CN115975017A - anti-GII.17 type norovirus monoclonal antibody and application thereof - Google Patents

anti-GII.17 type norovirus monoclonal antibody and application thereof Download PDF

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CN115975017A
CN115975017A CN202111204371.8A CN202111204371A CN115975017A CN 115975017 A CN115975017 A CN 115975017A CN 202111204371 A CN202111204371 A CN 202111204371A CN 115975017 A CN115975017 A CN 115975017A
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葛君
李建强
王晓东
谭昌耀
周童
任苏林
顾月
陈悦
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Yuanda Weixin Life Science Nanjing Co ltd
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Abstract

The invention provides an anti-GII.17 type norovirus monoclonal antibody and application thereof. The invention provides an anti-GII.17 norovirus monoclonal antibody 2F11-Ab, wherein the monoclonal antibody is prepared from a monoclonal antibody with a preservation number of CCTCC NO: c2021171, hybridoma cell line 2F11. The invention provides a hybridoma cell strain 2F11, which has a preservation number of CCTCC NO: C2021171. the invention provides a device for detecting GII.17 type norovirus in a sample, which comprises the anti-GII.17 type norovirus monoclonal antibody 2F11-Ab. The anti-GII.17 type norovirus monoclonal antibody 2F11-Ab provided by the invention has higher blocking activity, the BT50 is higher than 2560, the potential advantage of treating GII.17 type norovirus infection is realized, and the anti-GII.17 type norovirus monoclonal antibody can be applied to preparation of corresponding detection vaccines and treatment medicines.

