An HEK-293T cell line with high expression of H2-type human histo-blood group antigen, and its construction method and application

Abstract

The present invention relates to the field of biotechnology, and specifically to a method for constructing an HEK-293T cell line with high expression of H2-type human histo-blood group antigen, comprising the following steps: constructing an FUT2 lentiviral plasmid, co-transfecting the HEK-293T cells with the FUT2 lentiviral plasmid, pMD.2G and psPAX2 packaging plasmids using Lipofectamine 2000, and harvesting lentiviral particles; infecting the HEK-293T cells in the logarithmic growth phase with the lentiviral particles, and performing puromycin pressure screening to obtain the HEK-293T / FUT2 cell line. The present invention for the first time establishes an HEK-293T cell line with stable high expression of H2-type human histo-blood group antigen, and based on this cell line, establishes a method for evaluating the binding activity of GI.1-type recombinant norovirus virus-like particles.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to an HEK-293T cell line with high expression of H2-type human histo-blood group antigen, and a construction method and application thereof. Background Art

[0002] Histo-blood group antigens (HBGAs) are a class of fucosylated proteins that are abundantly distributed on the mucosal epithelial cells of the respiratory tract, urogenital tract, and intestine, and also exist in saliva, milk, and blood in the form of free oligosaccharides. They are divided into ABH antigens and Lewis antigens. The fucosylation of all proteins in cells is completed under the catalytic action of fucosyltransferases (FUTs). FUTs are responsible for transferring fucose to sugar, glycoprotein, or glycolipid molecules. Among them, FUT1-2 is related to the synthesis of α-1,2 fucosidic bonds; FUT3-7 and 9 are related to the synthesis of α-1,3 / 4 fucosidic bonds; FUT8 is related to the synthesis of α-1,6 fucosidic bonds. The blood group precursor substance, under the catalysis of α-1,2 fucosyltransferase, preferentially binds fucose to form H antigen, and the H antigen forms A antigen and B antigen under the action of A enzyme and B enzyme, which are the ABH blood group antigens. The Lewis antigen is that the blood group precursor substance forms H antigen under the action of α-1,2 fucosyltransferase, and then is catalyzed by α-1,3 / 4 fucosyltransferase to form Lewis b and Lewis y antigens.If the gene mutation is inactivated, the blood group precursor substance cannot be catalyzed by the α-1,2 fucosyltransferase expressed by FUT2 to form H antigen. Instead, it is directly acted on by α-1,3 / 4 fucosyltransferase to form Lewis a antigen (Archana P, Geetha P, Avinash L M T, et al. Histo-Blood Group Antigens in Oral Cancer and Potentially Malignant Disorders[J]. Asian Pacific journal of cancer prevention: APJCP, 2020, 21(4). Zhong Kan. Chemical Enzymatic Synthesis of Complex Glycans Related to Tissue Blood Group Antigens[D]. Shandong University, 2021. DOI: 10.27272 / d.cnki.gshdu.2021.000536. Hu MY, Zhang X Y, Li J Z, et al. Fucosyltransferase 2: A Genetic Risk Factor for Intestinal Diseases[J]. Frontiers in Microbiology, 2022, 13. DOI: 10.3389 / FMICB.2022.940196. Louisa P, Aisleen B, C K J, et al. Nonsecretor Histo-blood Group Antigen Phenotype Is Associated With Reduced Risk of Clinical Rotavirus Vaccine Failure in Malawian Infants.[J]. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America, 2019, 69(8).).

[0003] Norovirus is the main non-bacterial pathogen for acute diarrhea and sporadic outbreaks globally. There are 267 million people infected globally every year, more than 1 million people are hospitalized, resulting in 219,000 deaths, and bringing a medical cost and social burden of more than 64.5 billion US dollars. [5]。Norovirus is divided into 10 genomes, which are then further subdivided into more than 40 genotypes. Genomes GI, GII, and GIV are commonly found in human infection cases. Volunteer challenge tests and a large number of basic studies have shown that HBGA is an adsorption factor and receptor for norovirus, which plays an important role in host susceptibility to norovirus, host range, and epidemics, and is an important host factor mediating norovirus susceptibility and drug resistance. Multiple research data indicate that the expression of HBGA is related to norovirus type-specific susceptibility. The vast majority of norovirus types exhibit an H-type HBGA-dependent susceptibility pattern (A C M, Duncan S, L M S. Norovirus diarrhea is significantly associated with higher counts of fecal histo-blood group antigen expressing Enterobacter cloacae among black South African infants[J]. Gut microbes, 2021, 13(1). Almand E A, Moore M D, Jaykus L A. Norovirus Binding to Ligands Beyond Histo-Blood Group Antigens[J]. Front Microbiol. 2017;8:2549. Esseili M A, Gao X, Boley P, et al. Human Norovirus Histo-Blood Group Antigen(HBGA) Binding Sites Mediate the Virus Specific Interactions with Lettuce Carbohydrates[J]. Viruses. 2019;11(9):833. Almand E A, Moore M D, Jaykus L A. Characterization of human norovirus binding to gut-associated bacterial ligands[J]. BMC Res Notes. 2019;12(1):607. Mallagaray A, J, Hansman G, et al. Attachment of norovirus to histo blood group antigens: a cooperative multistep process[J]. Angew Chem Int Ed Engl. 2015;54(41):12014 - 12019.)。

