CN114736292B

CN114736292B - Nano antibody targeting norovirus protein and application thereof

Info

Publication number: CN114736292B
Application number: CN202210651337.3A
Authority: CN
Inventors: 叶其壮; 王梓杨; 白晓康; 陈锦德; 肖利群
Original assignee: Guangzhou Ming Pharmaceutical Technology Co ltd
Current assignee: Guangzhou Ming Pharmaceutical Technology Co ltd
Priority date: 2022-06-10
Filing date: 2022-06-10
Publication date: 2023-03-07
Anticipated expiration: 2042-06-10
Also published as: CN114736292A

Abstract

Norovirus is a virus that causes acute gastroenteritis. The invention discloses two nano antibodies targeting norovirus capsid proteins, and expression, purification and identification thereof. The nano-antibody targeting norovirus can be used for environmental monitoring of norovirus and clinical diagnosis and treatment of norovirus infection.

Description

Nano antibody targeting norovirus protein and application thereof

Technical Field

The invention relates to the field of antibody technology and biomedicine, in particular to monitoring norovirus transmission and diagnosing and treating norovirus infection diseases.

Background

Norovirus (Norovirus) was discovered by Kapikian in 1972 when feces from acute gastroenteritis patients who developed in Norwalk city (Norwalk) in the united states in 1968 were examined by using immunoelectron microscopy. Today, norovirus has become a major cause of non-bacterial acute diarrhea, and 60% -90% of all outbreaks of non-bacterial diarrhea in the united states are caused by norovirus every year. In China, the detection rate of norovirus in children with diarrhea under 5 years old is about 15%. However, no specific drug effective for treating norovirus infection is available on the market to date. Therefore, the research on the rapid and accurate norovirus early detection method has profound significance for preventing and treating the norovirus.

Antibodies are a key raw material for immunoassays, which rely on specific binding of an antigen to an antibody. The monoclonal antibody for immunodetection is mainly prepared by taking an experimental mouse as a host, infecting the mouse with norovirus, taking the spleen of the mouse which is successfully immunized under the aseptic condition, fusing the spleen with myeloma cells, and screening out positive clones by an ELISA method and the like. Cloning the screened cells to form stable cell strain, culturing the cell strain in vitro or inducing ascites in mouse, purifying from culture medium or ascites to obtain norovirus resisting monoclonal antibody, and final protein blotting and other steps.

The norovirus specific antibody prepared by the means is a traditional monoclonal antibody, and the monoclonal antibody has the following defects: long production period, high preparation cost, high immunogenicity, difficult removal in the body and weak tissue penetration capacity. The nano antibody is a fragment of an alpaca animal antibody, is a simplification of a conventional antibody, reserves a part of the antibody combined with an antigen, reserves the affinity and specificity of the conventional antibody to the antigen, has the potential of partially replacing the conventional antibody, and particularly belongs to the field of diagnostic reagents. Three nanobodies against norovirus have been described (Ruoff, k., et al, J Virol 93. The nano antibody is a novel antibody material and is still in a development stage, nano antibody products are rarely available on the market at present, and the nano antibody products targeting norovirus are more vacant.

Norovirus is a single-stranded RNA non-enveloped virus belonging to the family caliciviridae. Norovirus proteins include structural proteins (VP 1 and VP 2) and non-structural proteins associated with viral replication. Phylogenetic trees are currently constructed separately based on the complete amino acid diversity of the VP1 gene and the nucleotide diversity of the region encoding RNA-dependent RNA polymerase (RdRp) on ORF1, and norovirus can be divided into different genomes (Genogroup, G) and polymerase groups (P), and further into multiple genotypes and P-types. Among them, GI, GII, GIV, GVIII and GIX infect humans, and GI and GII are two major genomes causing infectious diarrhea in humans. Therefore, the development of diagnostic reagents for identifying norovirus needs to take into account the diversity of norovirus, the diversity of epitopes bound by norovirus and antibodies, and the diversity of conventional antibodies and nanobodies targeting norovirus, so as to meet the requirements of norovirus-related diagnosis and treatment.

