Monoclonal antibody against norovirus GI genotype VP1 protein and application thereof

Abstract

The application provides an anti-Norovirus GII genotype VP1 antigen monoclonal antibody and an application thereof. The anti-Norovirus GII genotype VP1 antigen monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a heavy chain CDR3 shown in SEQ ID NO. 3, and the light chain variable region comprises a light chain CDR3 shown in SEQ ID NO. 6. The monoclonal antibody is a rabbit-derived monoclonal antibody, the monoclonal antibody has good specificity and high affinity, and solves the problem of a mouse-derived monoclonal antibody in actual application. The anti-Norovirus GII genotype VP1 antigen monoclonal antibody has important application value in the preparation of a product for detecting Norovirus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a monoclonal antibody against Norovirus GII genotype VP1 protein and application thereof. BACKGROUND

[0002] Norovirus infection diarrhea is popular in the whole world, and can occur throughout the year. The infection objects are mainly adults and school-age children, and the infection is high in cold season. In the United States, 60%-90% of all non-bacterial diarrhea outbreaks are caused by Norovirus every year. Similar results are also obtained in developed countries such as the Netherlands, the United Kingdom, Japan and Australia. In China, the detection rate of Norovirus in children under 5 years old with diarrhea is about 15%, and the serum antibody level investigation shows that the infection of Norovirus in Chinese population is also very common.

[0003] Norovirus belongs to Norovirus genus of Caliciviridae family, has a diameter of about 26-35 nanometers, no envelope, rough surface, spherical, symmetrical icosahedron, and is a single-stranded positive strand RNA virus. Electron microscopy shows that the virus has a typical feather-like edge, and is a small round structure with a notch on the surface. The full length of the genome of Norovirus is about 7.7 kb, and the virus is divided into GⅠ-GⅦ groups according to the difference of the 1 nucleotide sequence of the capsid protein. Strictly speaking, only GⅠ, GⅡ and GⅣ can infect humans, and are commonly known as human Norovirus. Since Norovirus caused the first world-wide epidemic in 1995-1996, the 4th genotype (GⅡ.4) in the GⅡ group has dominated the epidemic in the world, and almost every two or three years, GⅡ.4 will cause a large-scale epidemic, and a new variant strain will appear at the same time. The genetic material genome of Norovirus is connected with the virus protein VPg at the 5' end, and exists polyadenylation at the 3' end, contains three open reading frames (ORF), and encodes eight virus proteins. ORF-2 and ORF-3 encode virus structural proteins, virus protein 1 (VP1) and VP2, respectively. The mature virus particle contains 90 VP1 dimers, forms an icosahedral symmetrical structure, and forms a hollow or cup-shaped structure on the virus surface. The VP1 protein has been proved to be the main immunogenic protein of Norovirus.

[0004] Monoclonal antibody is an antibody produced by a single B cell clone, which is highly uniform and only directed against a certain specific antigen epitope, and is called monoclonal antibody. It is usually prepared by using hybridoma technology. The hybridoma antibody technology is based on cell fusion technology, and the sensitized B cells with the ability to secrete specific antibodies are fused with myeloma cells with unlimited reproduction capacity to form B cell hybridoma. A single hybridoma cell with such characteristics is cultured into a cell group to prepare specific antibodies against an antigen epitope, i.e. monoclonal antibodies. At present, the most widely used monoclonal antibody is a mouse-derived monoclonal antibody, but the mouse-derived monoclonal antibody has the problems of weak affinity and poor specificity. In recent years, rabbit monoclonal antibody technology has developed rapidly, and compared with mouse monoclonal antibody, rabbit monoclonal antibody has the advantages of recognizing more sites, stronger affinity and specificity, etc.

[0005] An antibody with excellent performance is the basis of immunodetection technology, and only with a good antibody, an immunodetection kit with excellent performance can be developed. Therefore, it is of great significance to provide a monoclonal antibody against norovirus GII genotype VP1 protein with high affinity for detecting norovirus. SUMMARY

[0006] The application adopts the following technical solutions:

[0007] In a first aspect, the application provides a monoclonal antibody against norovirus GII genotype VP1, which comprises a heavy chain variable region and a light chain variable region.

