consecutive days. Use GraphPad Prism software to statistically analyze the survival rate, and analyze the statistical significance through the Log-rank (Mantel-Cox) test built in the software. Collect the nasal turbinates, lungs, brains, spleens and kidneys of the mice in the first and second subgroups on the 3rd day and the 5th day respectively for virus titration. Put 1 g of tissue into 1 ml of normal saline, and prepare homogenates of each tissue respectively. The titration method is the same as that in step (IV) of Example 2, except that the homogenate is used as the sample to be tested.

[0236] For the change situation of the resistance of mGluR2 gene knockout mice to different influenza viruses, see Figure 7 . For the results of intranasal inoculation of 16 wild-type C57BL / 6J mice and mGluR2 - / - mice with H1N1 virus, see A, B, C; for the corresponding results of H5N6 virus, see D, E, F; for the corresponding results of H7N9 virus, see G, H, I. In each group, 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. The results are shown in A, D, G. The body weight changes of the remaining 10 mice in each group were evaluated (results shown in B, E, H) and the 2-week survival rate was evaluated (results shown in C, F, I). The data in Figures A, D, and G are SD ± mean. Student's t-test was used for statistical analysis of virus titers. The log-rank (Mantel-Cox) test was used to analyze the statistical differences in the survival rates of wild-type and mGluR2 - / - mice. **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 H1N1 virus was detected only in the nasal turbinates and lungs of mGluR2 - / - mice, and the titers were significantly lower than those 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 - / - mice also lost nearly 29% of their body weight, four of which died on day 9, but the other six mice survived the infection ( Figure 7 B, C).

[0238] Combining the results of the first and second groups, H5N6 virus was detected in all 5 tested organs of wild-type mice and 4 tested organs other than the brain of mGluR2 - / - mice. The titers in the nasal turbinates, lungs, and spleens of mGluR2 - / - mice were significantly lower than those of 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 - / - mice lost about 12% of their body weight, three of which 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 groups, H7N9 virus was detected in four of the five tested organs except the spleen of wild-type mice, but H7N9 virus was detected only in the nasal turbinates and lungs of mGluR2 - / - mice. The titers of mGluR2 - / - 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 after infection; mGluR2 - / -The body weight of the mice decreased by approximately 8%, and 3 of them died between the 8th and 10th days after infection, while the other 7 survived. Figure 7 H, I). These animal studies indicate that mGluR2 plays an important role in the replication and lethality of influenza virus in vivo.

[0240] Thus, compared with wild-type mice, mGluR2 - / - mice have fewer types of susceptible target organs after infection with different influenza viruses. For the target organs that can be infected by all, the virus titer decreases, and the mortality rate is significantly reduced. mGluR2 - / - mice have significantly better resistance to influenza virus than wild-type mice.

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

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

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

[0244] Figure 8 The situation where mGluR2 affects the infection of DF1 cells by influenza virus is shown. After knocking down the expression of mGluR2, the infection of chicken-derived cells DF1 by H1N1 (A), H5N6 (B), H7N9 (C), and H9N2 (D) viruses was significantly inhibited. *p < 0.05, **p < 0.01.

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

[0246] To investigate whether mGluR2 affects the infection of other important livestock and poultry viruses, in this invention, the porcine acute diarrhea syndrome coronavirus (SADS-CoV) SADS-CoV / GDWT-P7 strain (GenBank accession number MK994934.1), porcine epidemic diarrhea virus (PEDV) LNCT strain (GenBank accession number KT323980.1), and adenovirus type 5 (purchased from Stratagene, catalog number 240010) were separately selected for experiments. After mGluR2 was knocked down, cells were infected with SADS-CoV and PEDV respectively, and then the supernatants were collected for titration. For SADS-CoV, Vero-E6 cells and IPI-2I cells were used for testing respectively, for PEDV, Vero-E6 cells were used for testing, and for Adenovirus type 5, A549 cells were used for testing. simGluR2 targeting mGluR2 was the same as the control siControl in Example 2, and siCLTC was the same as in Example 6. The RNAi experiments were carried out using the same steps and methods as in Examples 2 and 6 respectively.

[0247] Figure 9 It shows that mGluR2 affects the infection of porcine acute diarrhea syndrome coronavirus and porcine epidemic diarrhea virus. After simGluR2 knocked down the expression of mGluR2, the infection of porcine acute diarrhea syndrome coronavirus (A) and porcine epidemic diarrhea virus (B) was significantly inhibited, but the infection of adenovirus type 5 was not affected (C). ns, no significant difference, *p < 0.05, **p < 0.01.