Disclosure of Invention

The invention aims to provide two nano antibodies targeting norovirus capsid proteins, which have high affinity for norovirus capsid protein antigen, can be used for separation, detection and treatment of norovirus, particularly research and development of detection kits, and have good application prospects.

Generally, the amino acid sequence of a nanobody consists of about 100-130 amino acid residues, with four framework regions (FM 1-4) and three complementarity determining regions (CDR 1-CDR 3), staggered in order. The invention discloses two norovirus-targeting nanobodies Nor2 and Nor3, which have the same four framework regions and different three complementarity determining region amino acid sequences. The complementary determining regions of the nano antibody Nor2 respectively correspond to CDR1 of SEQ ID NO. 1, CDR2 of SEQ ID NO. 2 and CDR3 of SEQ ID NO. 3. Similarly, the complementary determining regions of the nano antibody Nor3 are respectively composed of SEQ ID NO. 4 corresponding to CDR1, SEQ ID NO. 5 corresponding to CDR2 and SEQ ID NO. 6 corresponding to CDR3.

The two nano antibodies Nor2 and Nor3 targeting norovirus proteins disclosed by the invention have decisive effects on the recognition of antigens by three complementarity determining regions, and the four framework regions have influences on the spatial structure of the complementarity determining regions so as to influence the recognition of the antigens. Thus, the complete amino acid sequences of the nanobodies Nor2 and Nor3 are also important bases for determining antigen recognition. The complete amino acid sequence of Nor2 is shown as SEQ ID NO. 7, and the complete amino acid sequence of Nor3 is shown as SEQ ID NO. 8.

The two nano antibodies targeting the norovirus protein disclosed by the invention both contain 122 amino acid residues, so that the core structure of the nano antibody is formed. The core structure of the nanobody has high affinity and specificity with the antigen protein without adding amino acid residues. In fact, the nanobody often exists in the form of a fusion protein, i.e., polypeptide and protein are added to the N-terminal or C-terminal of the core structure of the nanobody to form the fusion protein, which endows the fusion protein with more characteristics and functions while maintaining the high affinity and high specificity of the original nanobody. For example, in the panning and identification of the nanobody of the present disclosure, the C-terminal end of the nanobody is fused with a polypeptide as a protein tag (e.g., histidine tag, human influenza hemagglutinin tag), so that it can be purified using a nickel column and identified using an anti-human influenza hemagglutinin antibody. The complete nucleic acid sequence of the fusion protein formed by the coding nano antibody Nor2, the histidine tag and the human influenza hemagglutinin tag is shown in SEQ ID NO. 9, and the complete nucleic acid sequence of the fusion protein formed by the coding nano antibody Nor3, the histidine tag and the human influenza hemagglutinin tag is shown in SEQ ID NO. 10.

The modification of the nano antibody at the protein gene level is an obvious advantage of the nano antibody compared with the conventional antibody, has a mature molecular biology method, and the fusion protein is also a form embodied by the common nano antibody. The polypeptide and protein capable of being fused with the nano-antibody are various, and comprise various protein tags (histidine tag His, human influenza virus hemagglutinin tag HA, FLAG tag, MBP tag, myc tag and the like), green fluorescent protein, alkaline phosphatase, luminescent enzyme, glutathione transferase, toxin protein, antibody Fc fragment and the like. The fusion protein enables the nano antibody with the function of targeting antigen to have more functions, such as fusion and color development with fluorescent protein for tracing, fusion with cytotoxic protein to form immunotoxins (immunotoxins) for treatment and the like.

Furthermore, the nano antibody or the fusion protein constructed by the nano antibody is chemically modified, so that more characteristics and functions can be endowed to the modified nano antibody protein. For example, as described in the examples, the nanobody disclosed in the present invention is biotinylated during the identification process, so that it can bind to avidin or streptavidin, and thus is more convenient for quantification.

