FUT2 high-expression cell strain and application and method thereof in vaccine quality control

Abstract

The invention discloses an FUT2 high-expression cell strain as well as application and a method of the FUT2 high-expression cell strain in vaccine quality control, and belongs to the technical field of vaccines. The technical problem to be solved is that a simple and easy-to-operate cell model is lacked in the prior art, and the cell model is used for evaluating the effectiveness of NoV vaccines on the cellular level. According to the key points of the technical scheme, the FUT2 high-expression cell strain is provided, the cell strain is an HEK 293T cell strain (HEK 293T-FUT2-EGFP) with EGFP fluorescence for overexpressing human alpha 1, 2-fucosyltransferase 2 (FUT2), and the cell strain stably expresses H, Lewis b and Lewis y type HBGA antigens; meanwhile, an alternative neutralization experiment of the cell level of the norovirus vaccine based on the cell strain is developed, a basis is provided for interaction research of the norovirus and host cells, and the cell strain has high practical application value.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, specifically relating to a FUT2 high-expression cell line and its application and method in vaccine quality control. Background Technology

[0002] For understanding the technical content of this invention: Human norovirus (HuNoV) belongs to the Caliciviridae family and is a major causative agent of sporadic and epidemic viral gastroenteritis. [1,2] It is divided into seven genotype groups: G1-G2VII. [2] Among them, the proportion of G1 and G2 infections is the most common. [3] Although vaccines are an effective means of preventing norovirus infection, there are technical bottlenecks in establishing suitable norovirus cell culture models. [4,5] The development and evaluation of vaccines are progressing slowly.

[0003] Currently, there are three in vitro evaluation methods for norovirus vaccines, including the blood group antigen (HBGA) blocking test. [6,7] Blood coagulation inhibition (HAI) test [8] And neutralization assays using human intestinal organoids (HIEs) derived from stem cells. Among these, the HIE-based neutralization assay is currently the only cell model used for norovirus vaccine evaluation. [5] However, due to the technical difficulties in obtaining stem cells from human intestinal tissue, this model has not yet been widely used. Therefore, a simple and easy-to-use cell model is still needed to evaluate the effectiveness of human norovirus vaccines at the cellular level.

[0004] Currently, the Histo-Blood Group Antigen (HBGA) blocking antibody detection method (BT50 method) is widely used to evaluate vaccine efficacy for norovirus vaccine efficacy. This method is based on the principle of ELISA, using coated HuNoV receptors to detect the mixture of human norovirus antigen and antiserum, indirectly detecting blocking antibodies in serum through competitive inhibition. HBGA is a type of fucosylated protein that acts as an adsorption factor and receptor for human norovirus. [9] It is widely distributed on the surface of intestinal mucosal epithelial cells, respiratory tract, etc., and is divided into ABO(H) and Lewis antigens. It can also be distributed as free oligosaccharides in secretions such as saliva and breast milk.

[10] The synthesis of HBGA is catalyzed by fucosyltransferases (FUTs). FUT1 and FUT2 catalyze the addition of fucose to the precursor molecule at the α-1,2 linkage site, forming the H antigen. FUT1 is predominantly responsible for H antigen synthesis on erythrocytes, while FUT2 is predominantly associated with the secretory phenotype, where ABH antigens are present in saliva and epithelial cells. The H antigen is then converted into A and B antigens by enzymes A and B, respectively.

[11] Lewis antigens are formed from blood group precursors through the action of FUT1 and FUT2 to create H antigens, which are then catalyzed by α-1,3 / 4 fucotransferase to form Lewis b (Leb) and Lewis y (Ley) antigens. Multiple studies have shown that HBGA expression is associated with type-specific susceptibility to human norovirus, with the vast majority of human norovirus types exhibiting an H-type HBGA-dependent susceptibility pattern.

[12] Furthermore, transient expression of FUT2 in cells can also lead to the expression of H2-type HBGA antigen. [13-15] and norovirus attachment [13,16] However, due to the lack of a universal cell assessment model, no cell line-based neutralization assay method has been established.

[0005] Relevant patent documents retrieved: This document, published in China (CN117867027A) on April 12, 2024, discloses the construction of the FUT2 lentiviral plasmid. HEK-293T cells were co-transfected with the FUT2 lentiviral plasmid and the pMD.2G and psPAX2 packaging plasmids to harvest lentiviral particles. These particles were then used to infect logarithmically growing HEK-293T cells, and the HEK-293T / FUT2 cell line was obtained through puromycin-assisted selection. This invention establishes a HEK-293T cell line with stable high expression of H2 type human tissue blood group antigen, and based on this cell line, a method for evaluating the binding activity of GI.1 recombinant norovirus virus-like particles was developed.

[0006] Relevant non-patent literature retrieved: The journal or book title is "Advances in Microbiology and Immunology," and the article title is "Construction and Application of HEK-293T Cell Line with High Expression of H2 Human Tissue Blood Group Antigen," volume number 52, published in 2024. This article discloses the construction of the FUT2 lentiviral plasmid. HEK-293T cells were co-transfected with the FUT2 lentiviral plasmid and the pMD.2G and psPAX2 packaging plasmids using Lipofectamine 2000. Lentiviral particles were harvested, and the sequence-correct lentiviral plasmid PLVX-IRES-Puro-FUT2 was successfully constructed. Lentiviral particles were obtained. The FUT2 mRNA level in HEK-293T / FUT2 cells was increased, and 99.9% of HEK-293T / FUT2 cells expressed H2 human tissue blood group antigen. The GI.1 recombinant norovirus VLP can bind well to HEK-293T / FUT2 cells with an EC50 of 2.007 μg / mL. A HEK-293T cell line with stable high expression of H2 human tissue blood group antigen was established, and a preliminary method for evaluating the binding activity of GI.1 recombinant norovirus VLP was established based on this cell line.