[0008] The heavy chain variable region comprises a heavy chain CDR3 as shown in SEQ ID NO. 3.

[0009] The light chain variable region comprises a light chain CDR3 as shown in SEQ ID NO. 6.

[0010] Preferably, the heavy chain variable region further comprises a heavy chain CDR1 as shown in SEQ ID NO. 1 and a heavy chain CDR2 as shown in SEQ ID NO. 2.

[0011] Preferably, the light chain variable region further comprises a light chain CDR1 as shown in SEQ ID NO. 4 and a light chain CDR2 as shown in SEQ ID NO. 5.

[0012] SEQ ID NO. 1: EYGMS.

[0013] SEQ ID NO. 2: GINWNGGTTGYSESVKG.

[0014] SEQ ID NO. 3: SRYFDFRGYFDY.

[0015] SEQ ID NO. 4: RTSQSLGSNYFA.

[0016] SEQ ID NO. 5: EASRRAT.

[0017] SEQ ID NO. 6: QQYWHSPLT.

[0018] The monoclonal antibody in the application has multiple antigen recognition sites, good specificity and high affinity; the monoclonal antibody is a rabbit-derived monoclonal antibody, has multiple antigen recognition sites; the monoclonal antibody does not produce cross-reactions with other genotypes VP1 (including G I, G III, G IV, G V, G VI, G VII), has strong specificity; the screened monoclonal antibody has strong binding capacity to GXM antigen, has high affinity, and the titer of the GXM antigen reaches 1:1280000 (OD value>0.5).

[0019] Preferably, the amino acid sequence of the heavy chain variable region comprises the sequence shown in SEQ ID NO. 7.

[0020] Preferably, the amino acid sequence of the light chain variable region comprises the sequence shown in SEQ ID NO. 8.

[0021] SEQ ID NO. 7:

[0022] METGLRWLLLVAVLKGVQCEQLTESGGGLSMPRLSLKLSCKASGFTLSEYGMSWVRQAPGKGLEWNGGINWNGGTTGYSESVKGRFTVSRFNIESGPDLQMSSLTKTDTSLYYGARSRYFDFRGYFDYWGQGTLVTVSS

[0023] SEQ ID NO. 8:

[0024] MDTRAPTQLLGLLLLWLPGATFAVTTHTPSPVSAAVGDTVTINCRTSQSLGSNYFAWYQQNPGQPPKLLIYEASRRATGVPSRFRGSGSGTQFTLTISGMKAEDVATYYCQQYWHSPLTFGGGTKVEIK

[0025] Preferably, the anti-norovirus VP1 antigen monoclonal antibody further comprises any one or a combination of at least two of rabbit-derived IgG1, IgG2, IgG3 or IgG4 constant regions, and preferably a rabbit-derived IgG1 constant region.

[0026] The present application selects New Zealand big-eared rabbits as experimental animals, and prepares a monoclonal antibody, thereby overcoming the defect of weak affinity of a mouse-derived antibody; the present application uses a norovirus VP1 antigen as an antigen for immunization, and obtains rabbit-derived monoclonal antibodies by fusing spleen cells with myeloma cells, the antibodies have good stability and have strong affinity to the norovirus VP1 antigen.

[0027] In a second aspect, the present application provides a nucleic acid molecule encoding the anti-norovirus VP1 antigen monoclonal antibody of the first aspect.

[0028] Preferably, the nucleotide sequence encoding the heavy chain variable region of the anti-norovirus VP1 antigen monoclonal antibody comprises the sequence shown in SEQ ID NO. 9; and the nucleotide sequence encoding the light chain variable region of the anti-norovirus VP1 antigen monoclonal antibody comprises the sequence shown in SEQ ID NO. 10.

[0029] SEQ ID NO. 9:

[0030] ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTGAGCAGCTGACCGAGTCCGGGGGAGGCCTGAGCATGCCTCGCCTGTCCCTGAAACTCTCCTGCAAAGCCTCTGGATTCACCCTCAGTGAGTATGGCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGAATGGAGGTATTAATTGGAATGGTGGTACCACAGGTTATTCAGAGTCTGTGAAGGGCCGATTCACCGTCTCCAGATTCAACATCGAGAGCGGCCCGGATCTGCAAATGAGCAGTCTGACAAAGACGGACACGTCCCTCTATTACGGTGCAAGGTCCCGATATTTTGACTTCAGGGGTTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA.