The two nano antibodies of the targeted norovirus protein disclosed by the invention have only 122 amino acid residues, have definite structures, and have definite positions of modifiable groups on the surface of the antibody protein, so that the nano antibody or the fusion protein constructed by the nano antibody disclosed by the invention can be chemically modified, and the commonly used modified sites are amino groups modified on lysine residues, carboxyl groups of glutamic acid and aspartic acid, and the like. In order to make modification more convenient and make modification positioning and quantification more accurate, a specific chemical modification group can also be introduced into the N-terminal, C-terminal or other specific part of the nanobody, and then the nanobody is chemically modified, for example, cysteine is introduced to obtain a thiol group of a specific modifiable site. Has various protein modification reactions with amino, carboxyl and sulfhydryl groups, and has mature technology. Such as reaction with Fluorescein Isothiocyanate (FITC) or activated biotin, on the amino group of the nano-antibody, so as to obtain the fluorescent or biotinylated nano-antibody. The material capable of chemically modifying the nano antibody provided by the invention is various and comprises affinity chromatography filler, quantum dots, magnetic beads, colored microspheres or fluorescent microspheres, protein (such as horseradish peroxidase), chemical small molecules and the like. The requirement for the chemically modified material is to have a reactive or activatable group such as carboxyl, amino, thiol, etc. that can be used for chemical modification.

The application scenes of the specific nano antibody are many. An example is that the nano antibodies Nor2 and Nor3 are combined with magnetic beads and used for concentrating and separating norovirus. Many commercially available magnetic beads have a modifiable group such as an amino group or a carboxyl group on the surface thereof, and since a nano antibody also has a modifiable group on the surface thereof, the nano antibody can be covalently linked to the magnetic bead by a mature protein coupling method (such as a diazo method, a glutaraldehyde method, a glutaric anhydride method, a carbodiimide method, etc.). In the solution, the nano antibody can recognize and bind to the norovirus, and the conjugate of the nano antibody and the magnetic beads can capture, enrich and separate the norovirus by using a magnetic field. The second example is to use the nano antibody Nor2 or Nor3 to construct a quantitative immunoassay kit. When preparing an enzyme-linked immunosorbent assay (ELISA) kit, coating an ELISA plate with norovirus antigen protein, adding a solution to be detected containing norovirus, and then adding biotinylated nano antibody protein. The antigen on the norovirus in the solution and the norovirus antigen coated on the enzyme label plate are combined with the biotinylated nano antibody protein competitively. After washing, the amount of the remaining nano antibody capable of being combined with the norovirus antigen coated on the ELISA plate reflects the norovirus content in the solution to be detected. The amount of the nanobody can be quantified by using avidin-horseradish peroxidase (HRP) coupled protein.

The nano antibody disclosed by the invention is subjected to protein fusion and chemical modification, and is endowed with additional functions such as color, fluorescence, magnetism, biological activity and the like on the basis of specific identification of the nano antibody on norovirus, so that the nano antibody has wide application prospects in aspects of virus separation and concentration, virus detection, virus removal and the like.

Drawings

FIG. 1 shows the protein electrophoresis results of the nano antibodies Nor2 and Nor 3.

FIG. 2 shows the result of the affinity analysis of the nano antibodies Nor2 and Nor3 for the viral protein antigen.

FIG. 3 shows the result of affinity analysis of the Nor2 nanobody on viral protein antigen in the presence or absence of Nor 3.

FIG. 4 shows the result of affinity analysis of the nano antibody Nor3 for viral protein antigen in the presence or absence of Nor 2.

FIG. 5 shows the amino acid full-length sequences and CDR regions of nanobodies Nor2 and Nor 3. The CDR1, CDR2 and CDR3 regions are underlined, respectively.

