CN115724958B

CN115724958B - Monoclonal antibody of anti-norovirus GII genomic capsid protein VP1 and application thereof

Info

Publication number: CN115724958B
Application number: CN202211355634.XA
Authority: CN
Inventors: 周逸; 张贺秋; 冯晓燕
Original assignee: Beijing Bioneovan Co ltd
Current assignee: Beijing Bioneovan Co ltd
Priority date: 2022-11-01
Filing date: 2022-11-01
Publication date: 2023-05-30
Anticipated expiration: 2042-11-01
Also published as: CN115724958A

Abstract

The invention discloses a monoclonal antibody of anti-norovirus GII genomic capsid protein VP1 and application thereof. In the monoclonal antibody, the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.2, the amino acid sequence of the light chain CDR2 is LVS, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 3; the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 are shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 respectively. The monoclonal antibody of the anti-norovirus GII capsid protein VP1 can cover the main genotypes of epidemic GII genome norovirus, has good specificity and affinity, and can be applied to the preparation of norovirus detection products and the preparation of products for treating norovirus patients.

Description

Monoclonal antibody of anti-norovirus GII genomic capsid protein VP1 and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a monoclonal antibody for resisting a norovirus GII genomic capsid protein VP1 and application thereof.

Background

Norovirus (NoV) is one of the major pathogens responsible for human non-bacterial acute gastroenteritis, and is widespread, placing a serious burden on global social medicine and public health. Norovirus is a non-enveloped single-stranded positive-strand RNA virus, which is of a large variety of Genotypes due to large nucleic acid sequence variations, and is divided into at least 10 genomes (genoeups) and nearly 48 Genotypes (Genotypes) based on the similarity of amino acids of the major capsid protein VP1 of the norovirus. The main infections in humans are GI, GII and GIV, with GII genomic norovirus being the most common strain worldwide, accounting for over 90%. In the genome of the norovirus GII, the GII.4 genotype is the dominant epidemic, and the GII.4 genotype is dominant since the mid 90 s of the last century. However, since 2015, the GII.17 genotype has also assumed a diffuse situation worldwide. Domestic related researches also find that the GII.17 genotype becomes a dominant epidemic strain which coexists with the GII.4 genotype.

At present, three methods for diagnosing norovirus infection clinically or in a laboratory are mainly an electron microscope detection method, a molecular biological detection method and an immunological detection method. Electron microscopy can be classified into direct electron microscopy and immunoelectron microscopy. The direct electron microscopy method can detect virus particles with diameters of about 30nm, has low sensitivity, and requires at least about 10 per milliliter of fecal sample ⁶ The virus particles can be detected, and the detection rate is about 11.0% -19.0%. The immune electron microscopy method utilizes the labeled antibody to capture virus particles through surface antigen, and then performs electron microscopy observation, and the detection rate is far higher than that of the direct electron microscopy method. However, the electron microscope method is complicated in operation, requires professional operation, is very expensive in equipment, cannot be widely popularized, and is not suitable for clinical detection application. The molecular biological detection method is currently identified as a gold standard for norovirus detection, has good specificity and sensitivity, and has relatively accurate detection results, but the technical level requirements are relatively strict, limited by the technical level and the restriction of an instrument field, and are difficult to popularize and use in basic medical institutions and field detection. The immunological detection method utilizes specific monoclonal antibody to detect the norovirus antigen, including enzyme-linked immunosorbent assay and immunochromatography, and has the advantages of simple and rapid operation and low cost.

However, the currently available methods for detecting norovirus antigens are still limited, mainly due to the lack of highly-covered and highly-active monoclonal antibodies that can be used to capture and detect different genotypes of norovirus, and the inability to cover most newly-generated variant or recombinant genotypes, especially the newly-dominant circulating strain GII.17 genotype. Clinically, the sensitivity of the commercial kit for norovirus detection is about 50-80%, the specificity is 80-90%, at least 20% of samples are missed, and at least 10% of samples are misdiagnosed.

The norovirus capsid protein VP1 is the primary target for norovirus antigen detection. There are two major domains in capsid protein VP1, the S domain and the P domain. The amino acid sequence of the S domain is 1 to 225 in the whole VP1, and the sequence is relatively conserved corresponding to the amino terminal of the capsid protein; the P domain is the main part of the virus contacting with the outside, and corresponds to the carboxyl terminal part of the capsid protein, the amino acid sequence of the P domain has larger change and is easy to recombine and mutate. The P domain is in turn divided into two sub-domains, the P1 region and the P2 region. P1 comprises amino acids 226 to 278 and 406 to 520 and P2 comprises amino acids 279 to 405.VP1 is an important antigen that elicits an immune response in the body, but its amino acid sequence has high variability. The homology of VP1 between different groups of norovirus is only 30-40%, and the homology of VP1 between different genotypes of the same group is 60-70%, and the difference brings great difficulty to the preparation of monoclonal antibodies which can specifically identify different groups of norovirus and can also highly cover different genotypes in the same group.