[0031] SEQ ID NO. 10:

[0032] ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCGTGACGACCCATACTCCATCCCCCGTGTCTGCAGCTGTGGGAGACACTGTCACCATCAATTGCAGGACCAGTCAGAGTCTTGGGAGCAACTATTTTGCCTGGTACCAGCAGAATCCAGGGCAGCCTCCCAAGCTCCTGATCTATGAAGCGTCCAGGAGGGCCACTGGGGTGCCTTCGCGATTCCGTGGCAGTGGATCTGGGACACAGTTCACTCTTACCATCAGCGGCATGAAGGCTGAAGATGTTGCCACCTATTACTGTCAGCAGTATTGGCACTCACCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Detection results of affinity activity of monoclonal antibody in Example 6. Embodiment

[0034] The technical solutions of the present application will be further illustrated by specific embodiments. It should be understood by those skilled in the art that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0035] Unless otherwise specified in the examples, the techniques or conditions are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be commercially available through regular channels.

[0036] Example 1 Preparation of antigen

[0037] The preparation method of the antigen is as follows:

[0038] (1) Norovirus GII genotype VP1 protein sequence (SEQ ID NO: 11) is as follows:

[0039] [GeneID=39105315] MKMASSDANPSDGSAANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFVQAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTAGKIIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYNQSNDSTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVESRTKPFSVPVLTVEEMTNSRFPIPLEKLFTGPSSAFVVQPQNGRCTTDGVLLGTTQLSPVNICTFRGDVTHITGSRNYTMNLASQNWNNYDPTEEIPAPLGTPDFVGKIQGMLTQTTRTDGSTRGHKATVYTGSADFAPKLGRVQFETDTDHDFEANQNTKFTPVGVIQDGSTTHRNEPQQWVLPSYSGRNTHNVHLAPAVAPTFPGEQLLFFRSTMPGCSGYPNMDLDCLLPQEWVQYFYQEAAPAQSDVALLRFVNPDTGRVLFECKLHKSGYVTVAHTGQHDLVIPPNGYFRFDSWVNQFYTLAPMGNGTGRRRVV

[0040] (2) Construction of expression vector of protein

[0041] According to the sequence results, appropriate restriction sites were set, and the heavy chain and light chain were synthesized respectively; the gene synthesis products were connected to the pcDNA3.4 expression vector respectively.

[0042] (3) Expression and purification of protein

[0043] Expression of protein:

[0044] (a) Preparation before transfection: cell counting and subculture treatment to ensure that the cells have transfection conditions;

[0045] (b) Cell transfection: after shaking the cell liquid, 50 μL of cell suspension was aseptically taken with a pipette, diluted 20 times with 950 μL of medium, and the cell counting plate was used for cell counting, the viable cell density was about (4.5-5.5) × 106 cells / mL, and the cell viability should be 95-99%;

[0046] (c) Preparation of transfection reagent and plasmid mixture, incubate at room temperature for 20 min;

[0047] (d) Slowly add the transfection reagent into the cell culture solution to be transfected, and place the flask in a cell culture incubator at 37.0 ± 0.2 °C, 120 rpm and 8% CO2;

[0048] (e) On the second day after transfection (18 to 22 hours after transfection, day 1), add the feed to the flask, and gently shake the flask during the addition. Place the flask in a cell culture incubator at 37.0 ± 0.2 °C, 120 rpm and 8% CO2, and continue to culture for 3-5 days.

[0049] (3) Purification of protein

[0050] (a) Harvesting of cell culture solution: centrifugal collection of supernatant of cell culture solution, and antibody purification using Protein A Beads;

[0051] (b) Antibody concentration determination after completion of purification.

[0052] Example 2 Animal immunization

[0053] Mix the norovirus genotype GII VP1 protein with Freund's complete adjuvant in equal volume to a suitable volume. After thorough emulsification, subcutaneously inject New Zealand rabbits at multiple points, with the immunization dose of each rabbit controlled at 0.1-0.8 mg. Take ear blood from the New Zealand rabbits 3 days before immunization, and separate the serum to serve as a negative control. Immunize every 2 weeks after the first immunization, with the same method as the first time. A total of 6 immunizations are performed.