[0004] However, there has been no report on a HEK - 293T cell line with high expression of H2 - type human histo - blood group antigens and its construction method and application. Summary of the Invention

[0005] The purpose of the present invention is to over - express the fucosyltransferase 2 (FUT2) gene in HEK - 293T cells, catalyze the formation of the norovirus receptor human histo - blood group antigens, and then mediate the interaction between norovirus virus - like particles and the receptor, providing a cell model for the in - vitro activity evaluation of norovirus vaccines at the cellular level and materials for the study of the interaction between norovirus and host cells.

[0006] To achieve the above - mentioned purpose, the present invention adopts the following technical solutions:

[0007] Construct the FUT2 lentiviral plasmid, co - transfect HEK - 293T cells with the FUT2 lentiviral plasmid, pMD.2G and psPAX2 packaging plasmids using Lipofectamine 2000, and harvest the lentiviral particles. Infect the HEK - 293T cells in the logarithmic growth phase with the lentiviral particles, screen with puromycin pressure to obtain the HEK - 293T / FUT2 cell line, and identify whether the FUT2 gene is integrated into the genome by PCR. Detect the FUT2 mRNA transcription level of HEK - 293T / FUT2 cells by RT - qPCR, analyze the expression of HBGAs in HEK - 293T / FUT2 cells by flow cytometry, and analyze the binding activity between HEK - 293T / FUT2 cells and norovirus virus - like particles by indirect immunofluorescence assay.

[0008] The present invention successfully constructed a lentiviral plasmid PLVX-IRES-Puro-FUT2 with the correct sequence and obtained lentiviral particles. After the lentiviral particles infected HEK-293T cells, the HEK-293T / FUT2 cell line was obtained through puromycin pressure screening. PCR verification showed that the FUT2 gene was successfully integrated into the genome of HEK-293T cells. The FUT2 mRNA level in HEK-293T / FUT2 cells increased by 330-fold. HEK-293T / FUT2 cells highly expressed the H2 type human histo-blood group antigen. The GI.1 recombinant norovirus virus-like particles could produce a good binding reaction with HEK-293T / FUT2 cells, and the half-maximal effective dose EC 50 was 2.007 μg / ml.

[0009] Based on the above technical solution, in the first aspect of the present invention, there is provided a method for constructing a HEK-293T cell line with high expression of the H2 type human histo-blood group antigen, including the following steps: constructing an FUT2 lentiviral plasmid, co-transfecting the FUT2 lentiviral plasmid, pMD.2G and psPAX2 packaging plasmids into HEK-293T cells using Lipofectamine 2000, and harvesting lentiviral particles; infecting HEK-293T cells in the logarithmic growth phase with the lentiviral particles, and obtaining the HEK-293T / FUT2 cell line through puromycin pressure screening.

[0010] Further, when constructing the FUT2 lentiviral plasmid, the PCR amplification primers for the human FUT2 gene are used, wherein the upstream primer contains an EcoRI restriction site, and the downstream primer contains an XbaI restriction site, and the nucleotide sequences are respectively shown in SEQ ID NO.1 and SEQ ID NO.2:

[0011] 5’-CCG GAATTC ATGCTGGTCGTTCAGATGCC-3’(SEQ ID NO.1);

[0012] 5’-CTAG TCTAGA TTAGTGCTTGAGTAAGGGGG-3’(SEQ ID NO.2).

[0013] Further, the construction method specifically includes the following steps:

[0014] (A) Construction of the FUT2 lentiviral plasmid:

[0015] Using the human FUT2 gene cDNA cloning plasmid as a template, the human FUT2 gene was amplified by PCR with the primers shown in SEQ ID NO.1 and SEQ ID NO.2. After agarose gel electrophoresis of the PCR product, the gel was cut and recovered using the QIAquickR Gel Extraction Kit; the pLVX-IRES-Puro lentiviral vector and the FUT2 gene were digested with EcoRI and XbaI double enzymes respectively. After agarose gel electrophoresis of the digestion products, the gel was cut and the target fragments were recovered. The digested pLVX-IRES-Puro lentiviral vector and the FUT2 gene were ligated using a rapid ligation kit, and the ligation product was recovered using the QIAquickR PCR purification kit, which was the pLVX-IRES-Puro-FUT2 plasmid; the pLVX-IRES-Puro-FUT2 plasmid was transformed into Escherichia coli TOP10, and a single colony was picked and inoculated into LB-Amp + The culture medium was cultured at 37 °C for 6 hours, and the bacterial liquid was taken for PCR verification. The plasmid was extracted using the QIAprepR spin miniprep kit and sequenced for verification;

[0016] (B) Construction of FUT2 overexpression cell line:

[0017] (b1) Exploration of puromycin screening concentration

[0018] HEK-293T cells were seeded at 4×10 3 cells / well in a 96-well plate. After 24 hours, the supernatant was discarded, and the culture medium containing gradient concentrations of puromycin (32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 μg / mL) was added to the 96-well plate. The fresh medium containing puromycin was changed every 2 days and cultured for 14 days. The lowest puromycin concentration at which all cells died was taken as the optimal screening concentration (1 μg / mL).