[0248] The results showed that compared with the control cells, the virus titers in mGluR2-silenced cells were significantly lower than those in the control cells ( Figure 9 A - B), indicating that mGluR2 is involved in the infection process of SADS-CoV and PEDV. Notably, reducing the expression of mGluR2 did not affect the expression of adenovirus type 5 ( Figure 9 C). The silencing of mGluR2 did not affect the efficiency of adenovirus type 5 infecting mGluR2-positive A549 cells, but the silencing of CLTC affected the efficiency of adenovirus type 5 infecting mGluR2-positive A549 cells, suggesting that mGluR2-mediated virus infection is virus species-specific.

[0249] Example 11: Experiment on the effect of mGluR2 on the infection of Japanese encephalitis virus

[0250] (I) Materials

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

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

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

[0254] Murine IgG2a antibody: Isotype control, no specific targeted molecule, purchased from Southern Biotech, catalog number 0103-01.

[0255] (2) Antibody blocking experiment

[0256] N2a cells were grown on a 96-well plate for 16 h with opti-MEM as the culture medium. First, the cells were placed on ice and allowed to stand for 15 min, then the supernatant in the wells was discarded. 40 μg / ml IgG2a and 40 μg / ml mAB-mGluR2 (antibody dilution with opti-MEM) were added to the N2a cells, 100 μl / well, and the cells were placed on ice for 1 hour. After discarding the supernatant, the wells treated as above were added with Japanese encephalitis virus (MOI = 0.1) diluted with 40 μg / ml IgG2a and 40 μg / ml mAB-mGluR2 (antibody dilution with opti-MEM), 100 μl / well, and the cells were kept on ice for another 1 hour. After 1 hour, the supernatant was discarded, and the cells were washed 3 times with PBS. The above wells were added with 40 μg / ml IgG2a and 40 μg / ml mAB-mGluR2 (antibody dilution with opti-MEM), 100 μl / well. After incubation for 24 hours, the virus titer (PFU) in the cell supernatant was measured. The statistical results of the titer are shown in Figure 9 D. It can be seen that compared with the murine IgG2a antibody, the mGluR2 antibody mAb-mGluR2 effectively inhibits the infection of Japanese encephalitis virus in N2a cells. It can be seen that mAb-mGluR2 can shield the receptor mGluR2 on the cell surface, thereby hindering virus infection, while IgG2a, as an isotype control of mAb-mGluR2, randomly binds non-specifically to cell membrane proteins, so it cannot significantly hinder virus infection.

[0257] It can be seen that the antibody targeting mGluR2 used in this example can effectively reduce the proliferation ability of Japanese encephalitis virus. Targeting mGluR2 is expected to be used to inhibit Japanese encephalitis virus in order to treat or slow down Japanese encephalitis.

[0258] Example 12: Experiment on the effect of knocking down mGluR2 on the infection of porcine reproductive and respiratory syndrome virus

[0259] (1) Materials

[0260] Cultured cells: MARC145.

[0261] Porcine reproductive and respiratory syndrome virus strain: HP-PRRSV HuN4, GenBank accession number EF635006.1.

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

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

[0264] (2) Antibody blocking experiment

[0265] Except that the cells were MARC145 and the antibody concentration was 10 μg / ml, the remaining operation steps were the same as in Example 11. The infection dose of porcine reproductive and respiratory syndrome virus was MOI = 0.05. At 72 hours after infection, the virus titer (TCID 50 ) in the cell supernatant was measured. The statistical results of the titer are shown in Figure 9 Figure E. The results showed that compared with the murine IgG2a antibody, the mGluR2 antibody mAb-mGluR2 effectively inhibited the infection of porcine reproductive and respiratory syndrome virus in MARC145 cells. Thus, it can be seen that mAb-mGluR2 can shield the receptor mGluR2 on the cell surface, thereby hindering virus infection.

[0266] Thus, it can be seen that the antibody targeting mGluR2 used in this example can effectively reduce the proliferation ability of porcine reproductive and respiratory syndrome virus. Targeting mGluR2 is expected to be used to inhibit porcine reproductive and respiratory syndrome virus in order to slow down porcine reproductive and respiratory syndrome.