Description

anti-GII.17 type norovirus monoclonal antibody and application thereof
Technical Field
The invention belongs to the field of immunology, and relates to an anti-GII.17 type norovirus monoclonal antibody and application thereof, in particular to application of a kit containing the antibody in detection of GII.17 type norovirus infection, and application of the antibody in preparation of a medicament for preventing GII.17 type norovirus infection.
Background
Norovirus (Norovirus) was first discovered and named Norovirus (NV) in the feces of diarrheal patients in Norwalk town by the american scholar Kapikian in 1972, and subsequently named Norovirus on the eighth international committee for virus naming, which is one of the major pathogens causing outbreaks of human infectious gastroenteritis epidemics and acute diarrhea in infants.
Norovirus is a single-stranded positive-stranded RNA virus belonging to Caliciviridae, has a diameter of about 26-35nm, is non-enveloped, has a rough surface, is spherical, and is icosahedral symmetric; is separated from feces of patients with acute gastroenteritis. The norovirus genome is approximately 7.7kb in length and contains 3 Open Reading Frames (ORFs). Depending on the phylogenetic nature of the gene sequences, noroviruses can be divided into 6 gene groups (genoglup, GI-GVI), each of which is divided into multiple genotypes (genotype).
Human norovirus infection is mostly caused by the GI and GII gene groups, wherein GII group is the main group; statistically, more than 75% of human NoV acute gastroenteritis are caused by GII group infection. The detection rate and the prevalence conditions of each genotype have large difference and change in different periods and different regions, and the equivalent types of GII.2, GII.4, GII.6, GII.7, GII.12 and GII.17 are detected in the last 20 years.
Norovirus has the characteristics of genotypic diversity and rapid variation, and no ideal cell culture system or animal model exists at present, so that the research and development of anti-norovirus vaccines and medicines are limited to a certain extent. Therefore, there is a lack of effective preventive measures, preventive vaccines or therapeutic drugs against norovirus, and a lack of corresponding means for detecting viruses.
Based on the above, the inventor provides a quantitative and reliable norovirus detection means, and an anti-norovirus vaccine or drug with good clinical application prospect, which has wide application prospect.
Disclosure of Invention
Therefore, in order to solve the disadvantages of the prior art, the present invention aims to provide an anti-GII.17 type norovirus monoclonal antibody, which has a specific blocking effect on GII.17 type norovirus and has no cross-reaction on GI.1, GII.2, GII.4 and GII.6 types; the invention also provides a kit for detecting GII.17 type norovirus, which is simple and convenient to operate, high in sensitivity, low in cost and suitable for mass production. The invention also provides a medicament for preventing GII.17 type norovirus infection.
The purpose of the invention is realized by the following technical scheme:
in one aspect, the invention provides an anti-GII.17 norovirus monoclonal antibody 2F11-Ab, wherein the monoclonal antibody is represented by a sequence with a collection number of CCTCC NO: c2021171 hybridoma cell line 2F11.
On the other hand, the invention provides a hybridoma cell strain 2F11 with the preservation number of CCTCC NO: C2021171.
the preservation number of the hybridoma cell strain 2F11 is CCTCC NO: c2021171 (the preservation date is 2021 year, 9 month and 1 day, the preservation number is CCTCC NO: C2021171, the preservation place is China center for type culture Collection, the address is university of Wuhan, china).
In a further aspect, the present invention provides a device for detecting the presence and/or level of a type gii.17 norovirus in a sample, said device comprising an anti-type gii.17 norovirus monoclonal antibody 2F11-Ab of the invention; preferably, the sample is a multivalent norovirus vaccine.
Preferably, the detection uses an immunological method, the device being a device for use in an immunological method; more preferably, the immunological method is selected from the group consisting of immunoblotting, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay, immunochemiluminometry, and the like. More preferably, the detection is by enzyme-linked immunosorbent assay or immunochemiluminometric assay.
Preferably, the device is a kit; more preferably, the kit further comprises a rabbit anti-gii.17 type norovirus polyclonal antibody and/or a goat anti-murine species antibody; further preferably, the kit further comprises one or more of a coating solution, a washing solution, a blocking solution, a diluting solution, a stop solution and a standard substance; even more preferably, the kit further comprises an elisa plate and/or a sealing plate membrane.
In a further aspect, the invention provides the use of the monoclonal antibody 2F11-Ab described above in the preparation of a reagent or kit for detecting the presence and/or level of norovirus type gii.17 in a sample.
The use according to the invention, wherein the sample is a multivalent norovirus vaccine.
The invention also provides a quantitative detection method of the GII.17 type norovirus, and the detection method uses an immunization method; more preferably, the immunological method is selected from immunoblotting, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay, immunochemiluminescence, or the like.
In still another aspect, the present invention provides a detection reagent comprising the monoclonal antibody of the present invention. The detection reagent may be prepared by any method known to those skilled in the art, for example, by mixing the monoclonal antibody of the present invention with a suitable carrier, such as water, saline, a buffer, etc. The detection reagent can be used for detecting the expression level of the GII.17 type norovirus in a sample, and can dynamically detect the change of the protein level of the GII.17 type norovirus.
The invention also provides a pharmaceutical composition for preventing and/or treating GII.17 type norovirus infection, which comprises the monoclonal antibody 2F11-Ab.