The invention screens dominant antigen epitope which has difference in GI genome and has high conservation in different types of epidemic strains of the norovirus GII genome aiming at the norovirus protein capsid protein VP1, prepares a high-coverage high-activity monoclonal antibody aiming at the antigen epitope, detects the main genotypes of the epidemic GII genome norovirus, including but not limited to the dominant epidemic strain GII.4 genotype and GII.17 genotype, and improves the sensitivity and the specificity of the existing detection reagent.

Disclosure of Invention

Therefore, the invention aims to provide an anti-norovirus GII genomic capsid protein VP1 monoclonal antibody prepared by utilizing a fused hybridoma cell strain, and the monoclonal antibody obtained through experiments can identify the main genotype of the norovirus GII genome, has no cross reaction with the norovirus GI genome, has good specificity and affinity, and can be used for detecting the norovirus GII genome or capsid protein VP1 thereof or preparing products for treating norovirus GII genome infection.

Accordingly, one aspect of the present invention relates to a monoclonal antibody against the genomic capsid protein VP1 of norovirus GII, or an antigen binding fragment thereof, comprising a light chain variable region comprising CDR1, CDR2 and CDR3 and a heavy chain variable region comprising CDR1, CDR2 and CDR3, wherein,

the amino acid sequence of the light chain CDR1 is a sequence shown as SEQ ID NO.2 or an amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown as SEQ ID NO.2, or an amino acid sequence containing the sequence;

the amino acid sequence of the light chain CDR2 is LVS or an amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence LVS or an amino acid sequence containing the sequence;

the amino acid sequence of the light chain CDR3 is a sequence shown as SEQ ID NO.3 or an amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown as SEQ ID NO.3, or an amino acid sequence containing the sequence;

the amino acid sequence of the heavy chain CDR1 is a sequence shown as SEQ ID NO.5 or an amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown as SEQ ID NO.5, or an amino acid sequence containing the sequence;

the amino acid sequence of the heavy chain CDR2 is a sequence shown as SEQ ID NO.6 or an amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown as SEQ ID NO.6, or an amino acid sequence containing the sequence;

the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO.7 or has 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.7, or comprises the amino acid sequence of the sequence.

In a further aspect, the invention also relates to a monoclonal antibody or antigen binding fragment thereof, the light chain variable region sequence is shown as SEQ ID NO.1, and the heavy chain amino acid sequence is shown as SEQ ID NO. 4.

The invention also relates to the monoclonal antibody or the antigen binding fragment thereof, wherein the antibody or the antigen binding fragment is Fab fragment, fab 'fragment, F (ab') ₂ Fragments, single chain antibodies or humanized antibodies which are capable of recognizing and binding to antigen due to the retention of the variable regions of the light and heavy chainsBinds to the norovirus gii genomic capsid protein VP1.

Furthermore, the present invention relates to a nucleic acid molecule comprising a nucleic acid encoding the above-described antibody or antigen binding fragment thereof, and an expression vector comprising the above-described nucleic acid molecule, said expression vector being capable of expressing the above-described antibody or antigen binding fragment thereof. The invention also relates to a recombinant comprising the above nucleic acid molecule or the above expression vector, which can produce the above antibody or antigen-binding fragment thereof.

In another aspect, the invention relates to a monoclonal antibody hybridoma cell line against the norovirus clii genomic capsid protein VP1, which secretes the monoclonal antibody described above. Further, the invention relates to a monoclonal antibody hybridoma cell strain of the anti-norovirus GII genomic capsid protein VP1, which is a mouse hybridoma cell strain 3A21 with a preservation number of CGMCC No.45322.

In a further aspect, the invention relates to the use of a monoclonal antibody as described above, or an antigen binding fragment thereof, for the preparation of a product for detecting norovirus or for treating norovirus infection. Further, the present invention relates to a kit for detecting norovirus, comprising the monoclonal antibody or antigen-binding fragment thereof described above, for recognizing and binding to the norovirus gii genomic capsid protein VP1.

Description of biological Material preservation

The monoclonal antibody hybridoma cell strain: the mouse hybridoma cell strain 3A21 is preserved in China general microbiological culture collection center (CGMCC), the registration number of the preservation center is CGMCC No.45322, and the preservation date is: 2022, 10 months and 10 days. The China general microbiological culture Collection center (China Committee) has the following addresses: beijing, chaoyang area, north Chenxi Lu No.1, 3, postal code 100101.