Detailed Description

In the research of the invention, the library capacity and diversity of the nano antibody library are the key for panning out the high-specificity and high-affinity nano antibody. The final library size of the synthetic libraries used in the present invention reaches 8X 10 ⁹ pfu, ensuring the diversity of the library. The antigen is the capsid protein VP1 of norovirus, and the cloning and expression of the antigenic protein can be found in the literature (Leuthold, M.M., et al. J. Vis. Exp. (110), e 53845). The invention adopts a solid phase panning mode, during panning, a polystyrene plastic flat plate is coated with a capsid protein antigen of norovirus, and biological panning is carried out by using a phage display nano antibody library. The enrichment of phage particles is influenced by factors such as the purity of antigen, the coating concentration of antigen, the type and concentration of blocking solution, the concentration of PBST, the input amount of phage, the binding time and the elution time. The screening process of the invention adopts modes of non-sealing and 5% milk alternate sealing, gradually increasing washing strength and the like to improve the specificity of biological elutriation, and obtains the nano antibody with high specificity and high affinity by optimizing elutriation conditions.

EXAMPLE I panning of phage display libraries

Affinity panning is carried out on the phage display library according to the modes of combination, washing, elution and amplification, norovirus capsid protein VP1 antigen is adopted to be coated to serve as the antigen of targeted norovirus nanobody for panning, and nonspecific reduction is carried out by a method of non-blocking and 5% milk alternating blockingAnd (4) adsorbing. The specific steps of panning are as follows. mu.L of virus protein antigen with a concentration of 10. Mu.g/mL (1. Mu.g/well) was added to each well of the eight-well enzyme standard strip and coated at 37 ℃ for 2 hours. The supernatant was discarded and the enzyme strips were washed 5 times with 0.05% PBST. For blocking, 200. Mu.L of 5% milk was added to each well and blocked at 37 ℃ for 2 hours. The blocking solution was discarded and the enzyme label strip was washed 5 times with 0.05% PBST. Add 100. Mu.L phage library suspension per well (8X 10 library capacity) ⁹ pfu, titer 1X 10 ⁹ cfu/mL), incubated for 1 hour at room temperature with shaking on a shaker. The supernatant was discarded and the enzyme strips were washed 5 times with 0.05% PBST. mu.L of 0.2M Gly-HCl elution buffer (pH 2.2) was added to each well for elution, and the eluate was collected into a 1.5 mL centrifuge tube after elution by standing at room temperature for 10 minutes. Add 120. Mu.L of 1M Tris-HCl buffer (pH 9.0) for neutralization, vortex and mix well, take 20. Mu.L to preserve as the assay titer sample, and the remaining samples are all amplified for the next round of panning.

Example two, identification of Positive clones

Single colonies were randomly picked from titer-determining plates for phage rescue purification, followed by identification of positive clones by ELISA. The method comprises the following specific steps. mu.L of virus protein antigen with a concentration of 10. Mu.g/mL (1. Mu.g/well) was added to each well of the eight-linked-well enzyme standard strip and coated at 37 ℃ for 2 hours. The supernatant was discarded and the enzyme label was washed 5 times with PBST. 200 μ L of 5% milk was added to each well and one more strip was used as a negative control and blocked at 37 ℃ for 2 hours. The PBST wash enzyme bars were 5 times. Add 100. Mu.L of amplified monoclonal phage suspension to each well and incubate for 1 h with shaking at room temperature. PBST wash plate 5 times. mu.L of mouse anti-human influenza Hemagglutinin (HA) primary antibody is added into each well, and incubated for 1 hour in a shaking table at room temperature. PBST wash plate 5 times. mu.L of goat anti-mouse secondary antibody was added to each well, and incubated for 45 minutes in a shaker at room temperature. The plates were washed 5 times with PBST and the liquid in the enzyme label strip was patted as dry as possible on paper. 100. Mu.L of TMB developing solution was added to each well, and color was developed to a suitable depth. The reaction was stopped by adding 100. Mu.L of 1M HCl solution to each well, and the OD was immediately read with a microplate reader ₄₅₀ Numerical values. And sequencing the positive sample to determine the amino acid sequence of the nano antibody. Nor2 and Nor3 are two positive plants obtained from panningSex cloning.