Preferably, the pharmaceutical composition is a vaccine.
The invention also provides application of the monoclonal antibody 2F11-Ab in preparing a pharmaceutical composition for preventing and/or treating GII.17 type norovirus infection.
Preferably, the pharmaceutical composition is a vaccine.
The invention has the beneficial effects that:
1. because norovirus exists in a multivalent form, a simple and rapid quantitative detection method for GII.17 norovirus does not exist at present, the monoclonal antibody 2F11-Ab for resisting GII.17 norovirus provided by the invention has high specificity, and an experimental result shows that no cross immune reaction exists on GI.1, GII.2, GII.4 and GII.6, the GII.17 can be quantitatively detected from antigens with multiple valences, and the method is suitable for being used as a quality monitoring means in the preparation of norovirus multivalent vaccines.
2. Various indexes of the anti-GII.17 type norovirus monoclonal antibody 2F11-Ab meet research and development requirements, repeated freeze thawing can be performed for multiple times, the antibody property is stable, the detection result is not influenced, and the anti-GII.17 type norovirus monoclonal antibody is suitable for storage and use in the reagent application process and is convenient for users to apply.
3. The anti-GII.17 norovirus monoclonal antibody 2F11-Ab provided by the invention has higher blocking activity, the BT50 is higher than 2560, the potential advantage of treating GII.17 norovirus infection is achieved, and the anti-GII.17 norovirus monoclonal antibody can be applied to preparation of related treatment medicines.
Biological preservation information
The preservation number of the hybridoma cell strain 2F11 is CCTCC NO: c2021171, date of deposit: 2021, 9 months and 1 day, and the preservation number is CCTCC NO: c2021171, depository: china center for type culture Collection, address: china, wuhan university.
Detailed Description
The present invention is further illustrated by the following description of specific embodiments, which are not intended to limit the invention, and various modifications and improvements can be made by those skilled in the art based on the basic idea of the invention, but within the scope of the invention, without departing from the basic idea of the invention.
Example 1 preparation of different valency forms of noro antigen
A plurality of valency types of norovirus VLP proteins are prepared respectively, and the amino acid sequences are shown as follows:
the amino acid sequence of the VLP protein of the GII.1 norovirus is shown as SEQ ID NO:1 is shown.
The amino acid sequence of the VLP protein of the GII.2 type norovirus is shown as SEQ ID NO:2, respectively.
The amino acid sequence of the norovirus VLP protein of GII.4 is shown as SEQ ID NO:3, respectively.
The amino acid sequence of the norovirus VLP protein of GII.6 type is shown as SEQ ID NO:4, respectively.
The amino acid sequence of the VLP protein of the norovirus GII.17 is shown as SEQ ID NO:5, respectively.
The preparation method comprises the following steps: optimizing the nucleic acid sequence of the target protein sequence according to the target protein sequence to ensure that the codon of the target protein sequence accords with a yeast expression system, and synthesizing a target gene; connecting the synthesized target gene with pMAUR (S.C) KARS1 plasmid by enzyme digestion connection, transforming Top 10 competence, selecting positive monoclonal, and performing sequencing verification on the positive monoclonal; amplifying a large amount of monoclonal thallus, and extracting a large amount of plasmids conforming to electrotransformation by using a plasmid extraction kit; the plasmid was transformed into uracil auxotrophic hansenula competent cells by electroporation and positive transformed cells were obtained using G418 resistance selection.
Inoculating the recombinant strain to a yeast extract peptone glucose culture medium for activation, transferring the activated strain to the yeast extract peptone glucose culture medium for amplification culture, and supplementing methanol to the culture medium for induction culture once every 24 hours and twice after the strain is cultured to a plateau period. And collecting thalli after induction, and breaking and purifying the thalli to obtain the protein.
Example 2 preparation and selection of hybridoma cells
2.1 mouse immunization and Tail blood detection
1) Immunization of mice
3 mice (female, BALB/c, numbered 1#, 2#, 3#, respectively) were immunized with the GII.17 type norovirus VLP protein prepared in example 1, and tail blood was taken on day 14 after immunization.
2) Evaluation of antibody titer in Tail blood
Coating an enzyme label plate with 1 mu g/mL of GII.17 type norovirus VLP protein, adding 100 mu L of the enzyme label plate to each well, and standing overnight at 4 ℃; washing the plate 3 times with PBS solution, blocking with 5% mil-PBS at room temperature for 1 hour; washing the plate for 1 time by using a PBS solution, adding the mouse tail blood diluted in a gradient manner, and reacting for 1 hour at room temperature; the plate was washed 3 times with PBS solution, and 1: HRP-labeled goat anti-mouse Fc secondary antibody (purchased from Sigma) diluted 2000, and reacted at room temperature for 1 hour; washing the plate with PBS solution for 5 times, adding equal volume of TMB color developing solution, and reacting at room temperature for 20min; adding 50 mu L of stop solution, mixing uniformly, and reading the OD450 value on a microplate reader.
The results showed that the tail blood antibody titer of the # 1 mouse was high.
TABLE 1GII.17 evaluation of mouse tail blood antibody titers on day 14 after protein immunization
Mouse numbering 1/500 1/1000 1/5000 1/1W 1/5W NC
1# 3.004 2.950 2.868 2.828 2.655 0.045
2# 3.180 2.990 2.937 2.901 1.565 0.096
3# 2.943 3.010 2.885 2.875 2.034 0.043
Note: NC as negative control, 2% milk-PBS
2.2 monoclonal screening
Selecting a No. 1 mouse for cell fusion, killing the mouse, taking splenocytes to fuse with SP2/0 cells, culturing the cells in a selective culture medium to obtain a monoclonal cell strain, and then selecting and cloning.