[0019] (b2) Preparation and transfection of lentivirus

[0020] HEK-293T cells were cultured adherently at 37 °C in DMEM medium containing 10% FBS under 5% CO2; pLVX-IRES-Puro-FUT2, psPAX2 and pMD2.G plasmids were co-transfected into HEK-293T cells at a ratio of 3:2:1 using Lipofectamine 2000. Lentiviral particles were harvested and filtered 72 hours after transfection; the lentiviral particles were used to infect HEK-293T cells in the logarithmic growth phase; 48 hours after infection, puromycin selection was carried out at the optimal screening concentration, and the culture medium was changed every 2 days; the cells that did not die after 14 days of culture were the HEK-293T / FUT2 cell line, which was cryopreserved after two passages.

[0021] In a second aspect of the present invention, there is provided an HEK-293T cell line with high expression of human histo-blood group antigen of type H2, which is constructed by the construction method as described above.

[0022] In a third aspect of the present invention, there is provided an application of the HEK-293T cell line with high expression of human histo-blood group antigen of type H2 as described above in the in vitro activity evaluation of norovirus vaccine at the cellular level.

[0023] In a fourth aspect of the present invention, there is provided a method for evaluating the binding activity of recombinant norovirus virus-like particles based on the HEK-293T cell line with high expression of human histo-blood group antigen of type H2 as described above.

[0024] Furthermore, the recombinant norovirus is of types GI.1, GI.2, GI.3, GI.7, GII.5, GII.10, GII.12, GII.23, GII.24, and GII.25.

[0025] Furthermore, the method for evaluating the binding activity of GI.1 type recombinant norovirus virus-like particles includes the following steps:

[0026] Dilute the GI.1 type recombinant norovirus virus-like particles with PBS to 1000 μg / ml and serially dilute them 12 times by 2-fold. Take 20 μL of each concentration of the GI.1 type recombinant norovirus virus-like particles and react with 180 μL of HEK-293T / FUT2 cells at a density of 2×10 6 cells / mL at 4°C for 1 hour. Wash the cells and resuspend them in 180 μL of PBS, add 20 μL of mouse anti-recombinant norovirus virus-like particle antibody and react at 4°C for 1 hour. Wash the cells and resuspend them in 180 μL of PBS, add 20 μL of 0.1 mg / mL rabbit anti-mouse IgG-FITC and react at 4°C for 1 hour. Wash the cells and resuspend them in 100 μL of PBS, and detect them on a flow cytometer to obtain the geometric mean fluorescence of each measurement sample. Using the concentration of GI.1 type recombinant norovirus virus-like particles as the abscissa and the geometric mean fluorescence as the ordinate, fit a four-parameter curve, and calculate the half-maximal effective dose EC 50 .

[0027] In a fifth aspect of the present invention, there is provided an application of the HEK-293T cell line with high expression of human histo-blood group antigen of type H2 as described above in the screening of anti-norovirus antibodies and drugs.

[0028] The advantages of the present invention are as follows:

[0029] 1. The present invention has established for the first time a HEK-293T cell line with stable and high expression of H2-type human histo-blood group antigens, and the expression rate of H2-type human histo-blood group antigens in this cell line is 99.9%.

[0030] 2. The present invention has established a method for evaluating the binding activity of GI.1 recombinant norovirus virus-like particles based on the constructed HEK-293T / FUT2 cell line. The evaluation model based on HEK-293T / FUT2 cells can be used for the evaluation of the binding activity of noroviruses of various types, and can also be used for the screening of anti-norovirus antibodies and drugs. Brief Description of the Drawings

[0031] Figure 1 . PCR-amplified human FUT2 gene;

[0032] Figure 2 . Double digestion of the pLVX-IRES-Puro lentiviral vector with EcoRI and XbaI;

[0033] Figure 3 . Double digestion of the FUT2 gene with EcoRI and XbaI;

[0034] Figure 4 . pLVX-IRES-Puro-FUT2 plasmid;

[0035] Figure 5 . PCR identification of HEK-293T / FUT2 cells;

[0036] Figure 6 . Detection of FUT2 mRNA transcription level by RT-qPCR;

[0037] Figure 7 . Expression of HBGAs in HEK-293T / FUT2 cells;

[0038] Figure 8 . Binding activity between HEK-293T / FUT2 cells and GI.1 norovirus VLP. Detailed Embodiments

[0039] The following detailed description of the specific embodiments provided by the present invention is made in conjunction with the examples.