EXAMPLE III fusion of Nanobodies Nor2 and Nor3 with histidine tag and human influenza Virus hemagglutinin tag

In the phage display library, the nanobody is fused with capsid protein P3 of M13 phage and displayed on the surface of phage particle. The base sequences of the obtained nano antibodies Nor2 and Nor3 are cut into gene fragments by using restriction enzymes NdeI and XhoI, the gene fragments are inserted into pET23a plasmid, and a histidine tag and a human influenza virus hemagglutinin tag are introduced into the C terminal of the nano antibodies, so that the recombinant proteins can be conveniently separated, purified and identified by using a nickel column. The C-terminal amino acid sequence of the fusion protein of the nano antibodies Nor2 and Nor3 is as follows: gqagqhhhhhhgaypydvpdyalehhhhhhhh.

Example four expression and purification of Nanobodies Nor2 and Nor3 in E.coli

mu.L of the constructed pET23a plasmid containing the target gene of the nano antibody is added into 50 mu.L of escherichia coli BL21 (DE 3) competent cells, and after the mixture is placed on ice for 30 minutes, the mixture is placed into a 42 ℃ water bath for heat shock for 60 seconds. After 5 minutes of standing on ice, 450. Mu.L of SOC culture medium was added to the culture broth. After mixing, placing the mixture in a shaking table at the temperature of 37 ℃ and shaking at the speed of 200 rpm for culture and recovery for 1 hour. Then, 200. Mu.L of the bacterial solution was pipetted and spread on an LB + Amp solid plate, the liquid was dried by blowing, and the plate was placed upside down in a 37 ℃ incubator and cultured overnight. On the next day, a single clone was picked from the overnight-cultured plate, placed in 5 mL LB + Amp culture solution, shake-cultured at 37 ℃ with 200 rpm shaking for 8 hours, and then the whole culture solution was transferred to 330 mL TB + phosphate + Amp culture solution, and cultured at 30 ℃ with 200 rpm shaking overnight. On the third day, the suspension was collected by centrifugation, resuspended in 1 XPhosphate buffer (20 mM, pH 7.4) and then sonicated. The conditions were 150 w of 75%, i.e., 112.5 w, and the crushing was carried out for 5 seconds with a 20-second pause. Then, after centrifugation at 20,000 rpm for 20 minutes at 4 ℃, the ultrasonic supernatant and the ultrasonic precipitate were collected respectively, and the ultrasonic supernatant was stored at-20 ℃. The protein with higher concentration and purity can be obtained by performing denaturation and renaturation on the ultrasonic precipitation. Resuspend the sonicated samples with 10 mL of 2M urea solution and add 2M urea solution to 40 mL to wash the inclusion bodies. Centrifuge at 8,000 rpm for 10 minutes at 4 ℃. The washed pellet was resuspended in 10 mL of 8M urea solution, and the inclusion bodies were solubilized by adding 8M urea solution to 35 mL, and then shaken at 80 rpm in a shaker at room temperature for hours. They were then centrifuged at 10,000 rpm for 20 minutes at 4 ℃. The supernatant (containing the denatured protein of interest) is collected and the pellet is retained. Renaturation is carried out by dialysis after protein denaturation, dialysis is carried out by reducing the urea concentration in a step mode, 15 mL of 8M urea denaturation supernatant (target protein sample) is taken and added into 15 mL of 1 XPBS rapidly, and the concentration of the 8M urea solution is reduced to 4M. Dialysis was performed with 600 mL of 2M urea solution (stirring the dialysate in a refrigerator at 4 ℃) for 1 hour. 200 mL of the dialysate was decanted, 200 mL of 1M urea solution was added, and then dialyzed overnight. The next day, 200 mL of the dialysate was decanted, 200 mL of PBS was added, and dialysis was continued for 1 hour. 200 mL of the dialysate was decanted, 200 mL of PBS was added, and dialysis was continued for 1 hour. 200 mL of the dialysate was decanted, 200 mL of PBS was added, and dialysis was continued for 1 hour. After dialysis, the sample was collected, and the renatured protein solution was centrifuged at 10000 rpm at 4 ℃ for 25 minutes. Collecting the supernatant, wherein the collected supernatant is the renatured protein solution. The results of the denatured and renatured nano antibodies after protein electrophoresis are shown in figure 1, the results show that the relative molecular mass of the nano antibodies Nor2 and Nor3 with labels at the C terminal is 15-20 kDa, the molecular mass is consistent with the theoretical molecular mass, and the purified bands have no obvious impurity bands, which shows that the purity of the two nano antibodies is high.