[0054] Select New Zealand white rabbits of suitable age and weight of about 1.5 kg, and raise them in a standard animal house for 3 days. If there are no abnormal conditions, start the immunization. Take 30 μg of the norovirus genotype GII VP1 protein prepared above, add to 0.5 mL of sterilized normal saline, and thoroughly mix using a micro vortex shaker. Then, use a syringe to push and pull the 0.5 mL of Freund's complete adjuvant, and thoroughly emulsify. Subcutaneously inject the New Zealand rabbits at multiple points on the back.

[0055] Two weeks later, take 30 μg of the norovirus genotype GII VP1 protein, add to 0.5 mL of sterilized normal saline, and thoroughly mix using a micro vortex shaker. Then, use a syringe to push and pull the 0.5 mL of Freund's incomplete adjuvant, and thoroughly emulsify. Perform a booster immunization. Thereafter, perform a booster immunization every other week, for a total of 6 immunizations. Starting from the third immunization, take 200-500 μL of ear marginal vein blood from the New Zealand rabbits one week after immunization, and determine the titer and affinity. Take the spleen after the last immunization.

[0056] The specific immunization steps are shown below:

[0057] (1) The first week: initial immunization, Norovirus GII genotype VP1 protein is diluted to 1 mg / mL with normal saline, and is fully emulsified with Freund's complete adjuvant 1:1. Two solutions are respectively filled into two syringes without generating bubbles. The two syringes are docked, and the antigen diluent and the adjuvant are gradually mixed from slow to fast. Finally, the mixed solution is made into a milky white water-in-oil emulsion, and then multi-point immunization is performed on the back subcutaneously, with a dose of 1 mL per rabbit.

[0058] (2) The third week: booster immunization, Norovirus GII genotype VP1 protein is diluted to 0.5 mg / mL with normal saline, and is fully emulsified with Freund's incomplete adjuvant 1:1. The immunization dose is 1 mL per rabbit.

[0059] (3) The fifth week: booster immunization, the immunization method is the same as the third week, and the immunization dose is 1 mL per rabbit.

[0060] (4) The sixth week: first serum titer detection; 200-500 μL of ear marginal venous blood of New Zealand rabbits after immunization is taken, and the serum is separated after centrifugation for 30 min, and the antiserum titer is determined.

[0061] (5) The seventh week: booster immunization, the immunization method is the same as step (3).

[0062] (6) The eighth week: second serum titer detection, step (4).

[0063] (7) The ninth week: booster immunization, the immunization method is the same as step (3).

[0064] (8) The tenth week: third serum titer detection, step (4).

[0065] (9) The eleventh week: booster immunization, the immunization method is the same as step (3).

[0066] The antiserum titer determination method is as follows:

[0067] The prepared antibody serum was gradiently diluted, and 100 μL was added to the enzyme-labeled plate, and incubated at 37°C for 1 h. Then, the plate was washed with PBST for three times, and each time was allowed to stand for 3 min. Then, HRP-labeled goat anti-rabbit IgG was used as an enzyme-labeled antibody, and was diluted at 1:5000, and 100 μL was added to each well, and incubated at 37°C for 1 h. Then, the plate was washed with PBST for three times, and each time was allowed to stand for 40 s. 100 μL of TMB was added to each well, and incubated at 37°C for 15 min. Then, 50 μL of a stop solution was added to each well to end the reaction, and the OD450 value was detected by using an enzyme-labeled instrument.

[0068] Example 3 Preparation of rabbit monoclonal antibody

[0069] The prepared rabbit antiserum was subjected to titer detection. When the titer was qualified, the spleen of a qualified New Zealand rabbit was used for cell fusion to prepare a monoclonal hybridoma cell strain. The specific preparation method is shown as follows:

[0070] (1) Preparation of immune spleen cells

[0071] The immunized New Zealand rabbit was sacrificed under sterile conditions, and the spleen was taken out. After being washed once with cell culture solution, the spleen was crushed, washed twice with cell culture solution, and centrifuged to obtain cells.