[0040] Example:

[0041] 1. Materials and Methods

[0042] 1.1 Cells, Bacteria, Plasmids and Antibodies

[0043] Human embryonic kidney 293T cells (HEK-293T cells) were purchased from the Cell Bank of Wuhan University; Escherichia coli TOP10 was purchased from Shanghai Yubo Biotechnology Co., Ltd.; the cDNA cloning plasmid of human FUT2 gene was purchased from Shanghai Shiji Biotechnology Co., Ltd.; the pLVX-IRES-Puro lentiviral vector, psPAX2 and pMD2.G lentiviral packaging plasmids were purchased from Novagen, Germany; monoclonal antibodies against human H1, H2, Lewis b (Le b ), Lewis y (Le y ) type human histo-blood group antigens were purchased from Abcam, UK; rabbit anti-mouse IgG-FITC was purchased from Sigma, USA; The preparation method of GI.1 recombinant norovirus virus-like particles was referred to the literature (Du Jialiang, Gu Qiong, Liu Yueyue, etc. Secretory expression of norovirus VP1 protein virus-like particles in Pichia pastoris [J]. Chinese Journal of Biologicals, 2020, 33(10): 1097-1103.); mouse anti-recombinant norovirus antibody was purchased from Shanghai Qiming Biotechnology Co., Ltd.

[0044] 1.2 Main reagents and instruments

[0045] DNA Marker, EcoRI, and XbaI were purchased from Takara; Trizol, PrimeScript RT Reverse Transcription Kit, and Taq enzyme were purchased from Invitrogen; Quick Ligation Kit and Vent DNA Polymerase were purchased from BioLabs; QIAquickR Gel Extraction Kit (250), QIAprepR spin miniprep kit (250), QIAquickRPCR purification kit (250), and QIAamp DNA Mini Kit (250) were purchased from QIAGEN; fetal bovine serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), puromycin, and 0.25% trypsin were purchased from Gibco, USA; Bovine Serum Albumin (BSA), LB medium, and ampicillin were purchased from Shanghai Sangon Biotech Co., Ltd.; paraformaldehyde was purchased from Sigma, USA; 96-well plates were purchased from CORNING, USA; flow cytometers were purchased from BD, USA; centrifuges were purchased from BECKMAN, Germany.

[0046] 1.3 Construction of FUT2 lentiviral plasmid

[0047] PCR amplification primers for the FUT2 gene of the designer, where the upstream primer contains an EcoRI restriction site and the downstream primer contains an XbaI restriction site:

[0048] 5’-CCG GAATTC ATGCTGGTCGTTCAGATGCC-3’(SEQ ID NO.1);

[0049] 5’-CTAG TCTAGA TTAGTGCTTGAGTAAGGGGG-3’(SEQ ID NO.2).

[0050] Using the cDNA (SEQ ID NO.13) cloning plasmid of the human FUT2 gene as a template, the human FUT2 gene was amplified by PCR with the above primers. After agarose gel electrophoresis of the PCR product, the gel was cut and recovered using the QIAquickR Gel Extraction Kit. The pLVX-IRES-Puro lentiviral vector and the FUT2 gene were digested with EcoRI and XbaI double enzymes respectively. After agarose gel electrophoresis of the enzyme digestion products, the gel was cut and the target fragments were recovered. The digested pLVX-IRES-Puro lentiviral vector and the FUT2 gene were ligated using a rapid ligation kit, and the ligation product was recovered using the QIAquickR PCR purification kit, which was the pLVX-IRES-Puro-FUT2 plasmid. The pLVX-IRES-Puro-FUT2 plasmid was transformed into Escherichia coli TOP10, and a single clone was picked and inoculated into LB-Amp + culture medium and cultured at 37°C for 6 hours. The bacterial solution was taken for PCR verification, and the plasmid was extracted using the QIAprepR spin miniprep kit for sequencing verification.

[0051] 1.4 Construction of FUT2 overexpression cell line

[0052] 1.4.1 Exploration of puromycin screening concentration

[0053] HEK-293T cells were seeded at 4×10 3 cells / well in a 96-well plate. After 24 hours, the supernatant was discarded, and the culture medium containing gradient concentrations of puromycin (32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 μg / mL) was added to the 96-well plate. The fresh medium containing puromycin was changed every 2 days, and the cells were cultured for 14 days. The lowest puromycin concentration at which all cells died was taken as the optimal screening concentration.