EXAMPLE V biotinylation modification of Nanobodies Nor2 and Nor3

The NHS-Biotin reagent was accurately weighed, dissolved in 10 mM solution using DMSO, and a sample of purified Nor2 or Nor3 protein was taken out, diluted to a concentration of 1 mg/mL using 1 XPBS (20 mM, pH 7.4), added at a molar ratio of 8. The sample is desalted and purified after being biotinylated, a Sephadex G-25 Resin column is used for purification, 1 XPBS (20 mM, pH 7.4) is used for balancing the purification column during purification, a biotinylated protein sample is added after balancing, coomassie brilliant blue R-250 is used for detecting effluent liquid, the effluent liquid is collected immediately when the effluent liquid is changed into blue when meeting Coomassie brilliant blue, the purification column is washed by PBS after collection, and finally 20% ethanol is added for storing the purification column. The purified and collected sample is the prepared biotinylated nano antibody protein sample which is named as Nor2-biotin and Nor3-biotin respectively.

EXAMPLE sixthly, detection of the affinity of Nanobodies Nor2 and Nor3 for viral protein antigens

The EC50 of the nanobodies Nor2 and Nor3 against viral protein antigens was determined by ELISA assay and plotted. 100. Mu.L of virus protein antigen at a concentration of 10. Mu.g/mL was added to each well of the eight-well enzyme-labeled strip and coated at 37 ℃ for 2 hours. The supernatant was discarded and the enzyme strips were washed 5 times with 0.05% PBST. 100 mu L of nano antibody sample (containing 0.05% Tween-20) with the concentration of 10 mu g/mL is added into each well, the sample is diluted by 2 times of gradient for 16 points, and the mixture is incubated for 1 hour in a shaking table at room temperature. The supernatant was discarded, the enzyme strips were washed 5 times with 0.05% PBST, 100 μ L of murine primary anti-histidine tag antibody diluted 1. The supernatant was discarded and the enzyme strips were washed 5 times with 0.05% PBST. And adding 100 mu L of goat anti-mouse secondary antibody with the dilution of 1. The supernatant was discarded, the enzyme strips were washed 5 times with 0.05% PBST, and the strips were patted dry as much as possible on absorbent paper. Then, 100. Mu.L of TMB developing solution was added to each well, and the developing time was determined according to the development condition, and it was generally 5 to 10 minutes. After reaching the proper color development depth, 100 mu L of 1M HCl is added into each hole to terminate the reaction, and OD is read by a microplate reader immediately after the reaction is terminated ₄₅₀ Finally, data analysis and plotting are performed by Microsoft excel. The ELISA results are shown in FIG. 2, the EC50 values of the nano antibodies Nor2 and Nor3 are 167 ng/mL and 79 ng/mL respectively, and the affinity performance is better.