Drawings

FIG. 1 is a graph showing the results of the identification of the subclass of anti-norovirus GII genomic capsid protein VP1 monoclonal antibody, identified as IgG2a.

FIG. 2 is a schematic diagram showing the judgment of the result of detecting capsid protein VP1 by anti-norovirus GII genomic capsid protein VP1 monoclonal antibody colloidal gold immunochromatography, wherein positive is that two mauve bands appear. One is located in the detection area (T line) and the other is located in the quality control area (C line). The color of the strip of the detection area (T line) can be dark or light, and the detection area (T line) is a positive result. And negative is that only a mauve band appears in a quality control area (C line), and no band appears in a detection area (T). The invalidation is that no mauve stripe appears in the quality control area (C line), and whether the detection area (T line) appears or not, the invalidation is the result.

Detailed Description

The invention aims to provide an anti-norovirus GII genomic capsid protein VP1 monoclonal antibody prepared by utilizing a fused hybridoma cell strain, wherein the anti-norovirus GII genomic capsid protein VP1 monoclonal antibody can identify the capsid proteins VP1 of the norovirus GII genome dominant epidemic strains GII.4 and GII.17 and other multiple norovirus GII genome epidemic strains. A monoclonal antibody is prepared by taking a norovirus GII genomic capsid protein VP1 conserved dominant epitope synthetic peptide as an immunogen to immunize a mouse, and monoclonal antibody hybridoma cell strains are screened by adopting the capsid proteins VP1 of norovirus GII genomic epidemic strains GII.4 and GII.17 and other various norovirus GII genomic epidemic strains. After screening, a mouse hybridoma cell strain which expresses a monoclonal antibody with high coverage and high activity is obtained and named as 3A21. The cell strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45322 and the preservation date of 2022 and 10 months. The preservation address is Beijing, chaoyang area North Chen Xiyu No.1, 3 and the postal code 100101.

The inventor carries out sequencing on a monoclonal antibody generated by the mouse hybridoma cell strain, the amino acid sequence of a light chain variable region of the monoclonal antibody is 109 amino acids, and the sequence is as follows: DIVMTQSPASLAVSLGQRATISYRASKSVSTSG YSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEEAPSWKS (SEQ ID NO. 1) in which the underlined sequences are CDR1, CDR2 and CDR3 in this order, wherein CDR1 is located at 27-36aa and the amino acid sequence isKSVSTSGYSY(SEQ IDNo. 2); CDR2 is located at 54-56aa and has the amino acid sequence ofLVSThe method comprises the steps of carrying out a first treatment on the surface of the CDR3 is located at 93-100aa and the amino acid sequence isQHIRELTR(SEQ ID NO. 3). The heavy chain variable region has an amino acid sequence of 103 amino acids and the sequence is as follows: VQPRGSLKLSCAVSGFTLNNNAMNWVRQAPGRGLKWVARLSSKFNNYATYYADSVKGRFTISRDDSQPILYLQMNNLKTEDTAMYYCVRDDYWGQGTPVTVSS (SEQ ID NO. 4) in which the underlined sequences are CDR1, CDR2 and CDR3 in this order, wherein CDR1 is located at 15-22aa and the amino acid sequence isGFTLNNNA(SEQ ID NO. 5); CDR2 is located at 40-49aa and has the amino acid sequence ofLSSKFNNYAT(SEQ ID NO.7SEQ ID NO. 6); CDR3 is located at 88-92aa and has the amino acid sequence ofVRDDY(SEQ ID NO. 7). The monoclonal antibody has high coverage, high specificity and high affinity to the capsid protein VP1 of the genome of the norovirus GII.

It is well known that antibody heavy and light chain CDR regions are important sequences for the recognition and binding of the corresponding antigen, and that 1 or 2 conservative amino acid substitutions in the amino acid sequence generally do not alter the properties of the protein. Thus, monoclonal antibodies or antigen binding fragments thereof obtained after 1 or 2 conservative amino acid substitutions of light chain CDR1 and/or light chain CDR2 and/or light chain CDR3 and/or heavy chain CDR1 and/or heavy chain CDR2 and/or heavy chain CDR3 are still capable of recognizing and binding to the norovirus group viii capsid protein VP1. Conservative amino acid substitutions refer to the replacement of an amino acid in a protein with another chemically similar amino acid, such as the replacement of aromatic amino acid Phe, trp, tyr with each other, the replacement of aliphatic amino acid Ala, gly, leu, ile, val with each other, the replacement of polar amino acids Gln, asn with each other, the replacement of basic amino acid Lys, arg, his with each other, the replacement of acidic amino acids Asp, glu with each other, and the replacement of hydroxy amino acids Ser, thr with each other, etc.