[0007] References: [1] Karst, SM; Wobus, CE; Goodfellow, IG; Green, KY; Virgin, HW Advances in norovirus biology. Cell Host Microbe 2014, 15, 668–680. [2] Bartsch, SM; Lopman, BA; Ozawa, S.; Hall, AJ; Lee, BYGlobal economic burden of norovirus gastroenteritis. PLoS ONE 2016, 11,e0151219. [3]MAGWIRA CA,STEELE D,SEHERI M L.Norovinus diarhea is significantly associated with higher counts of fecal histo-bloodroup antigen expressing Enterobacter cloucae among black SouthAfrican infants[J].Gut microbes,2021,13(1):1979876. [4]Jones, M.K.; Watanabe, M.; Zhu, S.; Graves, C.L.; Keyes, L.R.;Grau, K.R.; Gonzalez-Hernandez, M.B.;Iovine, N.M.; Wobus, C.E.; Vinje, J.; etal. Enteric bacteria promote human and mouse norovirus infection of B cells.Science 2014, 346, 755–759. [5]Ettayebi, K.; Crawford, S.E.; Murakami, K.; Broughman, J.R.;Karandikar, U.; Tenge, V.R.; Neill, F.H.;Blutt, S.E.; Zeng, X.L.; Qu, L.; etal. Replication of human noroviruses in stem cell-derived humanenteroids.Science 2016, 353, 1387–1393. [6] Harrington, P.R.; Lindesmith, L.; Yount, B.; Moe, C.L.; Baric,R.S. Binding of Norwalk virus-like particles to ABH histo-blood groupantigens is blocked by antisera from infected human volunteers orexperimentally vaccinated mice. J. Virol. 2002, 76, 12335–12343. [7] LoBue, AD; Lindesmith, L.; Yount, B.; Harrington, PR;Thompson, JM; Johnston, RE; Moe, CL; Baric, RS Multivalent norovirus vaccines induce strong mucosal and systemic blocking antibodies against multiple strains. Vaccine 2006, 24, 5220–5234. [8] Czako, R.; Atmar, RL; Opekun, AR; Gilger, MA; Graham, DY; Estes, MK Serum hemagglutination inhibition activity correlates with protection from gastroenteritis in persons infected with Norwalk virus. Clin. Vaccine Immunol. 2012, 19, 284–287. [9] ALMANDEA, MOORE MD, JAYKUS LA. Norovirus binding toligands beyond histo-blood group antigens[J]. Front Microbiol, 2017.8:2549.

[10] POKALA A, PARAMKUSAM G, TEJASWI MLA, et al. Histo-blood groupantigens in oral cancer and potentially malignant disor-ders[J]. Asian Pac JCancer Prev, 2020, 21(4): 1163-1166.

[11] Zhong Kan. Study on chemical enzymatic synthesis of complex sugar chains related to tissue blood group antigens [D]. Jinan: Shandong University, 2021.

[12] ALMANDE A,MOORE M D,JAYKUS LA.Characterization ofhuman norovirusbinding to gut-associated bacterial ligands[J].BMC Res Notes,2019,12(1):607.

[13] Silva, L.M.; Carvalho, A.S.; Guillon, P.; Seixas, S.; Azevedo,M.; Almeida, R.; Ruvoen-Clouet, N.; Reis, C.A.; le Pendu, J.; Rocha, J.; etal. Infection-associated FUT2 (Fucosyltransferase 2) genetic variation andimpact on functionality assessed by in vivo studies. Glycoconj. J. 2010, 27,61–68. [CrossRef][PubMed]

[14] Marionneau, S.; Airaud, F.; Bovin, N.V.; Le Pendu, J.; Ruvoen-Clouet, N. Influence of the combined ABO, FUT2, and FUT3 polymorphism onsusceptibility to Norwalk virus attachment. J. Infect. Dis. 2005, 192,1071–1077.

[15] Lofling, JC; Hauzenberger, E.; Holgersson, J. Absorption of anti-blood group A antibodies on P-selectin glycoprotein ligand-1 / immunoglobulin chimeras carrying blood group A determinants: Core saccharidechain specificity of the Se and H gene encoded α1,2 fucosyltransferases indifferent host cells. Glycobiology 2002, 12, 173–182.

[16] Guix, S.; Asanaka, M.; Katayama, K.; Crawford, SE; Neill, FH; Atmar, RL; Estes, MK Norwalk virus RNA is infectious in mammaliancells. J. Virol. 2007, 81, 12238–12248. Summary of the Invention The purpose of this invention is to provide: A FUT2-overexpressing cell line and related technologies are provided to address the technical problem of the lack of a simple and easy-to-use cell model for evaluating the effectiveness of norovirus vaccines at the cellular level, or a combination thereof.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] The definition of the standard chemical term can be found in the reference "Molecular Cloning: A Laboratory Manual (4th Edition)": Science Press Co., Ltd.: March 2017: 1st Edition."

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as plasmid construction and identification, lentivirus preparation, qPCR, Western blot, and immunofluorescence detection methods, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The term "genetic engineering," also known as gene splicing technology and DNA recombination technology, is a genetic technology that uses molecular genetics as its theoretical basis and modern methods of molecular biology and microbiology as its means to construct hybrid DNA molecules in vitro according to a pre-designed blueprint using genes from different sources. These molecules are then introduced into living cells to change the original genetic characteristics of organisms, obtain new varieties, and produce new products.

[0014] The term "nucleotide" refers to a small molecule composed of a pentose sugar, a phosphate group, and a nitrogenous base. It is the basic unit of nucleic acids (DNA and RNA) and plays a crucial role in genetic information storage, energy metabolism, and cell signal transduction. In a nucleotide: the pentose sugar provides the backbone structure for RNA or DNA; DNA contains deoxyribose, and RNA contains ribose. The phosphate group is used to link sugar molecules (pentose sugars) to form phosphodiester bonds in the nucleic acid chain. The nitrogenous bases are purines (adenine, guanine) and pyrimidines (cytosine, thymine, uracil), which pair through hydrogen bonds (such as AT, CG). Different nucleotides are sometimes represented by the nitrogenous bases on them.

[0015] The term "vector" is a nucleic acid molecule capable of transporting another nucleic acid. Vectors can be, for example, plasmids, granules, viruses, or bacteriophages. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells.

[0016] The term "expression vector" is a vector that, when present in a suitable environment, can direct the expression of one or more transgenic proteins carried by the vector.

[0017] The term "virus-like particles" (VLPs) refers to hollow protein particles formed by the self-assembly of one or more viral structural proteins. These particles retain the natural conformation of viral antigens but do not contain genetic material and are neither infectious nor pathogenic. These particles have a regular surface structure (20-200 nanometers in diameter), possess nanomaterial properties, and can be mass-produced using animal cells or microbial expression systems. The term "plasmid vector" refers to a relatively small, circular DNA molecule (typically 1-200 kb in size) that exists independently of chromosomal DNA. Plasmids can be transferred, replicate independently, or integrate into chromosomal DNA and replicate along with it.