EXAMPLE seventhly, competitive assay of Nanobodies Nor2 and Nor3

The specificity of the nanobody binding to the viral protein antigen was determined by competition ELISA experiments. 100. Mu.L of virus protein antigen at a concentration of 10. Mu.g/mL was added to each well of the eight-well enzyme-labeled strip and coated at 37 ℃ for 2 hours. The supernatant was discarded and the enzyme strips were washed 5 times with 0.05% PBST. 100 μ L of a medium in a concentration of 10 μ g/well are added separatelymL of the nano antibody Nor2 or Nor3, then adding 100 mu L of Nor3-biotin and Nor2-biotin (i.e. biotinylated Nor2 and Nor3 antibodies) samples with the concentration of 10 mu g/mL into each well, diluting the samples for 16 points in a 2-fold gradient, and incubating for 1 hour in a shaking table at room temperature. The supernatant was discarded and the enzyme strips were washed 5 times with 0.05% PBST. mu.L of streptavidin-HRP antibody with a dilution of 1 10000 was added to each well and incubated for 1 hour with shaking at room temperature in a shaker. The supernatant was discarded, the enzyme strips were washed 5 times with 0.05% PBST, and the strips were patted dry as much as possible on absorbent paper. Then 100. Mu.L of TMB developing solution is added into each well, and the developing time is controlled according to the developing condition, and is generally 5-10 min. After reaching the proper color development depth, 100 mu L of 1M HCl is added into each hole to terminate the reaction, and OD is read by a microplate reader immediately after the reaction is terminated ₄₅₀ Finally, data analysis and plotting are performed by Microsoft excel. The ELISA results are shown in FIG. 3, after the Nor2 antibody is added, the Nor2 antibody is combined with the viral protein antigen before the viral protein antigen is combined, the subsequent combination curve of the Nor3 antibody and the viral protein antigen is obviously shifted to the right, and the EC50 is increased; similarly, after addition of the Nor3 antibody, which precedes binding of the viral protein antigen, the subsequent binding curve of the Nor2 antibody to the viral protein antigen shifts significantly to the right and the EC50 increases. The results show that the nano antibodies Nor2 and Nor3 specifically bind to the same epitope of the viral protein antigen.

The above description is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are also within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Sequence listing

<110> Guangzhouming medical technology Co., ltd

<120> norovirus protein-targeted nano antibody and application thereof

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Claims

1. The nano antibody of the targeted norovirus protein is characterized in that the complementarity determining regions CDR1, CDR2 and CDR3 of the amino acid sequence of the nano antibody are respectively shown by SEQ ID NO 1-3; or shown by SEQ ID NO 4-6 respectively.

2. The nano antibody targeting the norovirus protein is characterized in that the amino acid sequence of the nano antibody is shown as SEQ ID NO. 7 or 8.

3. The fusion protein of the nano antibody constructed by the fusion of the nano antibody, polypeptide and protein is characterized by comprising the following components in percentage by weight: (ii) (a) the nanobody of claim 1 or 2; and (b) a fusion moiety of a polypeptide and a protein selected from the group consisting of: protein tags, alkaline phosphatase, glutathione transferase, toxin proteins, and antibody Fc fragments.

4. The nanobody fusion protein of claim 3, wherein the fusion portion of the fusion protein is a histidine protein tag and a human influenza hemagglutinin protein tag.

5. A nucleic acid sequence encoding a nanobody fusion protein, wherein (a) comprises the nanobody fusion protein of claim 4, (b) lacks a signal peptide, resulting in expression of the fusion protein within the cytoplasm, and (c) the nucleic acid sequence is set forth in SEQ ID No. 9 or 10, respectively.

6. An expression vector comprising the nucleic acid sequence of claim 5; the host cell of the expression vector is Escherichia coli.

7. A derivative produced by chemically modifying a nanobody or a nanobody fusion protein, which comprises: (a) The nanobody of claim 1 or2, or the nanobody fusion protein of claim 3, and (b) a derivative portion of a modified nanobody selected from the group consisting of: affinity chromatography filler, quantum dots, magnetic beads, colored microspheres or fluorescent microspheres and chemical small molecules; the chemical small molecule comprises biotin; the nanobody is coupled to the derivatizing moiety via a covalent bond.

8. Use of the nanobody of claim 1 or2, the nanobody fusion protein of claim 3, or the nanobody derivative of claim 7, for the preparation of (a) an isolation and concentration medium, (b) detection and diagnostic reagents, and (c) a therapeutic antibody for norovirus.