Furthermore, it is well known in the art that in antibodies or antigen binding fragments thereof, the framework regions are outside each CDR of the light or heavy chain, and that the addition of a few amino acids, e.g. 1 or 2 amino acids, between the CDR sequences and the framework region sequences has less effect on the steric structure of the antibody or antigen binding fragment thereof, and thus still allows recognition and binding of the corresponding antigen. Thus, the inventionThe monoclonal antibody or antigen binding fragment thereof light chain CDR1 sequence may be a sequence comprising the sequence shown in SEQ ID No.2 or a sequence having 1 or 2 conservative amino acid substitutions compared thereto. Similarly, the light chain CDR2 sequences of the monoclonal antibodies or antigen binding fragments thereof of the invention are other than the sequencesLVSOr a sequence having 1 or 2 conservative amino acid substitutions as compared with the above sequence, may be a sequence comprising the above sequence; the light chain CDR3 sequence of the monoclonal antibody or antigen binding fragment thereof of the present invention may be a sequence comprising the sequence shown in SEQ ID NO.3 or a sequence having 1 or 2 conservative amino acid substitutions as compared with the sequence; the heavy chain CDR1 sequence of the monoclonal antibody or antigen-binding fragment thereof of the present invention may be a sequence comprising the above-mentioned sequence in addition to the sequence shown in SEQ ID NO.5 or a sequence having 1 or 2 conservative amino acid substitutions as compared with it, and the heavy chain CDR2 sequence of the monoclonal antibody or antigen-binding fragment thereof of the present invention may be a sequence comprising the above-mentioned sequence in addition to the sequence shown in SEQ ID NO.7SEQ ID NO.6 or a sequence having 1 or 2 conservative amino acid substitutions as compared with it; the heavy chain CDR3 sequence of the monoclonal antibody or antigen binding fragment thereof of the present invention may be a sequence comprising the sequence shown in SEQ ID NO.7 or a sequence having 1 or 2 conservative amino acid substitutions as compared with the sequence.

Various antibody fragments, i.e., antigen-binding fragments, capable of binding to the norovirus GII genomic capsid protein VP1, such as, but not limited to, fab ', F (ab') ₂ . Fab fragments are regions of an antibody structure that bind antigen and consist of a complete variable region VH and constant region CH1 domain (Fd segment) of the light and heavy chains, with a constant region and a variable region both present, and disulfide linkages between the light and heavy chains. Antigen binding fragments can be prepared, for example, by enzymatic cleavage using papain, whereby the antibody IgG is degraded into two Fab fragments and one Fc fragment (crystalline fragment). Under the action of pepsin, the antibody IgG is degraded into a F (ab') ₂ Fragments and a pFC 'fragment, F (ab') ₂ The fragment was further reduced to form two Fab' fragments. The antigen binding fragments can be used for preparing products for detecting the genome or capsid protein of the norovirus GII and therapeutic products for treating patients with the norovirus.

Single chain antibodies (scFv) may also be prepared from the monoclonal antibodies of the invention by techniques known in the art. The single chain antibody is formed by connecting a heavy chain variable region and a light chain variable region of the antibody through a short peptide linker of a plurality of amino acids, and only one chain is an artificially synthesized antibody. The length and amino acid composition of the short peptide linker are well known in the art, and the short peptide linker that can be used against the monoclonal antibody of the invention can be determined by simple repeated experiments. The single chain antibody may be expressed in, for example, E.coli by genetic engineering techniques. The single-chain antibody has the advantages of small molecular weight, strong penetrating power, weak antigenicity and the like, and can be applied to Yu Nuo such as detection of a virus GII genome or capsid protein thereof and diagnosis and treatment products of norovirus patients.

The light chain constant region and the heavy chain constant region of the monoclonal antibody of the invention can be substituted for the amino acid sequence of the adult antibody by the prior art in the field, so that the humanized antibody is obtained by humanized modification of the murine monoclonal antibody of the invention, and the humanized antibody can be used for antibody treatment of human norovirus patients so as to reduce the immune side reaction of the murine antibody to human bodies. Therefore, the humanized monoclonal antibodies of the invention can be used in Yu Nuo such as detection of viral GII genome or capsid proteins thereof, diagnosis and therapeutic products for patients with norovirus.