[0018] The term "viral vector" refers to a gene delivery tool developed by modifying the natural infection mechanism of viruses, which can introduce exogenous genetic material into host cells. This technology is widely used in cancer immunotherapy (such as the myvac™ platform combined with AI-predicted neoantigens), treatment of gene defect diseases, and vaccine development. Major types include lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors. Viral vectors must meet core indicators such as gene carrying capacity, transfer efficiency, and biosafety, and can achieve gene function regulation through integration or transient expression. The term "host cell" refers to a cell that can receive foreign genes (target genes) and express them within itself to produce the required proteins.

[0019] The term "protein expression" refers to a molecular biology technique that uses model organisms such as bacteria, yeast, animal cells, or plant cells to express exogenous gene proteins.

[0020] The term "stable expression" refers to the phenomenon where a foreign gene is integrated into the host cell genome through transfection technology, forming a stable and heritable transformed line. Its core characteristics are long-lasting and heritable expression of the foreign gene. This process often faces obstacles such as gene silencing, involving molecular mechanisms such as repetitive sequences, high copy numbers, and methylation levels. Currently, techniques such as genome editing, gene structure modification, and enhancer addition are mainly used to improve the efficiency of stable expression, which has significant application value in gene library construction and functional screening.

[0021] To achieve the above objectives, the present invention provides the following technical solution: On the one hand, the present invention provides a cell line with high FUT2 expression, namely human embryonic kidney cells HEK293T-FUT2-EGFP, which was deposited at the China Center for Type Culture Collection on December 2, 2025, with accession number CCTCC NO: C2025344.

[0022] On the other hand, the present invention provides applications of the above-mentioned cell lines, including evaluation of norovirus-like particle binding activity or evaluation of the effectiveness of norovirus vaccines.

[0023] On the other hand, the present invention provides a method for evaluating the binding activity of norovirus-like particles, including detecting the binding of the cell line described above with the norovirus-like particles.

[0024] Preferably, the detection method includes Western blot detection, ELISA detection, immunofluorescence detection, or RT-qPCR detection.

[0025] On the other hand, the present invention provides a method for evaluating the effectiveness of norovirus vaccines, using the aforementioned cell lines for evaluation.

[0026] Preferably, the method includes the following steps: Antiserum was obtained after immunizing test animals with norovirus vaccine. The antiserum was used to inhibit the adsorption of norovirus-like particles onto the cell line, and the efficacy of the norovirus vaccine was evaluated based on the inhibition effect.

[0027] Preferably, the method includes the following steps: The antiserum was mixed with norovirus-like particles, incubated, and then added to a culture medium containing the cell line. The adsorption of norovirus-like particles to the cell line was measured by an immunoassay.

[0028] Preferably, the immunization method is a cell ELISA detection method.

[0029] Preferably, the method includes the following steps: The antiserum was serially diluted and mixed with an equal volume of norovirus-like particles to obtain a mixture, which was then incubated at 37°C for 1 hour. The mixture was then added to the culture medium of the cell line and incubated at 4°C for 1 hour. OD was detected by ELISA. 450nm Optical density value at a given wavelength.

[0030] Preferably, the culture medium is serum-free DMEM medium, and the ELISA method is a cell ELISA detection method.

[0031] Preferably, the norovirus vaccine is selected from one or more of the following types of vaccines: GI.1, GI.2, GI.3, GI.7, GII.4, GII.5, GII.10, GII.12, GII.23, GII.24, and GII.25.

[0032] In some specific embodiments, the norovirus vaccine includes a G1.1 vaccine or a G2.4 vaccine.

[0033] The present invention has at least the following beneficial effects: This invention constructs a HEK 293T cell line overexpressing human α1,2-fucosyltransferase 2 (FUT2) with EGFP fluorescence (HEK 293T-FUT2-EGFP), enabling this cell line to stably express H, Lewis b, and Lewis y type HBGA antigens. Virus-like particles from the prototype strain GI.1 and the predominant circulating strain GII.4 of the human norovirus vaccine can attach to and bind to this cell line in a dose-dependent manner. Furthermore, antiserum from GI.1 and GII.4 VLPs vaccines can effectively inhibit their attachment. A cell-level alternative neutralization assay for human norovirus vaccines has been successfully developed and established. This not only provides new data support for establishing and improving a combined in vivo and in vitro vaccine efficacy evaluation system for recombinant norovirus vaccines, but also lays the foundation for research on the interaction between norovirus and host cells, possessing high practical application value.

[0034] This invention utilizes the overexpression of the FUT2 gene with an EGFP fluorescent tag in HEK 293T cells to screen for stable HEK293T-FUT2-EGFP cell lines, catalyzes the expression of H, Lewis b, and Lewis y human tissue blood group antigens, and then uses this cell line to develop for the first time a cell-level substitution and neutralization assay method for recombinant norovirus GI.1 and GII.4 VLP vaccines that have entered Phase III clinical trials. A cell model for evaluating the in vitro activity of norovirus vaccines has been established, providing a novel and practical method for vaccine development and evaluation, greatly accelerating the vaccine development process, and improving the quality control system for vaccine evaluation.

[0035] Preservation Instructions Preserved cell line: Human embryonic kidney cells HEK293T-FUT2-EGFP Homo sapiens; The cell line is accompanied by a scientific description; Accession number: CCTCC NO: C2025344; Preservation period: December 2, 2025; Depository: China Center for Type Culture Collection; Address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, inside Wuhan University. Attached Figure Description

[0036] Figure 1The results show the identification of the FUT2 lentiviral plasmid; Note: M represents DL 2000 DNA Marker; 1 indicates PCR amplification of the FUT2 gene with primers positioned upstream of the 5' end of the FUT2 fragment and downstream of the 3' end of the FUT2 fragment; 2 indicates PCR amplification of the FUT2 gene with primers positioned upstream of the 5' end of the FUT2 fragment and downstream of the 3' end of the EGFP sequence; 3 indicates PCR amplification of the FUT2 gene with primers positioned upstream of the CMV promoter sequence and downstream of the 3' end of the FUT2 fragment; 4 indicates PCR amplification of the FUT2 gene with primers positioned upstream of the CMV promoter sequence and downstream of the 3' end of the EGFP sequence; 5 indicates the negative control.