[0072] (2) Cell fusion

[0073] The logarithmic phase SP2 / 0 myeloma cells were mixed with the obtained spleen cells, washed once with cell culture solution without fetal bovine serum, centrifuged, and the supernatant was discarded. After the addition of polyethylene glycol solution and incubation at 37°C for about 90 s, the reaction was terminated by using cell culture solution without fetal bovine serum, and centrifuged. The cells were resuspended with HAT selection culture solution containing 20% fetal bovine serum, and added to a 96-well plate, which was placed in a cell culture box and incubated at 37°C and 5.0% CO2.

[0074] (3) Cell monoclonalization and screening

[0075] The 96-well plate with well-grown cells was diluted with cell culture solution to 1-3 cells / mL, and added to a 96-well plate, which was placed in a cell culture box and incubated at 37°C and 5.0% CO2. Each cell strain was numbered, and the cell strain with positive supernatant was selected for expansion culture. Finally, a hybridoma cell strain was obtained.

[0076] (4) Screening of hybridoma cells

[0077] The obtained hybridoma cells are screened using ELISA method to find at most 10 wells of polyclonal antibody cell strains against Norovirus GII genotype VP1 protein, and the cell strains can produce specific monoclonal antibodies with high affinity to respective antigens.

[0078] After the cell fusion step, two kinds of parent cells and three kinds of randomly fused cells exist in the culture medium, and in order to obtain hybridoma cell strains secreting the target antibody, the successfully fused hybridoma cells need to be separated from the numerous cells. The B lymphocytes cannot survive for a long time in vitro, and only the myeloma cells and their own fused cells need to be removed, so the fused cells need to be cultured in HAT medium to selectively retain the hybridoma cells.

[0079] On the 5th day after fusion, the growth of the cells can be observed, and on the 10th to 14th day, the cell culture supernatant can be detected by indirect ELISA method to screen positive hybridoma cell strains, and the positive hybridoma cell strains are cultured by cloning. The positive hybridoma cells with the highest titer are expanded to a cell positive rate of 100% to establish a strain. The titer of the culture supernatant of the hybridoma cell strain is measured by ELISA, and the expanded monoclonal hybridoma cell strain is frozen in liquid nitrogen.

[0080] The hybridoma cell supernatant is tested for specificity and affinity, and suitable hybridoma cells are selected for pairing; the selected four-well polyclonal hybridoma cell strains are recombined to express monoclonal antibodies; the purified antibodies obtained are paired by sandwich method to establish a sandwich detection system for Norovirus GII genotype VP1 protein, and finally the best paired monoclonal antibodies are obtained.

[0081] Example 4 Sequence determination of rabbit monoclonal antibodies

[0082] (1) Isolation of total RNA from hybridoma cells

[0083] After homogenization of the hybridoma cells, TRIzol is added, and the homogenate can be divided into a transparent upper aqueous phase layer (containing RNA), an interface, and a red lower organic layer (containing DNA and proteins). Then, isopropanol is used to precipitate RNA from the aqueous phase layer. Ethanol is used to precipitate DNA from the organic layer. Isopropanol is used to precipitate proteins from the phenol-ethanol supernatant. The precipitated RNA is washed to remove impurities, resuspended, and used.

[0084] (2) Reverse transcription of total RNA into cDNA

[0085] With dNTP as substrate, RNA as template, tRNA as primer, a single strand of cDNA complementary to the RNA template is synthesized on the 3'-end of tRNA in 5'→3' direction, which forms an RNA-cDNA hybrid with the RNA template. Then the RNA strand is hydrolyzed under the action of reverse transcriptase, and a second DNA strand is synthesized with the cDNA as template. Thus, total RNA is reverse transcribed into cDNA.

[0086] (3) Rapid amplification of cDNA ends (RACE)

[0087] The antibody fragments of heavy chain and light chain are amplified according to the standard operating procedure of rapid amplification of cDNA ends (RACE). The amplified antibody fragments are respectively cloned into standard cloning vectors. Colony PCR is performed to screen clones with correct size of inserts. The antibody sequences are obtained. The nucleotide sequence of the antibody is as follows:

[0088] The nucleotide sequence encoding the heavy chain variable region of the anti-Norovirus GI genotype VP1 protein monoclonal antibody comprises the sequence shown in SEQ ID NO. 9; the nucleotide sequence encoding the light chain variable region of the anti-Norovirus GI genotype VP1 protein monoclonal antibody comprises the sequence shown in SEQ ID NO. 10.