The skilled person can design and synthesize a nucleic acid molecule encoding the monoclonal antibody based on the amino acid sequence of the variable region of the anti-norovirus GII genomic capsid protein VP1, or insert the synthesized nucleic acid molecule into a nucleic acid vector to construct an expression vector capable of expressing the monoclonal antibody or antigen binding fragment thereof against the norovirus GII genomic capsid protein VP1. The person skilled in the art is also able to introduce the synthesized nucleic acid molecules or constructed expression vectors into organisms such as cells, bacteria, yeasts etc. to obtain recombinants and to express the antibodies or antigen binding fragments thereof of the invention via the recombinant expression as described above, the antibodies or antigen binding fragments thereof thus expressed being able to bind and recognize the norovirus GII genomic capsid protein VP1, and the nucleic acid molecules, expression vectors and recombinants as described above are therefore within the scope of the claims of the invention. And all of the above techniques are well known in the art and can be carried out by those skilled in the art without the need for creative effort.

As described above, the antibodies or antigen binding fragments thereof of the present invention are capable of recognizing and binding to the norovirus GII genomic capsid protein VP1 and thus can be used to prepare a kit for detecting norovirus or capsid protein thereof, which can be any kit utilizing the binding reaction of the antibodies or antigen binding fragments thereof of the present invention with the norovirus GII genomic capsid protein VP1, such as, but not limited to, a kit of the colloidal gold immunochromatography, fluorescence immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, immunohistochemical type.

Since the monoclonal antibody or antigen-binding fragment thereof of the present invention is capable of specifically binding to the norovirus GII genomic capsid protein VP1, the antibody or antigen-binding fragment thereof or humanized antibody can be used for preparing a product for treating norovirus infection, such as a drug. The treatment may be, for example, but is not limited to, targeted therapy in which a monoclonal antibody or antigen-binding fragment thereof or a humanized antibody of the invention is conjugated to a norovirus therapeutic drug, and the conjugated drug is targeted to the norovirus by an antibody or antigen-binding fragment thereof or a humanized antibody of the invention, thereby enhancing the therapeutic effect.

In order to describe the technical contents of the technical solution in detail, the achieved objects and effects, the following description will be made with reference to specific embodiments.

Example 1: screening and synthesis of dominant epitope peptide of capsid protein VP1 of norovirus G II genome

The full-length amino acid sequence of capsid protein VP1 of the following popular strains of published norovirus was downloaded from the Genebank database (https:// www.ncbi.nlm.nih.gov /): the dominant epidemic strains of the GII genome are the GII.17 genotype (with the gene accession number of ANW12068.1 and the full-length amino acid sequence of SEQ ID NO. 8) and the GII.4 genotype (with the gene accession number of AIV 43156.2), the other 7 epidemic strains of the GII genome are the GII.1 genotype (with the gene accession number of QTK 16413.1), the GII.2 genotype (with the gene accession number of AOW 69244.1), the GII.3 genotype (with the gene accession number of AZJ 17746.1), the GII.6 genotype (with the gene accession number of APX 56331.1), the GII.11 genotype (with the gene accession number of ACC 69026.1), the GII.12 genotype (with the gene accession number of AGT 39203.1), the GII.21 genotype (with the gene accession number of AOW 69241.1), and the GI genome of the norovirus (with the gene accession number of AOO 95025.1). Multiple sequence alignment was performed using BLAST to determine the highly conserved amino acid segments between different strains of the norovirus gii genome, then epitope analysis prediction was performed using Goldkey bioinformatics software, dominant epitope segments were screened, and finally screening was performed to determine the amino acid sequence VESKTKPFS (SEQ ID No. 9) with high score, conserved in the norovirus gii genome-prevalent strain and not present in the norovirus GI genome-prevalent strain, which was located at positions 220 to 228 of the sequence shown in SEQ ID No.8, predicted as B cell linear epitopes, with a score of 2.97011, as dominant epitopes. The synthesis of the dominant epitope peptide of the capsid protein VP1 of the Gai genome of the norovirus and the KLH coupling are entrusted to Shanghai De Fin Biotechnology Co.

Example 2: preparation of monoclonal antibody of dominant epitope peptide of capsid protein VP1 of norovirus GII genome

Taking KLH coupled dominant epitope polypeptide as an immunogen, and adopting 8-week-old BALB/c female mice, adding equivalent Freund complete adjuvant to the antigen, and injecting the mice into the back and abdominal cavity (50 mug/mouse); the same dose of immunization was performed at weeks 2 and 3 of week four and eight, with incomplete Freund's adjuvant, and spleen cells were taken 3 days later for fusion. The SP20 myeloma cells were resuscitated and cultured until they were in the log phase of growth. Taking immunized BALB/c mice, removing eyeball blood as positive control serum, killing the mice at cervical dislocation, disinfecting the body surface with 75% alcohol for 3-5min, taking spleen, and preparing spleen cell suspension.