Coating an enzyme label plate with 1 mu g/mL of GII.17 type norovirus VLP protein, adding 100 mu L of the enzyme label plate to each well, and standing overnight at 4 ℃; wash the plate 3 times with PBS solution, block for 1 hour at room temperature with 5% mil-PBS; wash the plate 1 time with PBS solution, monoclonal cell supernatant with 5% mil-PBS 1:1, mixing, adding into a hole, and reacting at room temperature for 1 hour; the plate was washed 3 times with PBS solution, and 1: reacting the goat anti-mouse Fc secondary antibody marked by HRP at 2000 dilution for 1 hour at room temperature; washing the plate with PBS solution for 5 times, adding equal volume of TMB color developing solution, and reacting at room temperature for 20min; adding 50 mu L of stop solution, mixing uniformly, and reading the OD450 value on a microplate reader.
The detection results are shown in Table 2, and the three screened positive clones of GII.17 have better specificity.
Specific detection of monoclonal antibody against norovirus of Table 2GII.17 type
Figure BDA0003306319830000051
Note: NC as negative control (2% mil-PBS), PC as positive control (GI.1, GII.4, GII.17 Noo antigen immunized mice heart blood)
2.3 ascites preparation and antibody purification
Selecting three clones of 5H9, 2E3 and 2F11 according to the above specificity detection results, preparing ascites by respectively adding about 1 × 10 7 Injecting each cell into abdominal cavity of 2 Balb/c mice pre-injected with IFA adjuvant, extracting ascites generated by each positive clone after injecting the cells for 10 days, centrifuging at 12000rpm for 15min at 4 ℃, collecting supernatant and purifying.
The sensitivity of the purified antibody was measured, and the results are shown in Table 3, in which the 2F11 clone produced the most sensitive antibody.
TABLE 3 GII.17 antibody sensitivity detection
Figure BDA0003306319830000061
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Note: NC is a negative control (2% mill-PBS), naN indicates OD >4.0.
EXAMPLE 3 Cross-blocking assay of monoclonal antibodies
The avidin plate was washed 1 time with 200. Mu.l/well wash; HBGA (Glycotech Co.) was diluted to 4. Mu.g/mL, 100. Mu.l/well was added to an avidin plate, and incubated at 25 ℃ for 1h; respectively carrying out multiple dilution on monoclonal antibodies generated by cloning 5H9, 2E3 and 2F11 in a 96-well plate, and incubating for 1H at 4 ℃; washing the HBGA-coated avidin plate 2 times with 200. Mu.l/well washing solution, sucking 100. Mu.l of diluted monoclonal antibody from a 96-well plate, adding the monoclonal antibody into the avidin plate, and incubating for 2h at 4 ℃; washing avidin plate with 200 μ l/well washing solution for 3 times, adding GII.17 type antigen rabbit polyclonal antibody, and incubating at 4 deg.C for 1h; washing avidin plate with 200 μ l/well washing solution for 3 times, adding goat anti-rabbit-HRP, and incubating at 37 deg.C for 40min; washing avidin plate with 200 μ l/hole washing solution for 3 times, adding TMB color developing solution 100 μ l/hole, and developing at 25 deg.C for 5-15min; and (4) terminating: adding stop solution, and stopping the reaction at 100 mu l/hole; reading: OD450nm values (corrected for OD630 nm) were determined.
Detection standard: blockade index% = (1-serogroup a value/positive control a value) × 100% BT50 calculated, i.e. the highest dilution of serum that is able to block 50% binding of vlps to HBGA.
TABLE 4 blocking Effect of monoclonal antibodies
Figure BDA0003306319830000062
Figure BDA0003306319830000071
Experimental results show that three monoclonal antibodies 5H9, 2E3 and 2F11 screened out have blocking activity on GII.17 noro antigen, the BT50 is 640, 1280 and not less than 2560, wherein the blocking effect of 2F11 is optimal, and the blocking effect is still achieved under the dilution multiple of 2560 times.
The above experiments demonstrated that 2F11 not only has high antibody sensitivity but also has a significant antibody blocking effect, and 2F11 was used as a preferred antibody in the following examples.
Example 4 GII.17 type norovirus detection kit
4.1 kit materials
The skim milk (skim milk) used in the invention is purchased from BD Biosciences, the TMB color developing solution is purchased from Thermo, and other chemical reagents are domestic analytical purity and above grade.
4.2 kit Assembly
The kit comprises the following antibodies:
GII.17 antigen rabbit polyclonal antibody (preparation of Shijingtian)
Horse radish peroxidase-labeled goat anti-mouse monoclonal antibody (hereinafter referred to as goat anti-mouse-HRP, SIGMA company)
Murine anti-GII.17 norovirus mab 2F11.
The kit further comprises the following reagents:
1) Coating solution reagent: 0.5% (g/100 mL) carbonate solution;
2) Washing solution reagent: 10g/L PBS +0.5mL/L Tween 20 (PBST);
3) Sealing liquid reagent: 5% skim milk PBST;
4) Diluent reagent: 2% skim milk PBST;
5) Stop solution reagent: 2M H 2 SO 4 ；
6) And (3) standard substance: GII.17 type norovirus antigen (homemade by Yuntan Sai Wen, already content-calibrated).
The kit also comprises an ELISA plate and a sealing plate film.
And (3) sequentially putting the antibody, the reagent, the ELISA plate and the sealing plate film into a packaging box to complete the assembly of the kit.
4.3 methods of use of the kits
1) Coating: diluting the rabbit polyclonal antibody with the GII.17 antigen to 1 mu g/mL by using a coating solution, coating an ELISA enzyme linked plate at 50 mu l/hole, and standing overnight at 4 ℃;
2) And (3) sealing: washing the enzyme label plate coated in the step 1) with a washing solution for 2 times, adding a sealing solution into the enzyme label plate, sealing the enzyme label plate at the temperature of 37 ℃ for 1 hour;
3) Diluting and loading: and taking the GII.17 antigen standard substance, performing serial dilution by using a diluent according to the content value of the marked protein, taking the concentration of 40 ng/mL-2.5 ng/mL as a standard series, and taking the diluent as a zero point of the standard series. The test sample was diluted to within the range of the standard series. Sequentially adding a standard substance with each concentration, a diluent and a diluted sample to be detected on the enzyme label plate sealed in the step 2), setting parallel experiments on all sample loading holes, and after sample loading, sealing the plate by using a sealing plate film and then incubating for 1 hour at 37 ℃;