[0089] SEQ ID NO. 9:

[0090] ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTGAGCAGCTGACCGAGTCCGGGGGAGGCCTGAGCATGCCTCGCCTGTCCCTGAAACTCTCCTGCAAAGCCTCTGGATTCACCCTCAGTGAGTATGGCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGAATGGAGGTATTAATTGGAATGGTGGTACCACAGGTTATTCAGAGTCTGTGAAGGGCCGATTCACCGTCTCCAGATTCAACATCGAGAGCGGCCCGGATCTGCAAATGAGCAGTCTGACAAAGACGGACACGTCCCTCTATTACGGTGCAAGGTCCCGATATTTTGACTTCAGGGGTTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA.

[0091] SEQ ID NO. 10:

[0092] ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCGTGACGACCCATACTCCATCCCCCGTGTCTGCAGCTGTGGGAGACACTGTCACCATCAATTGCAGGACCAGTCAGAGTCTTGGGAGCAACTATTTTGCCTGGTACCAGCAGAATCCAGGGCAGCCTCCCAAGCTCCTGATCTATGAAGCGTCCAGGAGGGCCACTGGGGTGCCTTCGCGATTCCGTGGCAGTGGATCTGGGACACAGTTCACTCTTACCATCAGCGGCATGAAGGCTGAAGATGTTGCCACCTATTACTGTCAGCAGTATTGGCACTCACCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA.

[0093] Example 5 Construction of monoclonal antibody expression vector and purification expression

[0094] (1) Construction of monoclonal antibody expression vector

[0095] According to the determination results of rabbit monoclonal antibody sequence, appropriate restriction sites were set, and the heavy chain and light chain were synthesized respectively; the gene synthesis products were respectively connected to pcDNA3.4 expression vector.

[0096] (2) Expression and purification of monoclonal antibody

[0097] Expression of monoclonal antibody:

[0098] (a) Preparation before transfection: cell counting and subculture treatment to ensure that the cells have transfection conditions;

[0099] (b) Cell transfection: After shaking the cell liquid, 50 μL of cell suspension was aseptically sucked with a pipette, diluted 20 times with 950 μL of medium, and the cell counting plate was used for cell counting. The viable cell density was about (4.5-5.5) × 106 cells / mL, and the cell viability should be 95-99%;

[0100] (c) Preparation of transfection reagent and plasmid mixture, incubate at room temperature for 20 min;

[0101] (d) Slowly add the transfection reagent to the cell culture solution to be transfected, and shake the bottle in a cell culture shaker at 37.0±0.2℃, 120 rpm and 8% CO2;

[0102] (e) On day 2 post-transfection (18-22 hours post-transfection, day 1), feed was added to the shake flasks and the shake flasks were gently shaken during the addition. The shake flasks were incubated in a cell culture incubator shaker at 37.0 ± 0.2 °C, 120 rpm and 8% C02for 3-5 days.

[0103] (3) Purification of monoclonal antibodies

[0104] (a) Cell culture fluid harvest: centrifugal collection of cell culture supernatant, antibody purification using Protein A Beads;

[0105] (b) Antibody concentration determination after completion of purification.

[0106] Example 6: Detection of the affinity activity (titer) of the monoclonal antibody to Norovirus GI genotype VP1 protein using the ELISA method

[0107] The detection of the affinity activity is as follows:

[0108] Norovirus GI genotype VP1 protein was diluted with PBS to 1 μg / mL, 100 μL per well was added to a 96-well enzyme-labeled plate, and coated overnight at 4 °C; the supernatant was discarded, the plate was washed with 0.01 M PBST 3 times, 3% BSA blocking solution was prepared with PBST, 100 μL per well was added, and blocked at 37 °C for 1 h; the supernatant was discarded, washed with PBST 5 times, the purified and concentrated antibody was gradient diluted, the concentration was diluted from 1:1000 to 1:2560000, 100 μL per well was added, and incubated at 37 °C for 1 h; the antibody dilution was discarded, washed with PBST 6 times, the HRP-labeled goat anti-rabbit IgG (secondary antibody) was diluted with 1:5000 blocking solution, 100 μL of secondary antibody was added per well, and incubated at 37 °C for 1 h; the secondary antibody dilution was discarded, washed with PBST 6 times, 100 μL of TMB was added per well, and placed in the dark at 37 °C for 15 min; 50 μL of 1 M dilute sulfuric acid was added per well to stop the reaction, and the absorbance was measured at 450 nm.