Taking spleen cells and myeloma cells according to a ratio of 5:1 in a serum-free DMEM medium, and centrifuging at 1500rpm for 5minFully sucking the supernatant, gently shaking the bottom of a centrifuge tube, shaking the cells, adding 1mL of preheated 50% PEG fused cells in 45-60 seconds, gently shaking the cells while adding the cells, standing for 90 seconds after the completion of the addition, adding serum-free DMEM medium to terminate the fusion (1 mL in the first minute, zhong Jia mL in the second minute, 8mL in the third minute), standing for 10min at 37 ℃, centrifuging at 1500rpm for 5 minutes, suspending the precipitate with HAT medium, subpackaging the precipitate into 96-well cell plates containing feeder cells, and standing at 37 ℃ and 5% CO ₂ Is cultured in a cell culture incubator. After 5 days of culture in a cell culture box, the HAT culture medium is used for changing the liquid once, the HAT culture medium is used for changing the liquid on the 10 th day, and when the fusion cells cover 10% -50% of the bottoms of the holes, the indirect ELISA method is also used for screening positive clones.

The capsid protein of the genomic epidemic strain GII.17 of norovirus from Huashi Biotechnology Co., ltd was diluted with a carbonate coating buffer at a concentration of 0.2. Mu.g/ml, 100. Mu.l per well, overnight at 4 ℃; wash plate 2 times with wash solution, 200 μl/well; adding 110 μl/well of blocking solution, and blocking at room temperature for 6 hr; the plate was washed 5 times with wash solution, 200. Mu.l/well. After 200. Mu.l of sample dilution was added to each well, 10. Mu.l of cell culture supernatant was added, incubated at room temperature for 30min, and the solution was discarded. The plate was washed with 1 Xwash solution, 200. Mu.L per well, and the wash was repeated 5 times. The washed ELISA plate is inverted on the absorbent paper and is beaten with force to remove the redundant washing liquid. 100 μl/well HRP-labeled anti-mouse IgG antibody was added and incubated for 20min at room temperature. The plate was washed 5 times. Freshly prepared substrate solution, 100. Mu.l/well, was added and incubated at room temperature for 10 minutes in the dark. Add 2M H ₂ SO ₄ The reaction was stopped by 50. Mu.L/well of stop solution. The detection wavelength of the enzyme label instrument is set to 450nm, the OD value of each hole is measured, and the reading is carried out within 10 minutes after termination.

After screening, 9 positive clones are obtained, and the detection value OD450nm is higher than 2.0.

Example 3: screening of high-coverage anti-norovirus GII genomic capsid protein VP1 monoclonal antibody

In order to obtain monoclonal antibodies capable of covering the genome dominant epidemic strains GII.4 genotype and GII.17 genotype of the norovirus and the capsid proteins VP1 of other multiple genome epidemic strains, detection is performed by respectively adopting capsid protein VP1 coated enzyme-linked plates of different epidemic strains of the norovirus GII genome.

The dominant strains GII, GII.S. of norovirus from Huashi Biotechnology Co.Ltd, were diluted with carbonate coating buffer, and 100. Mu.l of capsid protein VP1, 0.2. Mu.g/ml, was coated per well, at 4℃overnight; wash plate 2 times with wash solution, 200 μl/well; adding 110 μl/well of blocking solution, and blocking at room temperature for 6 hr; the plate was washed 5 times with wash solution, 200. Mu.l/well. After 200. Mu.l of sample dilution was added to each well, 10. Mu.l of cell culture supernatant was added, incubated at room temperature for 30min, and the solution was discarded. The plate was washed with 1 Xwash solution, 200. Mu.L per well, and the wash was repeated 5 times. The washed ELISA plate is inverted on the absorbent paper and is beaten with force to remove the redundant washing liquid. 100 μl/well HRP-labeled anti-mouse IgG antibody was added and incubated for 20min at room temperature. The plate was washed 5 times. Freshly prepared substrate solution, 100. Mu.l/well, was added and incubated at room temperature for 10 minutes in the dark. Add 2M H ₂ SO ₄ The reaction was stopped by 50. Mu.L/well of stop solution. The detection wavelength of the enzyme label instrument is set to 450nm, the OD value of each hole is measured, and the reading is carried out within 10 minutes after termination.

As shown in Table 1, only 3A21 and 5E17 of the prepared 9 anti-norovirus GII genomic capsid protein VP1 monoclonal antibodies can recognize the detected 9 norovirus GII genotypes, and simultaneously do not recognize the epidemic genotypes of the norovirus GI genomes, and have high coverage and high specificity for the norovirus GII genotypes. The detection value of the monoclonal antibody of the clone 3A21 is obviously higher than that of the monoclonal antibody of the clone 5E17, so that the monoclonal antibody of the clone 3A21 is determined to be the monoclonal antibody with the optimal performance.