[0054] 1.4.2 Preparation and transfection of lentivirus

[0055] HEK-293T cells were cultured in DMEM containing 10% FBS under adherent culture conditions at 37 °C with 5% CO₂. pLVX-IRES-Puro-FUT2, psPAX2, and pMD2.G plasmids were co-transfected into HEK-293T cells at a ratio of 3:2:1 using Lipofectamine 2000. Lentiviral particles were harvested and filtered 72 hours after transfection. The lentiviral particles were used to infect HEK-293T cells in the logarithmic growth phase. Puromycin at the optimal screening concentration was used for pressure screening 48 hours after infection, and the culture medium was changed every 2 days. Cells that survived for 14 days were the HEK-293T / FUT2 cell line, which was cryopreserved after two passages.

[0056] 1.4.3 Verification of FUT2 overexpressing cell line

[0057] Three pairs of verification primers were designed. For the first pair, the upstream primer was designed at the 5'-end of the FUT2 fragment and the downstream primer was designed at the 3'-end of the FUT2 fragment:

[0058] 5’-ATGCTGGTCGTTCAGATGCC-3’ (SEQ ID NO.3);

[0059] 5’-TTAGTGCTTGAGTAAGGGGG-3’ (SEQ ID NO.4);

[0060] For the second pair, the upstream primer was designed in the CMV promoter sequence and the downstream primer was designed at the 3'-end of the FUT2 fragment:

[0061] 5’-CGCAAATGGGCGGTAGGCGTG-3’ (SEQ ID NO.5);

[0062] 5’-TTAGTGCTTGAGTAAGG GGG-3’ (SEQ ID NO.6);

[0063] For the third pair, the upstream primer was designed at the 5'-end of the FUT2 fragment and the downstream primer was designed in the IRES sequence:

[0064] 5’-ATGCTGGTCGTTCAGATGCC-3’ (SEQ ID NO.7);

[0065] 5’-CCTCACATTGCCAAAAGACG-3’ (SEQ ID NO.8).

[0066] The genomic DNA of the HEK-293T / FUT2 cell line was extracted using the QIAamp DNA Mini Kit. Using the genomic DNA as a template, 3 pairs of verified primers were used for PCR amplification of the target bands, and the size of the target bands was judged by agarose gel electrophoresis of the PCR products.

[0067] 1.5 Cloning of FUT2 overexpressing cell lines

[0068] The HEK-293T / FUT2 cell line was seeded at 0.5 cells per well in a 96-well plate, and 200 μL of DMEM medium containing 1 μg / mL puromycin and 10% FBS was added and cultured for 14 days. Whether cell colonies were formed was observed under a microscope. For the wells with cell colonies formed, 20 μL of 0.25% trypsin was added to digest the cells, and each monoclonal cell was transferred to a 24-well plate, and 2 mL of DMEM medium containing 1 μg / mL puromycin and 10% FBS was added to continue the culture. After the confluence rate of each monoclonal cell reached more than 80%, 100 μL of 0.25% trypsin was added to each well to digest the cells, and each monoclonal cell was transferred to a 6-well plate, and 4 mL of DMEM medium containing 1 μg / mL puromycin and 10% FBS was added to continue the culture. Using the same method, each monoclonal cell was successively expanded in cell flasks of 25 cm 2 、75 cm 2 and 225 cm 2 for subsequent detection.

[0069] 1.6 Detection of FUT2 mRNA transcription level by RT-qPCR

[0070] Total RNA of HEK-293T cells, HEK-293T / FUT2 cell line and each monoclonal HEK-293T / FUT2 cell was extracted using Trizol, and then 1 μg of each of the above RNAs was reverse transcribed into cDNA using the PrimeScript RT reverse transcription kit.

[0071] Primers were designed for FUT2 respectively:

[0072] 5’-ATTGGGACGTTCGGGATCTG-3’(SEQ ID NO.9)

[0073] 5’-GTCGGGGAGGGTGTAATTGG-3’(SEQ ID NO.10)

[0074] and human β-actin:

[0075] 5’-GGACTTCGAGCAAGAGATGG-3’(SEQ ID NO.11)

[0076] qPCR primers for 5’-AGCACTGTGTTGGCGTACAG-3’ (SEQ ID NO.12). Using cDNA as a template, qPCR amplification was performed with the above primers, and human β-actin was used as an internal reference, and the 2 -ΔΔCT method was used for data analysis.

[0077] 1.7 Flow cytometry analysis of the expression of HBGAs in HEK-293T / FUT2 cells