[0267] It is known by common technical knowledge that the present invention can be implemented by other embodiments that do not depart from its spiritual essence or essential features. Therefore, the above-disclosed embodiments are illustrative in all aspects and are not the only ones. All changes within the scope of the present invention or within the scope equivalent to the present invention are encompassed by the present invention.

Claims

1. Use of a biomaterial in the preparation of a preparation for inhibiting virus proliferation, preventing a disease, treating a disease, slowing down a disease or controlling a disease; The biomaterial is selected from any one or a combination of the following M2, M3, M4, M5, M6 and M7; M2: siRNA targeting the mGluR2 gene; the double-stranded sequences of the siRNA targeting the mGluR2 gene are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively; M3: Monoclonal antibody against the extracellular domain of the protein encoded by the mGluR2 gene; The protein epitope recognized by the monoclonal antibody against the extracellular domain of the protein encoded by the mGluR2 gene falls within the 19th - 567th positions of the protein sequence encoded by the mGluR2 gene; M4: Truncated peptide or fusion peptide containing the extracellular domain of the protein encoded by the mGluR2 gene; the truncated peptide containing the extracellular domain of the protein encoded by the mGluR2 gene is the protein shown in the 19th - 567th positions of the protein sequence encoded by the mGluR2 gene; The fusion peptide containing the extracellular domain of the protein encoded by the mGluR2 gene is the protein shown in the 19th - 567th positions of the protein sequence encoded by the mGluR2 gene and a tag peptide for protein isolation and purification and / or for western blot detection; M5: Gene expression cassette The gene expression cassette contains an sgRNA gene sequence targeting the mGluR2 gene and a CAS9 protein gene sequence; the target sequences of the sgRNA gene targeting the mGluR2 gene are SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.20; The promoter of the gene expression cassette is a constitutive expression promoter, a tissue-specific expression promoter or an artificially inducible expression promoter; M6: Gene engineering vector The gene engineering vector contains the gene expression cassette described in M5; M7: Host cell The host cell contains the gene 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 application according to claim 1, characterized in that, Selected from any one or a combination of the following S5, S6 and S7; S5: The influenza is mammalian influenza or avian influenza; S6: The influenza virus is selected from 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 numbers of the protein sequences encoded by the mGluR2 gene are 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 application according to claim 2, characterized in that, The mammalian influenza includes human influenza, swine influenza, and murine influenza.

4. Use of a genetic engineering operation method in the preparation of a new animal strain with enhanced disease resistance in animals; The animals are selected from: mice, pigs, chickens, ducks, geese, and quails; The diseases are selected from: Influenza; Porcine acute diarrhea syndrome coronavirus disease; Porcine epidemic diarrhea; Japanese encephalitis; Porcine reproductive and respiratory syndrome; The method is: through genetic manipulation, making the animals into a strain with inactivated or weakened mGluR2 gene.

5. The application according to claim 4, characterized in that, The animals are selected from pheasants and turkeys.

6. The application according to claim 4, characterized in that, Selected from any one of B2 and B3 or a combination thereof; B2: The protein sequence encoded by the mGluR2 gene is as shown in SEQ ID NO.1 or SEQ ID NO.16, or the GenBank numbers of the protein sequences encoded by the mGluR2 gene are 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, making the animals into a homozygous strain with inactivated or weakened mGluR2 gene.

7. The application according to claim 4, wherein The genetic engineering operation method is to make the animals into a strain with inactivated or weakened mGluR2 gene by the Crispr-CAS9 method.

8. The application according to any one of claims 4 to 7, characterized in that The genetic engineering operation method includes the following steps: P1: Design an sgRNA target sequence targeting the mGluR2 gene, insert the DNA sequence corresponding to the sgRNA target sequence into a plasmid to obtain a recombinant plasmid containing a CAS enzyme gene expression cassette and an sgRNA gene expression cassette; P2: Perform in vitro transcription on the recombinant plasmid, microinject the in vitro transcribed Cas9 mRNA and sgRNA into mouse fertilized eggs, and transplant the mouse fertilized eggs into the mouse uterus to obtain 4 groups of F0 generation mice; P3: Through the identification of the mGluR2 gene sequence of the F0 generation mice, screen and obtain positive F0 generation mice with mGluR2 gene knockout.

9. The application according to claim 8, wherein The positive F0 generation mice with mGluR2 gene knockout are self-crossed to obtain positive homozygous mice with mGluR2 gene knockout.

10. The application according to claim 8, wherein, The target sequences of the sgRNA gene are SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, or SEQ ID NO.20.

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