TABLE 2 screening of high-coverage anti-norovirus GII genomic capsid protein VP1 monoclonal antibodies

Example 4: preparation of anti-norovirus GII genomic capsid protein VP1 monoclonal antibody 3A21 and subtype analysis thereof

The 3A21 hybridoma cell line was cultured in 1640 medium containing 10% fetal bovine serum. Each BALB/c male mouse was intraperitoneally injected with 0.5mL liquid paraffin. Cells were collected after 10 days, resuspended in 10mL of physiological saline, and each mouse was intraperitoneally injected with 0.5mL (cell density approximately 1X 10) ⁷ and/mL). After 2 weeks, ascites was collected. Antibody purification was performed using Thermo company Melon Gel Monoclonal IgG Purification Kit kit, and the purified antibodies were stored at-20 ℃ after packaging.

Antibody subtype identification using Pierce Papid Isotyping Kit-Mouse kit, first diluting the antibody to 100ng/mL with sample diluent, then adding 150 μl of diluted antibody per well, and observing and recording after 5-10 min. The results show that this monoclonal antibody is of the mouse IgG2a subtype, as shown in figure 1.

Example 5: variable region sequence determination of anti-norovirus GII genomic capsid protein VP1 monoclonal antibody

After culturing a mouse hybridoma cell strain 3A21 and extracting total RNA of the hybridoma cells by a Trizol method, reversely transcribing cDNA by a High Capacity cDNA Rever Transcription Kit kit of Thermo Fisher company, designing and synthesizing a heavy and light chain primer of the antibody by Beijing qing biological science and technology Co., ltd according to a primer sequence of mouse monoclonal antibody in recombinant antibody (scientific and Press, shen Beifen, published 2005), carrying out PCR amplification (amplification program: preheating for 1min at 95 ℃, carrying out 30 cycles (30 seconds at 95 ℃, 30 seconds at 58 ℃, 45 seconds at 72 ℃) and extending for 5min at 72 ℃), connecting a PMD18-T vector, expressing E.coli HB109, and selecting positive clones for sequencing. The sequences were aligned on the BLAST website (https:// www.ncbi.nlm.nih.gov/igblast /) to the CDR region sequences of the monoclonal antibodies of murine origin.

After sequence analysis, the amino acid sequence of the light chain variable region is 109 amino acids, and the sequence is as follows: DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEEAPSWKS (SEQ ID NO. 1) in which the underlined sequences are CDR1, CDR2 and CDR3 in this order, wherein CDR1 is located at 27-36aa and the amino acid sequence isKSVSTSGYSY(SEQ ID NO. 2); CDR2 is located at54-56aa, amino acid sequence isLVS() The method comprises the steps of carrying out a first treatment on the surface of the CDR3 is located at 93-100aa and the amino acid sequence isQHIRELTR(SEQ ID NO.3)。

The heavy chain variable region has an amino acid sequence of 103 amino acids and the sequence is as follows: VQPRGSLKLSCAVSGFTLNNNAMNWVRQAPGRGLKWVARLSSKFNNYATYYADSVKGRFTISRDDSQPILYLQMNNLKTEDTAMYYCVRDDYWGQGTPVTVSS (SEQ ID NO. 4) in which the underlined sequences are CDR1, CDR2 and CDR3 in this order, wherein CDR1 is located at 15-22aa and the amino acid sequence isGFTLNNNA(SEQ ID NO. 5); CDR2 is located at 40-49aa and has the amino acid sequence ofLSSKFNNYAT(SEQ ID NO.7SEQ ID NO. 6); CDR3 is located at 88-92aa and has the amino acid sequence ofVRDDY(SEQ ID NO.7)。

Example 6: detection of clinical samples of norovirus infected individuals

The invention uses the monoclonal antibody and polyclonal antibody with high coverage and high specificity to detect the capsid protein VP1 of the norovirus GII genome in human fecal samples by adopting the colloidal gold immunochromatography technology. Goat anti-mouse IgG polyclonal antibody (Beijing heat landscape biotechnology Co., ltd.) (C line) and rabbit anti-norovirus GII genomic capsid protein polyclonal antibody (Fei Peng Bio Co., ltd.) (T line) are coated on a nitrocellulose membrane in sequence, and colloidal gold-labeled anti-norovirus GII genomic capsid protein VP1 monoclonal antibody 3A21 of the invention is immobilized on a gold-labeled pad. The preparation process of the detection card comprises the following steps: 1) Coating buffer solution: PBS (pH 7.4) was used as coating buffer; 2) Coating concentration: the coating concentration of the rabbit anti-norovirus GII genomic capsid protein polyclonal antibody is 2.0mg/mL, and the coating concentration of the goat anti-mouse IgG polyclonal antibody is 1.0mg/mL; 3) NC film drying time: drying for more than 24 hours after coating the NC film; 4) Optimum marking amount: the labeling concentration of the anti-norovirus GII genomic capsid protein VP1 monoclonal antibody 3A21 is 25 mug/mL; 5) Metal spraying amount: performing metal spraying on the gold mark pad according to the concentration of 4 mu L/cm; 6) Gold mark pad drying time: 6-8 hours; 8) Film strip width: the width of the cut film strip should be not less than 3.0mm.