[0078] HEK-293T cells and HEK-293T / FUT2 cell lines in the logarithmic growth phase were digested with trypsin and centrifuged at 25 °C and 150 g for 10 min. The supernatant was discarded, and the cells were resuspended in 1 mL of cell washing solution (10 mmol / L PBS + 2% BSA), centrifuged at 25 °C and 150 g for 10 min, and repeated 2 times. The supernatant was discarded, and the cell density was adjusted to 1×10 6 cells / mL with 10 mmol / L PBS. A U-bottom 96-well plate was taken, and 200 μL of cell suspension was added to each well. 20 μL of mouse anti-human H1, H2, Le b 、Le y antibodies were added, and the reaction was carried out at 4 °C for 1 h. Centrifuged at 4 °C and 130 g for 10 min, the supernatant was discarded, and the cells were resuspended in 200 μL of cell washing solution, and repeated 2 times. The supernatant was discarded, 20 μL of rabbit anti-mouse IgG-FITC was added, and the reaction was carried out at 4 °C for 1 h. Centrifuged at 4 °C and 130 g for 10 min, the supernatant was discarded, and the cells were resuspended in 200 μL of cell washing solution, and repeated 2 times. The supernatant was discarded, and the cells were resuspended in 100 μL of cell fixing solution (10 mmol / L PBS + 1% paraformaldehyde), and detected by flow cytometry. First, analyze the fluorescence signal map of HEK-293T cells. On the FL1 histogram, a single-line gate was drawn with the right side of the peak as the limit, and the graph was divided into two regions. The left side is the negative region, and the right side is the positive region, and the ratio of negative and positive cells is automatically calculated. Then, measure the fluorescence signal map of HEK-293T / FUT2 cells, and the proportion of cells in the right region of the experimental group is the expression rate of the corresponding tissue blood group antigen.

[0079] 1.8 Analysis of the binding activity between HEK-293T / FUT2 and recombinant norovirus virus-like particles

[0080] HEK-293T / FUT2 cells were digested with trypsin, centrifuged at 150 g at room temperature for 5 minutes, the supernatant was discarded, and the cells were resuspended in 5 mL of cell washing solution (10 mM PBS + 2% BSA), centrifuged at 150 g at room temperature for 5 minutes again, and the supernatant was discarded. After repeating twice, the cell density was adjusted to 2×10 6PFU / mL. The recombinant norovirus GI.1 virus-like particles were diluted to 1000 μg / ml with PBS and serially diluted 12-fold. The diluted recombinant norovirus GI.1 virus-like particles were added to a U-bottom 96-well plate, with 3 parallel wells for each concentration, 20 μL added to each well. Then, 180 μL of the above HEK-293T / FUT2 cells were added to each well, and wild-type HEK-293T cells were used as a control. After reacting the recombinant norovirus VLP and HEK-293T / FUT2 cells at 4 °C for 1 hour, they were centrifuged at 150 g for 5 minutes at 4 °C. The supernatant was discarded, and each well was added with 300 μL of cell wash solution to resuspend the cells. Then, they were centrifuged at 150 g for 5 minutes at 4 °C again, and this was repeated twice. After that, each well was added with 180 μl of cell wash solution to resuspend the cells. 20 μl of mouse anti-recombinant norovirus virus-like particle antibody was added to each well. After reacting at 4 °C for 1 hour, they were centrifuged at 150 g for 5 minutes at 4 °C. The supernatant was discarded, and each well was added with 300 μL of cell wash solution to resuspend the cells. Then, they were centrifuged at 150 g for 5 minutes at 4 °C again, and this was repeated twice. After that, each well was added with 180 μl of cell wash solution to resuspend the cells. 20 μl of rabbit anti-mouse IgG-FITC (the antibody was diluted to 0.1 mg / mL with PBS in advance) was added to each well. After reacting at 4 °C for 1 hour, they were centrifuged at 150 g for 5 minutes at 4 °C. The supernatant was discarded, 300 μl of cell wash solution was added to resuspend the cells. Then, they were centrifuged at 150 g for 5 minutes at 4 °C again, and this was repeated 2 times. After that, each well was added with 100 μl of PBS to resuspend the cells. The geometric mean fluorescence of each measurement sample was obtained by detecting on a flow cytometer. The concentration of recombinant norovirus virus-like particles was used as the abscissa, and the geometric mean fluorescence was used as the ordinate to fit a four-parameter curve.

[0081] 1.9 Statistical analysis

[0082] The binding activity between HEK-293T / FUT2 and norovirus VLP was analyzed using Graphpad prism 8.0 software. The final concentration of norovirus VLP in the reaction was used as the abscissa, and the geometric mean of the fluorescence signal measured by the flow cytometer was used as the ordinate. The abscissa was logarithmically transformed, and the Sigmodial dose response (variable scope) was selected under the non-linear fitting function to fit an S-shaped curve to obtain the half-maximal effective concentration EC 50 .

[0083] 2. Results

[0084] 2.1 Construction of FUT2 lentiviral plasmid

[0085] Using the human FUT2 gene cDNA cloning plasmid as a template, the human FUT2 gene was amplified by primer PCR at around 1000 bp, as Figure 1As shown. The pLVX-IRES-Puro lentiviral vector and the FUT2 gene were digested with EcoRI and XbaI. After digestion, the fragment sizes were consistent with expectations, as Figure 2 and Figure 3 shown. The digested pLVX-IRES-Puro lentiviral vector and the FUT2 gene were ligated to obtain the pLVX-IRES-Puro-FUT2 plasmid. The sequencing results showed that the sequence was correct, as Figure 4 shown.