4) And (3) incubation of the detection antibody: washing the enzyme label plate incubated in the step 3) for 3 times by using a washing solution, and adding a diluent according to a volume ratio of 1: murine anti-GII.17 norovirus mab 2F11 after 2000 dilutions, 50. Mu.l per well. Sealing the plate with a sealing plate membrane, and incubating at 37 ℃ for 1 hour;
5) And (3) detecting secondary antibody incubation: washing the enzyme label plate incubated in the step 4) for 3 times by using a washing solution, and adding a diluent according to a volume ratio of 1:10000 diluted goat anti-mouse-HRP, 50. Mu.l per well. Sealing the plate with a sealing plate membrane, and incubating at 37 ℃ for 1 hour;
6) Color development: washing the enzyme-linked immunosorbent assay plate incubated in the step 5) for 3 times by using a washing solution, adding 50 mu l of TMB (from Thermo) into each hole, sealing the plate by using a sealing plate membrane, and then developing for 10-20 minutes at 37 ℃;
7) Terminating the reading of the plate: after the color development was completed, 50. Mu.l of stop buffer was added to all wells. The absorbance (OD value) was read on a microplate reader at a wavelength of 450nm with 630nm as a reference wavelength.
4.4 antigen content calculation
And subtracting the blank control average OD detection value from the average OD detection value of each dilution sample of the standard substance to obtain the OD detection value of the dilution sample. And (3) taking the concentration value (ng/mL) of each dilution of the standard as an abscissa and the OD detection value as an ordinate, and performing four-parameter fitting by using ELISAcalc software. And (3) blank deducting the OD detection value of the sample, substituting the blank into a fitting curve, and calculating to obtain the antigen content (ng/mL) of the sample solution:
antigen content (μ g/mL) = antigen content value (ng/mL) × dilution factor/1000
Example 5 Effect verification of GII.17 type norovirus assay kit
5.1 Attribute validation
The norovirus type GII.2, GII.4 and GII.6 VLP proteins prepared in example 1 were diluted to 500ng/mL and the norovirus type GII.17 VLP proteins were diluted to 100ng/mL, respectively, and BL was set (blank control). The kit prepared in example 4 was used for detection, and the results are shown in Table 5. Wherein the OD values of the blank control, GII.2, GII.4 and GII.6 are all less than 0.05 which is 2.1 times of the BL value; GII.17 has an OD mean of 2.66, which is greater than 1.0 of the limit OD.
The test proves that the kit provided by the invention has good specificity when used for detecting the GII.17 type norovirus antigen.
TABLE 5 results of the Attribute verification
Sample price form GII.2 GII.4 GII.6 GII.17 BL 2.1BL
OD value 0.03 0.03 0.03 2.66 0.02 0.05
5.2 accuracy verification
Taking four parts of GII.17 samples to be detected, respectively diluting the four parts to 10^5 times, taking three parts of the samples, respectively adding 150 microlitres of the three parts into 150 microlitres of GII.17 type standard solutions with antigen contents of 40ng/mL, 20ng/mL and 5ng/mL, and detecting the samples together with the solutions without the standard solutions, wherein the results are shown in a table 6. The result shows that the recovery rate of the GII.17 sample by adding the standard is 88-96 percent, and the recovery rate is within the range of 80-125 percent of the limit, thereby meeting the requirements.
Proved by verification, the kit provided by the invention has good accuracy when detecting the antigen content of the GII.17 type norovirus.
Table 6 accuracy verification results
Adding standard concentration ng/mL OD average value The antigen content detection value ng/mL Recovery (%)
40 0.678 23.50 88
20 0.421 14.92 90
5 0.223 8.29 96
Without adding marks 0.327 11.78 ——
5.3 repeatability verification
A GII.17 sample to be tested is taken, and 6 tubes are parallelly measured to be used as a repeatability test. The Relative Standard Deviation (RSD) of the results was calculated for six times and the results are shown in table 7. Through calculation, the RSD of the parallel 6-tube detection result of the GII.17 sample is 8.58 percent and is less than 10 percent of the limit value of the repetitive RSD, thereby meeting the requirement.
The test proves that the kit provided by the invention has good precision when being used for detecting the content of the GII.17 type norovirus antigen.
TABLE 7 results of repeatability verification
Figure BDA0003306319830000091
5.4 kit linearity and Range validation
The VLP protein of GII.17 type norovirus prepared in example 1 was diluted to 40ng/mL, 20g/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, respectively, and each group was loaded in 3 wells. Calculating to obtain R of the standard curve 2 Is 0.99995, at the limit R 2 The range is more than or equal to 0.99; the average recovery rate of each point of the standard curve is 101.49-115.65%, and the limit is 80% -125%; the RSD of each concentration point is 0.75-3.16%, and the RSD is less than or equal to 10%, which meets the requirement.
The verification proves that the kit provided by the invention has good linearity when used for determining the content of the GII.17 type norovirus antigen, and the detection range is 40 ng/mL-2.5 ng/mL.
TABLE 8 Linear and Range verification results
Figure BDA0003306319830000101
5.5 Key antibody stability validation
Three portions of mouse anti-GII.17 type norovirus monoclonal antibody 2F11, which are respectively marked as monoclonal antibody-1, monoclonal antibody-2 and monoclonal antibody-3, are preserved in a refrigerator at the temperature of-20 ℃, wherein the monoclonal antibody-1 is taken out once in the first day and the second day respectively, and is preserved in the refrigerator at the temperature of-20 ℃ after being melted; the monoclonal antibody-2 is taken out once in the next day, and is put back into a refrigerator at the temperature of-20 ℃ for storage after being melted; the freeze-thaw interval is more than 12 hours. And on the third day, taking out and melting the monoclonal antibody-1, the monoclonal antibody-2 and the monoclonal antibody-3, and carrying out subsequent detection. Through calculation, after freezing and thawing 2F11 for 1 time, 2 times and 3 times, the RSD of the antigen content of the sample is measured to be 3.87 percent and less than 10 percent of the limit.