When a sample to be measured is dropped into the sample well of the test card, the sample will move forward along the test card by capillary action. If the sample contains the norovirus GII genomic capsid protein VP1, the sample is combined with a colloidal gold labeled mouse anti-norovirus GII genomic capsid protein VP1 monoclonal antibody 3A21, and the complex is captured by a coating immobilized on a nitrocellulose membrane and a rabbit anti-norovirus GII genomic capsid protein polyclonal antibody, so that a mauve T line is formed. The C-line region forms a mauve band regardless of whether the sample contains the norovirus GII genomic capsid protein VP1. The result judgment criteria are shown in fig. 2:

positive: two purplish red bands appear. One is located in the detection area (T line) and the other is located in the quality control area (C line). The color of the strip of the detection area (T line) can be dark or light, and the detection area (T line) is a positive result.

Negative: only the quality control area (C line) appears a mauve band, and no band appears in the detection area (T).

Invalidation: no mauve stripe appears in the quality control area (C line), no matter whether the stripe appears in the detection area (T line), the result is invalid, and the detection card needs to be taken again for retesting.

A total of 76 clinical stool samples for norovirus-associated diarrhea, wherein 62 cases of norovirus GII genome-positive samples, including the norovirus GII genome-epidemic strains GII.17, GII.4, GII.1, GII.2, GII.3, GII.6, GII.11, GII.12, GII.21, etc., were tested; the number of the norovirus GI genome positive samples is 14, and the norovirus GI genome epidemic strains GI.1 and GI.8 are covered. In addition, 50 stool samples containing other common intestinal cross pathogens were tested, including rotavirus, enterovirus EV71, adenovirus, zae virus, astrovirus, salmonella enteritidis, enterococcus, diarrheagenic escherichia coli, and the like.

The detection result shows that 62 cases of norovirus GII genome positive samples are detected to be positive, 14 cases of norovirus GI gene positive samples are detected to be negative, 50 cases of samples containing other common digestive tract cross pathogens are detected to be negative, and the detection sensitivity and the specificity of the detection technology are 100%.

As described above, the anti-norovirus GII genomic capsid protein VP1 monoclonal antibody 3A21 with high coverage and high activity based on the invention can be used for detecting infection of viruses such as Yu Nuo, has very high sensitivity and specificity for the dominant epidemic strains GII, 17, GII, 4, GII, 1, 2, GII, 3, GII, 6, GII, 11, GII, 12 and GII.21 of the norovirus.

Claims

1. A monoclonal antibody against the capsid protein VP1 of the Gatevirus G II genome or an antigen-binding fragment thereof, comprising a light chain variable region comprising CDR1, CDR2 and CDR3 and a heavy chain variable region comprising CDR1, CDR2 and CDR3, characterized in that,

the amino acid sequence of the light chain CDR1 is a sequence shown as SEQ ID NO. 2;

the amino acid sequence of the light chain CDR2 is LVS;

the amino acid sequence of the light chain CDR3 is a sequence shown as SEQ ID NO. 3;

the amino acid sequence of the heavy chain CDR1 is a sequence shown in SEQ ID NO. 5;

the amino acid sequence of the heavy chain CDR2 is a sequence shown in SEQ ID NO. 6;

the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 7.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region sequence is set forth in SEQ ID No.1 and the heavy chain variable region sequence is set forth in SEQ ID No. 4.

3. The monoclonal antibody according to claim 2, which is secreted by the mouse hybridoma cell line 3a21 with a collection number of cgmccno.45322.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment is a Fab fragment, fab 'fragment, F (ab') ₂ Fragments, single chain antibodies or humanized antibodies.

5. A nucleic acid molecule comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 4.

6. An expression vector comprising the nucleic acid molecule of claim 5.

7. A recombinant comprising the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. The monoclonal antibody hybridoma cell strain resisting the norovirus GII genomic capsid protein VP1 is characterized in that the monoclonal antibody hybridoma cell strain is a mouse hybridoma cell strain 3A21, and the preservation number is CGMCC No.45322.

9. Use of a monoclonal antibody or antigen binding fragment thereof according to any one of claims 1 to 4 for the manufacture of a product for detecting norovirus or for treating norovirus infection.

10. A kit for detecting norovirus, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.