[0086] 2.2 Construction of FUT2 overexpressing cell line

[0087] HEK-293T cells were cultured in medium containing 32, 16, 8, 4, 2, 1 μg / mL puromycin for 14 days, and all the cells died. Then the optimal screening concentration of puromycin was 1 μg / mL. Lentiviral particles were obtained 72 hours after co-transfecting HEK-293T cells with pLVX-IRES-Puro-FUT2, psPAX2 and pMD2.G plasmids. The lentiviral particles were used to infect HEK-293T cells, and the HEK-293T / FUT2 cell line was obtained by puromycin pressure screening. Genomic DNA of the HEK-293T / FUT2 cell line was extracted, and the target bands were amplified by PCR using 3 pairs of verification primers. The sizes of the target bands were consistent with expectations, as Figure 5 shown. The results showed that the FUT2 gene was successfully integrated into the genome of HEK-293T cells.

[0088] 2.3 Detection of FUT2 mRNA transcription level by RT-qPCR

[0089] Compared with wild-type HEK-293T cells, the FUT2 mRNA level in HEK-293T / FUT2 cells increased by 300 - 800 times (the expression of wild-type HEK-293T cells was set as 1). The HEK-293T / FUT2 cell line increased by 330 times, and the 3# and 4# monoclonal cell lines increased by 785 and 655 times respectively, which were higher than the HEK-293T / FUT2 mixed cell line, as Figure 6 shown.

[0090] 2.4 Expression of HBGAs in HEK-293T / FUT2 cells

[0091] A single-line gate was drawn on the FL1 histogram. The expression rates of H1, H2, Le b and Le y type histo-blood group antigens in HEK-293T cells were all ≤ 0.5%. The expression rates of H1, H2, Le b and Le yThe expression rates of type O histo-blood group antigens were 0.1%, 99.9%, 0.6% and 0.0% respectively, as Figure 7 shown. The results showed that HEK-293T / FUT2 cells highly expressed H2 histo-blood group antigen.

[0092] 2.5 Binding activity of HEK-293T / FUT2 cells to norovirus VLP

[0093] Wild-type HEK-293T cells did not bind to different concentrations of recombinant norovirus GI.1 VLP, while HEK-293T / FUT2 cells could all produce good binding reactions with recombinant norovirus GI.1 VLP, forming a classic S-shaped curve under four-parameter equation fitting, as Figure 8 shown. The half-maximal effective dose (EC 50 50) of HEK-293T / FUT2 cells binding to recombinant norovirus GI.1 VLP was 2.007 μg / ml.

[0094] 3 Conclusion

[0095] The present invention for the first time established a HEK-293T cell line with stable and high expression of H2 histo-blood group antigen, and the expression rate of H2 histo-blood group antigen in this cell line is 99.9%. Based on the constructed HEK-293T / FUT2 cell line, the present invention established a method for evaluating the binding activity of GI.1 recombinant norovirus virus-like particles. The histo-blood group antigen bound to the cell membrane is restricted by the interaction of membrane lipids and membrane proteins. Compared with free histo-blood group antigens, it can better simulate the state of norovirus host cells. Therefore, this model can better exhibit the binding characteristics of noroviruses. Literature reports that GI.2, GI.3, GI.7, GII.5, GII.10, GII.12, GII.23, GII.24 and GII.25 bind to H2 histo-blood group antigens. It can be reasonably assumed that the evaluation model based on HEK-293T / FUT2 cells can be used for the evaluation of the binding activities of multiple types of noroviruses, and can also be used for the screening of anti-norovirus antibodies and drugs (Muslum Y, Abdulkadir K. Molecular Dynamics Studies of Histo-Blood Group Antigen Blocking Human Immunoglobulin A Antibody and Escape Mechanism in Noroviruses Upon Mutation. [J]. Journal of computational biology: a journal of computational molecular cell biology, 2019, 26(9). Tamminen K, Malm M, Vesikari T, et al. Norovirus-specific mucosal antibodies correlate to systemic antibodies and block norovirus virus-like particles binding to histo-blood group antigens [J]. Clinical Immunology, 2018, 197. Nasir, Waqas, Frank, et al. Histo-blood group antigen presentation is critical for norovirus VLP binding to glycosphingolipids in membranes [J]. Glycobiology, 2016, 26(12).).

[0096] The preferred embodiments of the present invention have been specifically described above. However, the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for constructing an HEK-293T cell line with high expression of H2-type human tissue blood group antigen, characterized in that, It includes the following steps: Construct a lentiviral plasmid of human FUT2 gene. Use Lipofectamine2000 to co-transfect HEK-293T cells with FUT2 lentiviral plasmid, pMD.2G and psPAX2 packaging plasmids, and harvest lentiviral particles; infect HEK-293T cells in the logarithmic growth phase with the lentiviral particles, and screen with puromycin pressure to obtain the HEK-293T / FUT2 cell line; the expression rate of H2 type human histo-blood group antigen in the said cell line is 99.9%.