[0037] Figure 2 Screening for Puro concentration in HEK 293T.

[0038] Figure 3 The expression of FUT2 in the HEK 293T-FUT2-EGFP cell line is shown in Figure A. Figure A shows the fluorescence image of the HEK 293T-FUT2-EGFP cell line, observed under an inverted fluorescence microscope at 100×. Figure B shows the RT-qPCR detection of the HEK 293T-FUT2-EGFP cell line, with expression set at 1 in wild-type HEK 293T cells. A4, A10, D7, E5, E8, F9, and G5 represent different HEK293T-FUT2-EGFP monoclonal cell lines. Figure C shows the Western blot detection of the HEK 293T-FUT2-EGFP cell line. Data are from independent triplicate experiments. Statistical significance was analyzed using a t-test in GraphPad Prism 8.0.2. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0039] Figure 4 The expression of HBGA in the HEK 293T-FUT2-EGFP cell line is shown in Figure A, where H-type HBGA expression, Ley-type HBGA expression, Leb-type HBGA expression, A-type HBGA expression, and B-type HBGA expression are shown in Figure E. The cells were observed using a 1000× laser confocal microscope.

[0040] Figure 5 The images show the binding of norovirus-like particles to the 293T-FUT2 cell line; A and B show the immunofluorescence analysis (1000× observation) of the binding of norovirus-like particles GI.1 and GII.4 to the HEK 293T-FUT2-EGFP cell line; C and D show the Western blot analysis of the binding of norovirus-like particles GI.1 and GII.4 to the HEK 293T-FUT2-EGFP cell line.

[0041] Figure 6 This study investigated the dose-dependent binding of norovirus GI.1 and GII.4 virus-like particles to the HEK 293T-FUT2-EGFP cell line. Figure A shows the binding of norovirus GI.1 virus-like particles to the HEK 293T-FUT2-EGFP cell line; Figure B shows the binding of norovirus GII.4 virus-like particles to the HEK 293T-FUT2-EGFP cell line. Data were presented as independent triplicate experiments, and statistical significance was analyzed using a t-test in GraphPad Prism 8.0.2. **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered statistically significant.

[0042] Figure 7 Alternative neutralization experiments for norovirus vaccine evaluation; A: Results of mouse serum inhibition of GI.1 VLPs attachment by anti-GI.1 VLPs; B: Results of mouse serum inhibition of GII.4 VLPs attachment by anti-GI.4 VLPs; VLPs were pre-incubated with serially diluted mouse antiserum at 37°C for 1 hour, and the resulting VLPs / antiserum mixture was added to HEK 293T / HEK 293T-FUT2-EGFP cells; attached VLPs were detected using VLP-specific rabbit antiserum; data are OD. 450nm The mean ± SD of the values; all experiments were repeated three times. Detailed Implementation

[0043] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0045] Example 1: Cell line construction and application (1) Materials and Methods 1.1 Cells HEK 293T cells were preserved by Lanzhou Institute of Biological Products Co., Ltd.

[0046] 1.2 Sample The recombinant human norovirus GⅠ.1 and GⅡ.4 VLPs vaccines were prepared and supplied by Lanzhou Institute of Biological Products Co., Ltd.

[0047] 1.3 Main Reagents and Instruments pLVX-IRES-Puro lentiviral vector, psPAX2, and pMD2.G lentiviral packaging plasmids were purchased from Novagen, Germany; monoclonal antibodies against human ABO, Lewis b (Leb), Lewis y (Ley), A, and B human tissue blood group antigens, anti-EGFP monoclonal antibody, anti-GAPDH monoclonal antibody, rabbit anti-mouse IgG H&L (HRP), goat anti-mouse IgG H&L (Alexa Fluor® 647), and DAPI staining solution were purchased from Abcam, UK; monoclonal antibody against human H type human tissue blood group antigen was purchased from Neobiotechnologies, USA; monoclonal antibody against human H1 type human tissue blood group antigen, rabbit anti-goat IgG (H&L) secondary antibody, and Lipofectamine 2000 were purchased from Invitrogen, USA; DL2000 DNA Marker, Taq enzyme, and reverse transcription kit were purchased from Takara, Japan; Plasmid Plus Midi Kit, RNEsay Mini Kit, and QIAamp DNA Mini Kit were also purchased. All kits were purchased from QIAGEN GmbH, Germany; fetal bovine serum (FBS), Dulbecco's modified eagle medium (DMEM), puromycin, and 0.25% trypsin were purchased from Gibco, USA; bovine serum albumin (BSA), 4S Red Plus nucleic acid staining agent, and ampicillin were purchased from Sangon Biotech (Shanghai) Co., Ltd.; BCA protein concentration assay kit, RIPA lysis buffer, QuickBlock WB blocking buffer, primary antibody dilution buffer, penicillin-streptomycin solution, and sealing film were purchased from Beyotime Biotechnology Co., Ltd.; Rainbow 180 broad-spectrum protein marker, universal tissue fixative, protein loading buffer, and electrophoresis buffer were purchased from Beijing Solarbio Biotechnology Co., Ltd. 6, 12, 24, and 96-well plates, T25, and T75 cell culture flasks were all purchased from Nunc, USA; the MiniAmp Plus PCR instrument, MULTISKAN FC microplate reader, HEARcell 150i CO2 cell culture incubator, and SORVALL ST8R centrifuge were all purchased from Thermo Fisher Scientific, USA; the IX71 fluorescence microscope was purchased from OLYMPUS, Japan; and the LSM-980 laser confocal microscope was purchased from Zeiss, Germany (provided by the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences).