[0109] The detection results of the affinity activity of the monoclonal antibody are shown in Table 1 and Figure 1 As the OD value remained unchanged, the greater the dilution factor of the monoclonal antibody, the stronger the binding ability of the antigen-antibody, and the selected monoclonal antibody had strong binding ability to Norovirus GI genotype VP1 protein, and the titer of GXM antigen reached 1:1280000 (OD value > 0.5).

[0110] Table 1

[0111] Antibody concentration (ng / mL) OD value 10000 4.332 1000 4.253 100 3.998 10 2.796 1 1.878 0.1 1.044 0.01 0.278

[0112] In conclusion, the application provides a norovirus GII genotype VP1 protein monoclonal antibody, which is a rabbit-derived monoclonal antibody, has multiple antigen recognition sites, good specificity and high affinity, solves the problems of mouse-derived monoclonal antibodies in practical application, and has important application value in the preparation of norovirus detection products.

[0113] The applicant states that the above is only a specific embodiment of the application, but the protection scope of the application is not limited thereto, and those skilled in the art should understand that any changes or replacements within the technical scope disclosed by the application, which can be easily thought of by any person skilled in the art, falls within the protection scope and disclosure scope of the application.

Claims

1. An anti-Norovirus GI genotype VP1 protein monoclonal antibody, characterized in that, The anti-Norovirus GI genotype VP1 protein monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises a heavy chain CDR3 as shown in SEQ ID NO. 3; The light chain variable region comprises a light chain CDR3 as shown in SEQ ID NO. 6; The heavy chain variable region further comprises a heavy chain CDR1 as shown in SEQ ID NO. 1 and a heavy chain CDR2 as shown in SEQ ID NO. 2; The light chain variable region further comprises a light chain CDR1 as shown in SEQ ID NO. 4 and a light chain CDR2 as shown in SEQ ID NO.

5.

2. The anti-Norovirus GI genotype VP1 protein monoclonal antibody according to claim 1, characterized by, The amino acid sequence of the heavy chain variable region is a sequence as shown in SEQ ID NO. 7; The amino acid sequence of the light chain variable region is a sequence as shown in SEQ ID NO.

8.

3. The anti-Norovirus GI genotype VP1 protein monoclonal antibody according to any one of claims 1-2, characterized in that, The anti-Norovirus GI genotype VP1 protein monoclonal antibody further comprises any one or a combination of at least two of rabbit-derived IgG1, IgG2, IgG3 or IgG4 constant regions.

4. The anti-Norovirus GI genotype VP1 protein monoclonal antibody of claim 3, characterized in that, The anti-Norovirus GI genotype VP1 protein monoclonal antibody further comprises a rabbit-derived IgG1 constant region.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-Norovirus GI genotype VP1 protein monoclonal antibody according to any one of claims 1-4.

6. The nucleic acid molecule of claim 5, wherein The nucleotide sequence encoding the heavy chain variable region of the anti-Norovirus GI genotype VP1 protein monoclonal antibody is a sequence as shown in SEQ ID NO. 9; and the nucleotide sequence encoding the light chain variable region of the anti-Norovirus GI genotype VP1 protein monoclonal antibody is a sequence as shown in SEQ ID NO.

10.

7. An expression vector comprising the nucleic acid of claim 1. The expression vector comprises the nucleic acid molecule according to any one of claims 5-6.

8. Use of any one or a combination of at least two of the anti-Norovirus GI genotype VP1 monoclonal antibody according to any one of claims 1-4, the nucleic acid molecule according to any one of claims 5-6, and the expression vector according to claim 7 in the preparation of a Norovirus infection disease detection product.

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