Proved by verification, when the kit provided by the invention is used for determining the content of the recombinant norovirus GII.17 antigen, the number of times of freezing and thawing of the detection antibody is 3, and the storage and the use in the practical application process are facilitated.
TABLE 9 stability verification results of the detection antibodies
Figure BDA0003306319830000102
In conclusion, the hybridoma cell strain 2F11 can secrete the anti-GII.17 norovirus monoclonal antibody. The antibody has a specific blocking effect on GII.17 type norovirus, does not have cross reaction on GI.1, GII.2, GII.4 and GII.6 types, can be used for preparing an antibody medicament for preventing GII.17 type norovirus infection, and can also be used for preparing a kit for detecting GII.17 type norovirus. The antibody has high stability, can be repeatedly frozen and thawed for multiple times, and the prepared kit is convenient for storage by a user.
Although the present invention has been described in detail above, those skilled in the art will appreciate that various modifications and changes can be made to the present invention without departing from the spirit and scope of the invention. The scope of the invention is not to be limited by the above detailed description but is only limited by the claims.
Sequence listing
<110> Yuxin Life sciences of Tanshima (Nanjing) Ltd
<120> anti-GII.17 type norovirus monoclonal antibody and use thereof
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<211> 542
<212> PRT
<213> Norovirus (Norovirus)
<400> 2
Met Lys Met Ala Ser Asn Asp Ala Ala Pro Ser Thr Asp Gly Ala Ala
1 5 10 15
Gly Leu Val Pro Glu Ser Asn Asn Glu Val Met Ala Leu Glu Pro Val
20 25 30
Ala Gly Ala Ala Leu Ala Ala Pro Val Thr Gly Gln Thr Asn Ile Ile
35 40 45
Asp Pro Trp Ile Arg Ala Asn Phe Val Gln Ala Pro Asn Gly Glu Phe
50 55 60
Thr Val Ser Pro Arg Asn Ala Pro Gly Glu Val Leu Leu Asn Leu Glu
65 70 75 80
Leu Gly Pro Glu Leu Asn Pro Tyr Leu Ala His Leu Ala Arg Met Tyr
85 90 95
Asn Gly Tyr Ala Gly Gly Met Glu Val Gln Val Met Leu Ala Gly Asn
100 105 110
Ala Phe Thr Ala Gly Lys Leu Val Phe Ala Ala Val Pro Pro His Phe
115 120 125
Pro Val Glu Asn Leu Ser Pro Gln Gln Ile Thr Met Phe Pro His Val
130 135 140
Ile Ile Asp Val Arg Thr Leu Glu Pro Val Leu Leu Pro Leu Pro Asp
145 150 155 160
Val Arg Asn Asn Phe Phe His Tyr Asn Gln Lys Asp Asp Pro Lys Met
165 170 175
Arg Ile Val Ala Met Leu Tyr Thr Pro Leu Arg Ser Asn Gly Ser Gly
180 185 190
Asp Asp Val Phe Thr Val Ser Cys Arg Val Leu Thr Arg Pro Ser Pro
195 200 205
Asp Phe Asp Phe Thr Tyr Leu Val Pro Pro Thr Val Glu Ser Lys Thr
210 215 220
Lys Pro Phe Thr Leu Pro Ile Leu Thr Leu Gly Glu Leu Ser Asn Ser
225 230 235 240
Arg Phe Pro Val Ser Ile Asp Gln Met Tyr Thr Ser Pro Asn Glu Ile
245 250 255
Ile Ser Val Gln Cys Gln Asn Gly Arg Cys Thr Leu Asp Gly Glu Leu
260 265 270
Gln Gly Thr Thr Gln Leu Gln Val Ser Gly Ile Cys Ala Phe Lys Gly
275 280 285
Glu Val Thr Ala His Leu His Asp Asn Asp His Leu Tyr Asn Val Thr
290 295 300
Ile Thr Asn Leu Asn Gly Ser Pro Phe Asp Pro Ser Glu Asp Ile Pro
305 310 315 320
Ala Pro Leu Gly Val Pro Asp Phe Gln Gly Arg Val Phe Gly Ile Ile
325 330 335
Ser Gln Arg Asp Lys His Asn Ser Pro Gly His Asn Glu Pro Ala Asn
340 345 350
Arg Gly His Asp Ala Val Val Pro Thr Tyr Thr Ala Gln Tyr Thr Pro
355 360 365
Lys Leu Gly Gln Ile Gln Ile Gly Thr Trp Gln Thr Asp Asp Leu Thr
370 375 380
Val Asn Gln Pro Val Lys Phe Thr Pro Val Gly Leu Asn Asp Thr Glu
385 390 395 400
His Phe Asn Gln Trp Val Val Pro Arg Tyr Ala Gly Ala Leu Asn Leu
405 410 415
Asn Thr Asn Leu Ala Pro Ser Val Ala Pro Val Phe Pro Gly Glu Arg
420 425 430
Leu Leu Phe Phe Arg Ser Tyr Ile Pro Leu Lys Gly Gly Tyr Gly Asn
435 440 445
Pro Ala Ile Asp Cys Leu Leu Pro Gln Glu Trp Val Gln His Phe Tyr
450 455 460
Gln Glu Ala Ala Pro Ser Met Ser Glu Val Ala Leu Val Arg Tyr Ile
465 470 475 480
Asn Pro Asp Thr Gly Arg Ala Leu Phe Glu Ala Lys Leu His Arg Ala
485 490 495
Gly Phe Met Thr Val Ser Ser Asn Thr Ser Ala Pro Val Val Val Pro
500 505 510
Ala Asn Gly Tyr Phe Arg Phe Asp Ser Trp Val Asn Gln Phe Tyr Ser
515 520 525
Leu Ala Pro Met Gly Thr Gly Asn Gly Arg Arg Arg Val Gln
530 535 540
<210> 3
<211> 540
<212> PRT
<213> Norovirus (Norovirus)
<400> 3
Met Lys Met Ala Ser Ser Asp Ala Asn Pro Ser Asp Gly Ser Ala Ala
1 5 10 15
Asn Leu Val Pro Glu Val Asn Asn Glu Val Met Ala Leu Glu Pro Val
20 25 30
Val Gly Ala Ala Ile Ala Ala Pro Val Ala Gly Gln Gln Asn Val Ile
35 40 45
Asp Pro Trp Ile Arg Asn Asn Phe Val Gln Ala Pro Gly Gly Glu Phe
50 55 60
Thr Val Ser Pro Arg Asn Ala Pro Gly Glu Ile Leu Trp Ser Ala Pro
65 70 75 80
Leu Gly Pro Asp Leu Asn Pro Tyr Leu Ser His Leu Ala Arg Met Tyr
85 90 95
Asn Gly Tyr Ala Gly Gly Phe Glu Val Gln Val Ile Leu Ala Gly Asn
100 105 110
Ala Phe Thr Ala Gly Lys Val Ile Phe Ala Ala Val Pro Pro Asn Phe
115 120 125
Pro Thr Glu Gly Leu Ser Pro Ser Gln Val Thr Met Phe Pro His Ile
130 135 140
Val Val Asp Val Arg Gln Leu Glu Pro Val Leu Ile Pro Leu Pro Asp
145 150 155 160
Val Arg Asn Asn Phe Tyr His Tyr Asn Gln Ser Asn Asp Pro Thr Ile
165 170 175
Lys Leu Ile Ala Met Leu Tyr Thr Pro Leu Arg Ala Asn Asn Ala Gly
180 185 190
Asp Asp Val Phe Thr Val Ser Cys Arg Val Leu Thr Arg Pro Ser Pro
195 200 205
Asp Phe Asp Phe Ile Phe Leu Val Pro Pro Thr Val Glu Ser Arg Thr
210 215 220
Lys Pro Phe Ser Val Pro Val Leu Thr Val Glu Glu Met Thr Asn Ser
225 230 235 240
Arg Phe Pro Ile Pro Leu Glu Lys Leu Phe Thr Gly Pro Ser Ser Ala
245 250 255
Phe Val Val Gln Pro Gln Asn Gly Arg Cys Thr Thr Asp Gly Val Leu
260 265 270
Leu Gly Thr Thr Gln Leu Ser Pro Val Asn Ile Cys Thr Phe Arg Gly
275 280 285
Asp Val Thr His Ile Thr Gly Ser Arg Asn Tyr Thr Met Asn Leu Ala
290 295 300
Ser Gln Asn Trp Asn Asn Tyr Asp Pro Thr Glu Glu Ile Pro Ala Pro
305 310 315 320
Leu Gly Thr Pro Asp Phe Val Gly Lys Ile Gln Gly Val Leu Thr Gln
325 330 335