[0048] 1.4 Construction and Identification of pLVX-IRES-Puro-FUT2-EGFP Lentiviral Plasmid The pLVX-IRES-Puro-FUT2-EGFP plasmid was synthesized by Genewiz Suzhou Co., Ltd. using the pLVX-IRES-Puro lentiviral vector as the vector backbone. Four pairs of validation primers were designed. The first pair of primers was designed upstream at the 5' end of the FUT2 fragment and downstream at the 3' end (upstream primer: 5'-ATGCTGGTCGTTCAGATGCC-3' (SEQ ID NO. 1); downstream primer: 5'-TTAGTGCTTGAGTAAGGGGG-3' (SEQ ID NO. 2)). The second pair of primers was designed upstream at the 5' end of the CMV promoter sequence and downstream at the 3' end of the FUT2 fragment (upstream primer: 5'-CGCAAATGGGCGGTAGGCGTG-3' (SEQ ID NO. 3); downstream primer: 5'-TTAGTGCTTGAGTAAGGGGG-3' (SEQ ID NO. 2)). The third pair of primers was designed upstream at the 5' end of the FUT2 fragment and downstream at the 3' end of the EGFP sequence (upstream primer: 5'-ATGCTGGTCGTTCAGATGCC-3' (SEQ ID NO. 1); downstream primer: The first primer pair consisted of 5'-CTTGTACAGCTCGTCCATGC-3' (SEQ ID NO.4); the second primer pair was designed upstream at the 5' end of the CMV promoter sequence and downstream at the 3' end of the EGFP fragment (upstream primer: 5'-CGCAAATGGGCGGTAGGCGTG-3' (SEQ ID NO.3); downstream primer: 5'-CTTGTACAGCTCGTCCATGC-3' (SEQ ID NO.4)). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The target band was amplified by PCR using the four pairs of validation primers, and the PCR products were analyzed by agarose gel electrophoresis to determine the correct size of the target band. The reaction mixture consisted of: 5.0 μL of 10× Buffer, 2.0 μL of 50 mmol Mg2+, 4.0 μL of 2.5 mmol dNTPs, 2.5 μL each of forward and reverse primers, 2.0 μL of template, 0.5 μL of Taq polymerase, and 31.5 μL of ddH2O. The amplification cycle was: 95 ℃ for 5 min; 95 ℃ for 5 s; 60 ℃ for 30 s; 72 ℃ for 40 s, for 30 cycles; and 72 ℃ for 10 min. The synthesized plasmid was sequenced, confirming its accuracy. The linear sequence of the pLVX-IRES-Puro-FUT2-EGFP plasmid is SEQ ID NO. 5.

[0049] 1.5 Construction of a stable HEK 293T-FUT2-EGFP cell line 1.5.1 Determination of puromycin screening concentration HEK 293T cells were used at a rate of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates. After 24 hours, the culture medium was discarded, and culture medium containing different gradient concentrations of puromycin (0.125 μg / mL, 0.250 μg / mL, 0.500 μg / mL, 1.000 μg / mL, 2.000 μg / mL, and 4.000 μg / mL) was added to the 6-well plates. Fresh puromycin-containing culture medium was replaced every 2 days. The cells were cultured for 14 days, and the lowest concentration of puromycin that resulted in complete cell death was taken as the optimal selection concentration for the cell line.

[0050] 1.5.2 Preparation and Transfection of Lentivirals HEK 293T cells were cultured in DMEM containing 10% FBS at 5% CO2 and adherent at 37°C. pLVX-IRES-Puro-FUT2-EGFP, psPAX2, and pMD2.G were co-transfected into HEK 293T cells at a ratio of 3:2:1 using Lipofectamine 2000. Lentiviral particles were harvested and filtered at 48 and 72 h post-transfection. These lentiviral particles were used to infect HEK 293T cells in logarithmic growth phase. Cells were selected 48 h post-infection using puromycin at the optimal selection concentration, with the culture medium changed every 2 days. Cells that did not die after 14 days of culture were identified as stable HEK 293T-FUT2-EGFP transfected cells, passaged twice, and then cryopreserved.

[0051] 1.6 Cloning of HEK 293T-FUT2-EGFP stable cell line HEK 293T-FUT2-EGFP cells were seeded at a rate of 1 cell / well in 96-well plates using a limiting dilution method. 200 μL of DMEM medium containing 1 μg / mL puromycin and 10% FBS was added, and the cells were cultured for 14 days. Cell colony formation was observed under a microscope. In wells with colony formation, the single-clone cells were transferred to 24-well plates, and 1 mL of DMEM medium containing 1 μg / mL puromycin and 10% FBS was added to each well for further culture. Once the cell density reached over 80%, the single-clone cells were transferred to 6-well plates, and 2 mL of DMEM medium containing 1 μg / mL puromycin and 10% FBS was added to each well for further culture. The same method was used to sequentially culture each single-clone cell at 25 cm⁻¹. 2 75cm 2 The cells were expanded and cryopreserved in cell flasks for subsequent testing.

[0052] 1.7 RT-qPCR detection of FUT2 expression in HEK 293T-FUT2-EGFP cell line Total RNA was extracted from HEK 293T cells, HEK 293T-FUT2 cell line, and each monoclonal cell line using the RNesay Mini Kit. Then, 1 μg of each RNA was reverse transcribed into cDNA using a reverse transcription kit. qPCR primers were designed for FUT2 (upstream primer: 5'-ATTGGGACGTTCGGGATCTG-3' (SEQ ID NO. 6); downstream primer: 5'-GTCGGGGAGGGTGTAATTGG-3' (SEQ ID NO. 7)) and for human β-actin (upstream primer: 5'-GGACTTCGAGCAAGAGATGG-3' (SEQ ID NO. 8); downstream primer: 5'-AGCACTGTGTTGGCCTACAG-3' (SEQ ID NO. 9)). qPCR amplification was performed using cDNA as a template and the above primers, with human β-actin as an internal control. Data analysis was performed using the 2-ΔΔCT method.

[0053] 1.8. Western Blot detection of FUT2 expression in HEK 293T-FUT2-EGFP cell line HEK 293T cells, HEK 293T-FUT2-EGFP stable transgenic mixed cells, and individual HEK 293T-FUT2-EGFP monoclonal cell lines were lysed at -4℃ using RIPA lysis buffer. After complete lysis, the cells were centrifuged at 12000 rpm for 5 min, and the supernatant was collected. 50 μL of the supernatant from each cell was used to determine protein concentration using a BCA protein assay kit. The remaining protein was boiled in Loading Buffer at 100℃ for 10 min before Western blotting. The primary anti-EGFP antibody was diluted 1:2000 and incubated overnight at 4℃ on PVDF membranes. The secondary antibody, rabbit anti-mouse IgG HRP, was diluted 1:5000 and incubated on a shaker at room temperature for 1 h on PVDF membranes. The membranes were then exposed and developed using ECL chemiluminescence working solution, with GAPDH as an internal control.