2. The construction method of the H2-type human histo-blood group antigen highly expressed HEK-293T cell line according to claim 1, characterized in that, When constructing the FUT2 lentiviral plasmid, the nucleotide sequences of the PCR amplification primers of the human FUT2 gene used are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

3. The construction method of the H2-type human histo-blood group antigen highly expressed HEK-293T cell line according to claim 1, characterized in that, The said construction method includes the following steps: (A) Construction of FUT2 lentiviral plasmid: Using the human FUT2 gene cDNA cloning plasmid as a template, the human FUT2 gene was amplified by PCR with the primers shown in SEQ ID NO.1 and SEQ ID NO.

2. After agarose gel electrophoresis of the PCR product, the gel was cut and the DNA was recovered using the QIAquickR Gel Extraction Kit. The pLVX-IRES-Puro lentiviral vector and the FUT2 gene were digested with EcoRI and XbaI, respectively. After agarose gel electrophoresis of the digested products, the gel was cut and the target fragments were recovered. The digested pLVX-IRES-Puro lentiviral vector and the FUT2 gene were ligated using a rapid ligation kit, and the ligation product was recovered using the QIAquickR PCR purification kit to obtain the pLVX-IRES-Puro-FUT2 plasmid. The pLVX-IRES-Puro-FUT2 plasmid was transformed into Escherichia coli TOP10, and a single colony was picked and inoculated into LB-Amp + culture medium and cultured at 37 °C for 6 hours. The bacterial solution was taken for PCR verification, and the plasmid was extracted using the QIAprepR spin miniprepkit and sequenced for verification; (B) Construction of FUT2 high-expression cell line (b1) Exploration of puromycin screening concentration Inoculate HEK-293T cells at a density of 4×10 3 cells per well in a 96-well plate. After 24 hours, discard the supernatant. Add culture medium containing puromycin at gradient concentrations of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg / mL to the 96-well plate. Replace the fresh puromycin-containing medium every two days and culture for 14 days. The lowest puromycin concentration at which all cells die is taken as the optimal screening concentration; (b2) Preparation and transfection of lentiviral particles HEK-293T cells are cultured adherently at 37°C in DMEM medium containing 10% FBS under 5% CO2; use Lipofectamine 2000 to co-transfect HEK-293T cells with pLVX-IRES-Puro-FUT2, psPAX2 and pMD2.G plasmids at a ratio of 3:2:

1. Harvest and filter to obtain lentiviral particles 72 hours after transfection; infect HEK-293T cells in the logarithmic growth phase with the lentiviral particles; perform puromycin pressure screening with the optimal screening concentration 48 hours after infection, and change the culture medium every 2 days; the cells that have not died after culturing for 14 days are the HEK-293T / FUT2 cell line, which is cryopreserved after subculturing twice.

4. An HEK-293T cell line with high expression of H2 type human histo-blood group antigen, characterized in that, It is constructed by using the construction method described in any one of claims 1-3.

5. Use of the HEK-293T cell line with high expression of H2 type human histo-blood group antigen described in claim 4 in the in vitro activity evaluation at the cell level of norovirus vaccine.

6. A method for evaluating the in vitro binding activity of recombinant norovirus virus-like particles based on the HEK-293T cell line with high expression of H2 type human histo-blood group antigen described in claim 4.

7. The evaluation method according to claim 6, characterized in that The said recombinant norovirus is of types GI.1, GI.2, GI.3, GI.7, GII.5, GII.10, GII.12, GII.23, GII.24 and GII.

25.

8. The evaluation method according to claim 7, wherein The evaluation method for the binding activity of GI.1 recombinant norovirus virus-like particles includes the following steps: Dilute the GI.1 recombinant norovirus virus-like particles with PBS to 1000 μg / ml, and sequentially dilute them 12 times by a factor of 2; Take 20 μL of the GI.1 recombinant norovirus virus-like particles at each concentration and react with 180 μL of HEK-293T / FUT2 cells at a density of 2×10 6 cells / mL at 4°C for 1 hour. Wash the cells and resuspend them in 180 μL of PBS, add 20 μL of mouse anti-recombinant norovirus virus-like particle antibody and react at 4°C for 1 hour. Wash the cells and resuspend them in 180 μL of PBS, add 20 μL of 0.1 mg / mL rabbit anti-mouse IgG-FITC and react at 4°C for 1 hour. Wash the cells and resuspend them in 100 μL of PBS, and detect them on a flow cytometer to obtain the geometric mean fluorescence of each measurement sample; Use the concentration of GI.1 recombinant norovirus virus-like particles as the abscissa and the geometric mean fluorescence as the ordinate to fit a four-parameter curve, and calculate the half-maximal effective dose EC 50 .

9. Use of the HEK-293T cell line with high expression of H2 type human histo-blood group antigen described in claim 4 in the screening of anti-GI.1 type norovirus antibodies and drugs.

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