Thr Thr Arg Thr Asp Gly Ser Thr Arg Gly His Lys Ala Thr Val Tyr
340 345 350
Thr Gly Ser Ala Asp Phe Ala Pro Lys Leu Gly Arg Val Gln Phe Glu
355 360 365
Thr Asp Thr Asp His Asp Phe Glu Ala Asn Gln Asn Thr Lys Phe Thr
370 375 380
Pro Val Gly Val Ile Gln Asp Gly Ser Thr Thr His Arg Asn Glu Pro
385 390 395 400
Gln Gln Trp Val Leu Pro Ser Tyr Ser Gly Arg Asn Thr His Asn Val
405 410 415
His Leu Ala Pro Ala Val Ala Pro Thr Phe Pro Gly Glu Gln Leu Leu
420 425 430
Phe Phe Arg Ser Thr Met Pro Gly Cys Ser Gly Tyr Pro Asn Met Asp
435 440 445
Leu Asp Cys Leu Leu Pro Gln Glu Trp Val Gln Tyr Phe Tyr Gln Glu
450 455 460
Ala Ala Pro Ala Gln Ser Asp Val Ala Leu Leu Arg Phe Val Asn Pro
465 470 475 480
Asp Thr Gly Arg Val Leu Phe Glu Cys Lys Leu His Lys Ser Gly Tyr
485 490 495
Val Thr Val Ala His Thr Gly Gln His Asp Leu Val Ile Pro Pro Asn
500 505 510
Gly Tyr Phe Arg Phe Asp Ser Trp Val Asn Gln Phe Tyr Thr Leu Ala
515 520 525
Pro Met Gly Asn Gly Thr Gly Arg Arg Arg Ala Val
530 535 540
<210> 4
<211> 547
<212> PRT
<213> Norovirus (Norovirus)
<400> 4
Met Lys Met Ala Ser Asn Asp Ala Ala Pro Ser Asn Asp Gly Ala Ala
1 5 10 15
Asn Leu Val Pro Glu Ala Thr Asn Glu Val Met Ala Leu Glu Pro Val
20 25 30
Val Gly Ala Ser Ile Ala Ala Pro Val Val Gly Gln Gln Asn Ile Ile
35 40 45
Asp Pro Trp Ile Arg Glu Asn Phe Val Gln Ala Pro Gln Gly Glu Phe
50 55 60
Thr Val Ser Pro Arg Asn Ser Pro Gly Glu Met Leu Leu Asn Leu Glu
65 70 75 80
Leu Gly Pro Glu Leu Asn Pro Tyr Leu Ser His Leu Ser Arg Met Tyr
85 90 95
Asn Gly Tyr Ala Gly Gly Met Gln Val Gln Val Val Leu Ala Gly Asn
100 105 110
Ala Phe Thr Ala Gly Lys Ile Ile Phe Ala Ala Val Pro Pro His Phe
115 120 125
Pro Val Glu Asn Ile Ser Ala Ala Gln Ile Thr Met Cys Pro His Val
130 135 140
Ile Val Asp Val Arg Gln Leu Glu Pro Val Leu Leu Pro Leu Pro Asp
145 150 155 160
Ile Arg Asn Arg Phe Phe His Tyr Asn Gln Glu Asn Thr Pro Arg Met
165 170 175
Arg Leu Val Ala Met Leu Tyr Thr Pro Leu Arg Ala Asn Ser Gly Glu
180 185 190
Asp Val Phe Thr Val Ser Cys Arg Val Leu Thr Arg Pro Ala Pro Asp
195 200 205
Phe Glu Phe Thr Phe Leu Val Pro Pro Thr Val Glu Ser Lys Thr Lys
210 215 220
Pro Phe Thr Leu Pro Ile Leu Thr Leu Gly Glu Leu Ser Asn Ser Arg
225 230 235 240
Phe Pro Ala Pro Ile Asp Met Leu Tyr Thr Asp Pro Asn Glu Ala Ile
245 250 255
Val Val Gln Pro Gln Asn Gly Arg Cys Thr Leu Asp Gly Thr Leu Gln
260 265 270
Gly Thr Thr Gln Leu Val Pro Thr Gln Ile Cys Ser Phe Arg Gly Thr
275 280 285
Leu Val Ser Gln Thr Ser Arg Ser Ala Asp Ser Thr Asp Ser Ala Pro
290 295 300
Arg Val Arg Ser His Pro Leu His Val Gln Leu Lys Asn Leu Asp Gly
305 310 315 320
Thr Pro Tyr Asp Pro Thr Asp Glu Val Pro Ala Val Leu Gly Ala Ile
325 330 335
Asp Phe Lys Gly Thr Val Phe Gly Val Ala Ser Gln Arg Asn Thr Thr
340 345 350
Gly Asn Ser Ile Gly Ala Thr Arg Ala His Glu Val His Ile Asp Thr
355 360 365
Thr Asn Pro Arg Tyr Thr Pro Lys Leu Gly Ser Val Leu Met Tyr Ser
370 375 380
Glu Ser Asn Asp Phe Asp Asp Gly Gln Pro Thr Arg Phe Thr Pro Ile
385 390 395 400
Gly Met Gly Ala Asp Asp Trp His Gln Trp Glu Leu Pro Glu Tyr Ser
405 410 415
Gly His Leu Thr Leu Asn Met Asn Leu Ala Pro Ala Leu Ala Pro Ala
420 425 430
Phe Pro Gly Glu Arg Ile Leu Phe Phe Arg Ser Val Val Pro Ser Ala
435 440 445
Gly Gly Tyr Gly Ser Gly His Ile Asp Cys Leu Ile Pro Gln Glu Trp
450 455 460
Val Gln His Phe Tyr Gln Glu Ala Ala Pro Ser Gln Ser Ala Val Ala
465 470 475 480
Leu Ile Arg Tyr Val Asn Pro Asp Thr Gly Arg Asn Ile Phe Glu Ala
485 490 495
Lys Leu His Arg Glu Gly Phe Ile Thr Val Ala Asn Ser Gly Asn Asn
500 505 510
Pro Ile Val Val Pro Pro Asn Gly Tyr Phe Arg Phe Glu Ala Trp Val
515 520 525
Asn Gln Phe Tyr Thr Leu Thr Pro Met Gly Thr Gly Gln Gly Arg Arg
530 535 540
Arg Val Gln
545
<210> 5
<211> 540
<212> PRT
<213> Norovirus (Norovirus)
<400> 5
Met Lys Met Ala Ser Asn Asp Ala Ala Pro Ser Asn Asp Gly Ala Ala
1 5 10 15
Gly Leu Val Pro Glu Gly Asn Asn Glu Thr Leu Pro Leu Glu Pro Val
20 25 30
Ala Gly Ala Ala Ile Ala Ala Pro Val Thr Gly Gln Asn Asn Ile Ile
35 40 45
Asp Pro Trp Ile Arg Thr Asn Phe Val Gln Ala Pro Asn Gly Glu Phe
50 55 60
Thr Val Ser Pro Arg Asn Ser Pro Gly Glu Ile Leu Leu Asn Leu Glu
65 70 75 80
Leu Gly Pro Asp Leu Asn Pro Tyr Leu Ala His Leu Ser Arg Met Tyr
85 90 95
Asn Gly Tyr Ala Gly Gly Val Glu Val Gln Val Leu Leu Ala Gly Asn
100 105 110
Ala Phe Thr Ala Gly Lys Ile Leu Phe Ala Ala Val Pro Pro Asn Phe
115 120 125
Pro Val Glu Phe Leu Ser Pro Ala Gln Ile Thr Met Leu Pro His Leu
130 135 140
Ile Val Asp Val Arg Thr Leu Glu Pro Ile Met Ile Pro Leu Pro Asp
145 150 155 160
Val Arg Asn Thr Phe Phe His Tyr Ser Asn Gln Pro Asn Ser Arg Met
165 170 175
Arg Leu Val Ala Met Leu Tyr Thr Pro Leu Arg Ser Asn Gly Ser Gly
180 185 190
Asp Asp Val Phe Thr Val Ser Cys Arg Val Leu Thr Arg Pro Thr Pro
195 200 205
Asp Phe Glu Phe Thr Tyr Leu Val Pro Pro Ser Val Glu Ser Lys Thr
210 215 220
Lys Pro Phe Ser Leu Pro Ile Leu Thr Leu Ser Glu Leu Thr Asn Ser
225 230 235 240
Arg Phe Pro Val Pro Ile Asp Ser Leu Phe Thr Ala Gln Asn Asn Val
245 250 255
Leu Gln Val Gln Cys Gln Asn Gly Arg Cys Thr Leu Asp Gly Glu Leu
260 265 270
Gln Gly Thr Thr Gln Leu Leu Pro Ser Gly Ile Cys Ala Phe Arg Gly
275 280 285
Arg Val Thr Ala Gln Ile Asn Gln Arg Asp Arg Trp His Met Gln Leu
290 295 300
Gln Asn Leu Asn Gly Thr Thr Tyr Asp Pro Thr Asp Asp Val Pro Ala
305 310 315 320
Pro Leu Gly Thr Pro Asp Phe Lys Gly Val Val Phe Gly Met Val Ser
325 330 335
Gln Arg Asn Val Gly Asn Asp Ala Pro Gly Ser Thr Arg Ala Gln Gln
340 345 350
Ala Trp Val Ser Thr Tyr Ser Pro Gln Phe Val Pro Lys Leu Gly Ser
355 360 365
Val Asn Leu Arg Ile Ser Asp Asn Asp Asp Phe Gln Phe Gln Pro Thr
370 375 380
Lys Phe Thr Pro Val Gly Val Asn Asp Asp Asp Asp Gly His Pro Phe
385 390 395 400
Arg Gln Trp Glu Leu Pro Asn Tyr Ser Gly Glu Leu Thr Leu Asn Met
405 410 415
Asn Leu Ala Pro Pro Val Ala Pro Asn Phe Pro Gly Glu Gln Leu Leu
420 425 430
Phe Phe Arg Ser Phe Val Pro Cys Ser Gly Gly Tyr Asn Gln Gly Ile
435 440 445
Ile Asp Cys Leu Ile Pro Gln Glu Trp Ile Gln His Phe Tyr Gln Glu
450 455 460
Ser Ala Pro Ser Gln Ser Asp Val Ala Leu Ile Arg Tyr Val Asn Pro
465 470 475 480
Asp Thr Gly Arg Thr Leu Phe Glu Ala Lys Leu His Arg Ser Gly Tyr
485 490 495
Ile Thr Val Ala His Ser Gly Asp Tyr Pro Leu Val Val Pro Ala Asn
500 505 510
Gly His Phe Arg Phe Asp Ser Trp Val Asn Gln Phe Tyr Ser Leu Ala
515 520 525
Pro Met Gly Thr Gly Asn Gly Arg Arg Arg Ala Gln
530 535 540