[0054] 1.9 Immunofluorescence assay for HBGA expression in HEK 293T-FUT2-EGFP cell line HEK 293T cells and the validated HEK 293T-FUT2-EGFP monoclonal A10 cell line were cultured at a rate of 1×10⁻⁶. 6Cells were seeded per well in 6-well plates with coverslips. After 24 hours, when the cell density reached 80%, the culture medium was discarded. Cell slides were fixed with 4% paraformaldehyde at room temperature, permeated with 0.1% Triton X-100 at room temperature for 5 minutes, and incubated with BSA at room temperature for 1 hour. Primary antibody was diluted 1:100 and the slides were incubated overnight at 4°C. Cells were then incubated with Alexa Fluor 647-bound secondary antibody and 4',6-diamino-2-phenylindole (DAPI) in PBS buffer. The slides were mounted and fluorescence was observed under a laser confocal microscope.

[0055] 1.10. Detection of binding of human norovirus VLPs to HEK 293T-FUT2-EGFP cell line 1.10.1 Western Blot detection of the binding of human norovirus VLPs to HEK 293T-FUT2-EGFP cell line HEK 293T cells and the validated HEK 293T-FUT2-EGFP monoclonal A10 cell line were mixed at a ratio of 1×10⁻⁶ cells / cells. 6 Four cells / well were seeded into each of the six wells. After 24 hours, when the cell density reached 80%, the culture medium was discarded. The culture medium in one well each of HEK 293T cells and A10 monoclonal cells was replaced with medium containing 2 μg G I.1 VLP. Then, one well of each cell line was replaced with medium containing 2 μg G II.4 VLP. The remaining four wells were kept unchanged. Cells were incubated at 4°C for 2 hours. Cells from eight wells were lysed using RIPA lysis buffer. After complete lysis, the cells were centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected. 50 μL of the supernatant from each well was used to determine the protein concentration using a BCA protein assay kit. The remaining protein was boiled in Loading Buffer at 100°C for 10 minutes before Western blotting. Anti-GⅠ.1 / GⅡ.4 VLP antibody was diluted 1:10000 and incubated overnight at 4°C on PVDF membranes. Secondary antibody rabbit anti-mouse IgG HRP was diluted 1:5000 and incubated on a shaker at room temperature for 1 hour. The membranes were then exposed and developed using ECL luminescent working solution. GAPDH was used as an internal control. Western blot was used to detect the binding ability of HEK 293T cells and monoclonal HEK 293T-FUT2-EGFP cell lines to VLP antigens.

[0056] 1.10.2. Immunofluorescence assay for the binding of human norovirus VLPs to HEK 293T-FUT2-EGFP cell line. HEK 293T cells and the validated HEK 293T-FUT2-EGFP monoclonal A10 cell line were cultured at a rate of 1×10⁻⁶. 6Two cells / well were seeded into each well of a 6-well plate containing coverslips. After 24 hours, when the cell density reached 80%, the culture medium was discarded. The culture medium in one well of each HEK 293T cell line and A10 monoclonal cell line was replaced with medium containing 2 μg GⅠ.1 VLP, and then the culture medium in one well of each was replaced with medium containing 2 μg GⅡ.4 VLP. The cells were incubated at 4°C for 2 hours and washed three times with PBS buffer. Subsequent procedures followed step 1.9, using immunofluorescence to detect the binding ability of HEK 293T cells and the monoclonal 293T-FUT2 cell line to VLP antigens.

[0057] 1.11 Establishment of a cell ELISA method for norovirus VLP attachment The day before the cell ELISA experiment, confirm cell status to ensure high cell viability (>90%). Measure viable cell density using a cell counter, and fill 96-well plates with 1 × 10⁶ cells per well. 6 Deploy cells into plates according to the specified quantity. Gently mix the cell suspension during plating to avoid clumping, and allow the plates to stand to ensure even cell distribution after plating. Aspirate the cell culture medium and add 100 µL of DMEM containing different doses of norovirus virus-like particles (0.5, 1.0, 2.0, 4.0, 8.0, 16.0 μg / mL) to each well, and incubate at 4°C for different times (0.5, 1, 1.5, 2 h). After incubation, centrifuge at 4000 rpm for 5 min and wash three times with 1% PBST buffer on a shaker. Fix with 100 µL of 4% paraformaldehyde to each well at room temperature for 30 min. Add 100 µL of ELISA blocking buffer of different concentrations to each well and block at 37°C for 1 h. Add 100 µL of NoV-specific antibody at different dilution gradients (1:10000, 1:20000) to each well in PBST buffer containing 1% PBST, and incubate at 37°C for 1 hour. After final color development, mix the solutions in the 96-well plate twice using a microplate reader, and measure the optical density at OD450 nm. Select the optimal experimental conditions to establish a cell ELISA method.

[0058] 1.12. Animal immunization with recombinant human norovirus GI.1 and GII.4 vaccines Recombinant norovirus GI.1 / GII.4 VLP antigen was diluted to 2 μg / ml using aluminum adjuvant diluent. Six- to eight-week-old female Balb / c mice were randomly assigned to groups of 10 each, with the aluminum adjuvant diluent serving as a control. 0.5 mL of the antigen / aluminum adjuvant mixture was administered intraperitoneally at weeks 0 and 3, respectively. Two weeks after the second immunization, blood was collected from the mice's eyes, and serum was separated at 4°C, 3000 rpm, and 5 min.

[0059] 1.13. Adhesion inhibition experiment of recombinant human norovirus GI.1 and GII.4 VLPs HEK 293T and HEK 293T-FUT2-EGFP monoclonal A10 cell lines were seeded into 96-well plates one day before use (1 × 10⁶ cells per well). 6 Each immune serum sample was serially diluted and mixed with an equal volume of 4 μg VLPs (serum dilution ratios: 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800), and incubated at 37°C for 1 h. The resulting VLP / serum mixture was then added to HEK 293T and HEK 293T-FUT2-EGFP monoclonal A10 cells, and co-incubated at 4°C for 1 h. The OD was detected using the cell ELISA method established in step 1.11. 450nm Optical density value at a given wavelength.