Claims (9)

1. An anti-GII.17 norovirus monoclonal antibody 2F11-Ab, wherein the monoclonal antibody is represented by a sequence with a collection number of CCTCC NO: c2021171, hybridoma cell line 2F11.
2. A hybridoma cell strain 2F11 with a preservation number of CCTCC NO: C2021171.
3. a device for detecting the presence and/or level of a type gii.17 norovirus in a sample, the device comprising an anti-type gii.17 norovirus monoclonal antibody 2F11-Ab of claim 1;
preferably, the sample is a multivalent norovirus vaccine.
4. The device of claim 3, wherein the device is a device for use in an immunization method;
preferably, the immunological method is selected from one or more of immunoblotting, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay, and immunochemiluminescence; more preferably, the detection is by enzyme-linked immunosorbent assay or immunochemiluminometric assay.
5. The device of claim 3 or 4, wherein the device is a kit;
preferably, the kit further comprises a rabbit anti-gii.17 type norovirus polyclonal antibody and/or a goat anti-murine species antibody;
more preferably, the kit further comprises one or more of a coating solution, a washing solution, a blocking solution, a diluting solution, a stop solution and a standard;
further preferably, the kit further comprises an enzyme label plate and/or a plate sealing membrane.
6. Use of the anti-GII.17 norovirus monoclonal antibody 2F11-Ab of claim 1 in the preparation of a reagent or kit for detecting GII.17 norovirus in a sample.
7. The use of claim 6, wherein the sample is a multivalent norovirus vaccine.
8. A pharmaceutical composition for preventing and/or treating gii.17 type norovirus infection, comprising the anti-gii.17 type norovirus monoclonal antibody 2F11-Ab of claim 1;
preferably, the pharmaceutical composition is a vaccine.
9. Use of the anti-GII.17 norovirus monoclonal antibody 2F11-Ab of claim 1 for the preparation of a pharmaceutical composition for the prevention and/or treatment of GII.17 norovirus infection;
preferably, the pharmaceutical composition is a vaccine.
CN202111204371.8A 2021-10-15 2021-10-15 anti-GII.17 type norovirus monoclonal antibody and application thereof Pending CN115975017A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111204371.8A CN115975017A (en) 2021-10-15 2021-10-15 anti-GII.17 type norovirus monoclonal antibody and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111204371.8A CN115975017A (en) 2021-10-15 2021-10-15 anti-GII.17 type norovirus monoclonal antibody and application thereof

Publications (1)

Publication Number Publication Date
CN115975017A true CN115975017A (en) 2023-04-18

Family

ID=85960680

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111204371.8A Pending CN115975017A (en) 2021-10-15 2021-10-15 anti-GII.17 type norovirus monoclonal antibody and application thereof

Country Status (1)

Country Link
CN (1) CN115975017A (en)

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