[0060] 1.14 Statistical Analysis Statistical analysis and graphing were performed using GraphPad Prism 8.0.2 software. The t-test was used to compare the two groups, and the results are shown in the graph as follows: ns≥0.05; *p<0.05; **p<0.01; and ***p<0.001.

[0061] (2) Results 2.1 Construction of pLVX-IRES-Puro-FUT2-EGFP lentiviral plasmid The target band was amplified by PCR using four pairs of validation primers. The molecular weight of the target band was determined by agarose gel electrophoresis of the PCR products. The results are as follows: Figure 1 As shown, the amplification product of the first primer pair FUT2-F and FUT2-R was 1032 bp; the amplification product of the second primer pair FUT2-F and EGFP-R was 1788 bp; the amplification product of the third primer pair CMV-F and FUT2-R was 1330 bp; and the amplification product of the fourth primer pair CMV-F and EGFP-R was 2086 bp. The electrophoresis results were consistent with the size of the target fragment, confirming that the plasmid was pLVX-IRES-Puro-FUT2-EGFP.

[0062] 2.2 Determination of Puromycin (Puro) Screening Concentration in HEK 293T Cell Line Puro concentration screening was performed as described in the experimental method, and the results are as follows: Figure 2 As shown, the final Puro screening concentration for HEK 293T was determined to be 1 ug / mL.

[0063] 2.3 Construction of a stable FUT2 overexpression cell line pLVX-FUT2-EGFP, psPAX2, and pMD2.G plasmids were co-transfected into HEK 293T cells to generate lentiviral particles. After 48 hours, lentiviral particles were collected and re-infected with HEK 293T cells in the logarithmic growth phase. After 24 hours, selection was performed using 1 μg / ml puromycin. A mixed cell line expressing green fluorescent protein was successfully screened. Seven monoclonal cell lines were then obtained from the mixed cell line using a limiting dilution method (see...). Figure 3 A).

[0064] Total RNA was extracted from HEK 293T-FUT2-EGFP hybrid cell lines and individual monoclonal cell lines, and analyzed by RT-qPCR. Compared with wild-type HEK 293T cells, the FUT2 RNA levels in monoclonal and hybrid cell lines were significantly increased by an average of 25-45 times (see...). Figure 3 Total protein was extracted from the HEK 293T-FUT2-EGFP mixed cell line and each monoclonal cell line. Western blot analysis of FUT2-EGFP expression at the protein level was performed using an EGFP antibody. The results were consistent with RT-qPCR, with the highest FUT2 expression level observed in the A10 monoclonal cell line (see B). Figure 3 (C). In subsequent experiments, the HEK293T-FUT2-EGFP cell line was selected as A10. The A10 cell line was deposited at the China Center for Type Culture Collection on December 2, 2025, with accession number CCTCC NO: C2025344.

[0065] 2.5. The HEK 293T-FUT2-EGFP cell line can successfully express H, Leb, and Ley type HBGA. Blood group precursors preferentially bind to fucose to form H antigens under the catalysis of FUT2. These antigens are subsequently catalyzed to synthesize Leb, Ley, A, and B antigens. To investigate the expression of HBGA in HEK 293T-FUT2-EGFP, HEK293T and HEK 293T-FUT2-EGFP cells were immobilized, and immunofluorescence analysis was performed using antibodies corresponding to H, Leb, Ley, A, and B HBGA types. Laser confocal microscopy observation showed that no red fluorescent signal was detected in HEK 293T cells, indicating the absence of HBGA in HEK 293T cells, while HEK 293T-FUT2-EGFP cells exhibited H, Ley, and Leb antigen-specific red fluorescent signals (see [link to study].) Figure 4 (A, B, C), but no specific fluorescent signal for antigens A and B (see A, B, C). Figure 4(D, E). The blue fluorescent signal represents DAPI-stained cell nuclei, and the green fluorescent signal represents EGFP expression in the constructed HEK 293T-FUT2-EGFP cell line. The results show that the HEK 293T-FUT2-EGFP cell line specifically expresses H, Ley, and Leb type HBGA.

[0066] 2.6 Human norovirus GI.1 and GII.4 virus-like particles can bind to HEK 293T-FUT2-EGFP cell line. Using our independently developed recombinant human norovirus bivalent (GI.1 / GII.4) vaccine technology, 1 μg of norovirus GI.1 and GII.4 virus-like particles were obtained and co-incubated with HEK 293T and HEK 293T-FUT2-EGFP cells at 4°C for 1 h. Immunofluorescence was performed using antibodies specific to the corresponding virus-like particles, and total cellular protein was extracted and analyzed using Western blot. Figure 5 As shown in Figures A and C, no red fluorescent signal was detected in HEK 293T cells, indicating that recombinant norovirus GI.1 and GII.4 virus-like particles do not bind to wild-type HEK 293T cells. However, significant red fluorescent signals were observed in HEK 293T-FUT2-EGFP cells, indicating that norovirus GI.1 and GII.4 virus-like particles can bind to cell lines overexpressing FUT2. The green fluorescent protein represents EGFP protein expression in the HEK 293T-FUT2-EGFP cell line. Similarly, as... Figure 5 As shown in Figures B and D, GAPDH was used as an internal control in the experiment. Western blot results showed that norovirus GI.1 and GII.4 virus-like particles could only be detected after co-incubation with the HEK 293T-FUT2-EGFP cell line. This indicates that the virus-like particles of both the prototype strain GI.1 and the circulating strain GII.4 of the recombinant norovirus bivalent (GI.1 / GII.4) vaccine developed by our unit can effectively attach to the HEK 293T-FUT2-EGFP cell line constructed in this invention, laying a good foundation for the subsequent development of cell replacement neutralization experiments.

[0067] 2.7 Establishment of a dose-dependent ELISA method for binding human norovirus GI.1 and GII.4 virus-like particles to HEK 293T-FUT2-EGFP cell line. To facilitate a simple and quantitative detection of the cell-adsorption characteristics of norovirus VLPs, this invention developed a cell immunoassay (ELISA). The effectiveness of this detection method was verified by co-incubating different doses of norovirus VLPs with HEK 293T-FUT2-EGFP cells and detecting them using specific antibodies. Figure 6 As shown, GI.1 and GII.4 virus-like particles (VLPs) can bind efficiently to HEK 293T-FUT2-EGFP cells in a dose-dependent manner. These results indicate that the cell ELISA developed on HEK 293T-FUT2-EGFP cells constructed in this invention can serve as a simple and rapid detection method for assessing the cell adsorption characteristics of norovirus VLPs.

[0068] 2.8 Development of Cell-Level Substitution Neutralization Experiments for Evaluating the Efficacy of Human Norovirus Vaccines Given that the human norovirus VLPs developed by our company can effectively bind to HEK 293T-FUT2-EGFP cells, this invention has developed an alternative neutralization assay based on cell ELISA. Figure 7 As shown, the results indicate that mouse antiserum containing GI.1 and GII.4 VLPs can inhibit the binding of the corresponding VLPs to HEK 293T-FUT2-EGFP cells in a dose-dependent manner, with neutralizing titers of GI.1 and GII.4 of 1:6400 and 1:12800, respectively. In contrast, control serum obtained by injecting mice with aluminum adjuvant could not inhibit the binding of GI.1 and GII.4 VLPs to HEK 293T-FUT2-EGFP cells. Therefore, this invention demonstrates the successful development of a cellular-level alternative neutralization assay for evaluating the efficacy of norovirus vaccines.

[0069] Because HuNoV lacks a suitable in vitro cell culture model, the level of vaccine-induced neutralizing antibodies cannot be directly measured through neutralization assays. This significantly limits the accurate evaluation of protective efficacy in HuNoV vaccine development. To address the limitations of existing evaluation methods, which are singular, restrictive, and unable to comprehensively assess vaccine efficacy, it is urgent to optimize and standardize these methods.

[0070] This invention successfully constructed a stable HEK 293T-FUT2-EGFP cell line for the first time, and established a cellular-level substitution neutralization assay for evaluating the efficacy of norovirus vaccines based on this cell line. Results showed that overexpression of FUT2 in HEK 293T cells induced high expression of blood group tissue antigens H, Lewis b, and Lewis y. The GI.1 and GII.4 VLPs of our company's world's first human norovirus vaccine to enter Phase III clinical trials could bind to this cell line in a dose-dependent manner. Furthermore, the specific antiserum for GI.1 and GII.4 VLPs vaccines effectively inhibited their attachment, thus successfully establishing a cellular-level substitution neutralization assay for norovirus vaccines. Furthermore, ten norovirus genotypes, including GI.2, GI.3, GI.7, GII.3, GII.6, GII.7, and GII.25, can bind to H2 and Lewis antigens. Therefore, it is reasonable to speculate that HEK293T-FUT2-EGFP can serve as a cell model for evaluating the effectiveness of norovirus vaccines against these strains.

[0071] Compared to the existing BT50 method for determining norovirus vaccine efficacy, the HEK 293T-FUT2-EGFP cell line expressing H, Lewis b, and Lewis y HBGA can be affected by the interaction and restriction of membrane lipids and membrane proteins.

[17] Compared to free HBGA, it can better simulate the state of norovirus host cells, thus this model can better simulate the binding characteristics of norovirus. Secondly, the stability of the BT50 method is highly dependent on the batch of HBGA purchased.

[18] The HEK 293T-FUT2-EGFP stable cell line constructed in this invention originates from the same monoclonal cell line and can stably express HBGA, thus exhibiting higher experimental consistency. Furthermore, compared to HIEs, the cell line developed in this invention is easier to obtain, the method is simpler to operate, and it can also be used for high-throughput screening of norovirus antibodies and drugs.

[0072] In summary, this invention successfully constructed a stable HEK 293T-FUT2-EGFP cell line and developed a cell-level substitution neutralization experiment for norovirus vaccine, establishing a cell model for evaluating the effectiveness of norovirus vaccine.

[0073] References:

[17] Nasir, W.; Frank, M.; Kunze, A.; Bally, M.; Parra, F.; Nyholm,PG; Hook, F.;

[18] Uusi-Kerttula, H.; Tamminen, K.; Malm, M.; Vesikari, T.; Blazevic, V. Comparison of human saliva and synthetic histo-blood groupantigens usage as ligands in norovirus-like particle binding and blocking assays. Microbes Infect. 2014, 16, 472–480. Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A cell line with high FUT2 expression, characterized in that, The cell line described is human embryonic kidney cells HEK 293T-FUT2-EGFP, which was deposited at the China Center for Type Culture Collection on December 2, 2025, with accession number CCTCC NO: C2025344.

2. The application of the cell line according to claim 1, characterized in that, The applications include evaluating norovirus-like particle binding activity or evaluating the effectiveness of norovirus vaccines.

3. A method for evaluating the binding activity of norovirus-like particles, characterized in that, This includes detecting the norovirus-like particles after the cell line of claim 1 has been bound to them.

4. The method according to claim 3, characterized in that, The detection methods include Western blot detection, ELISA detection, immunofluorescence detection, or RT-qPCR detection.

5. A method for evaluating the effectiveness of a norovirus vaccine, characterized in that, The cell line described in claim 1 was used for evaluation.

6. The method according to claim 5, characterized in that, Includes the following steps: Antiserum was obtained after immunizing test animals with norovirus vaccine. The antiserum was used to inhibit the adsorption of norovirus-like particles onto the cell line, and the efficacy of the norovirus vaccine was evaluated based on the inhibition effect.

7. The method according to claim 6, characterized in that, Includes the following steps: The antiserum was mixed with norovirus-like particles, incubated, and then added to a culture medium containing the cell line. The adsorption of norovirus-like particles to the cell line was measured by an immunoassay.

8. The method according to claim 7, characterized in that, Includes the following steps: The antiserum was serially diluted and mixed with an equal volume of norovirus-like particles to obtain a mixture, which was then incubated at 37°C for 1 hour. The mixture was then added to the culture medium of the cell line and incubated at 4°C for 1 hour. OD was detected by ELISA. 450nm Optical density value at a given wavelength.

9. The method according to any one of claims 5-8, characterized in that, The norovirus vaccine is selected from one or more of the following types of vaccines: GI.1, GI.2, GI.3, GI.7, GII.4, GII.5, GII.10, GII.12, GII.23, GII.24, and GII.

25.

10. The method according to claim 9, characterized in that, The norovirus vaccine includes either type G1.1 or type G2.4.

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