Nanometer antibody targeting G4 genotype rotavirus and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a G4 genotype rotavirus targeting nano antibody and application thereof. In order to solve the problem of rotavirus detection, a complementary determining region CDR of the nano antibody comprises a CDR1 sequence, a CDR2 sequence and a CDR3 sequence: (1) the amino acid sequence of the CDR1 is shown as SEQ ID NO.11, and (2) the amino acid sequence of the CDR2 is shown as SEQ ID NO.21; and (3) the amino acid sequence of the CDR3 is as shown in SEQ ID NO. 31. The nano antibody disclosed by the invention has very high affinity and activity, can specifically recognize and combine with the G4 genotype rotavirus, and can be used for quantitative detection of the G4 genotype rotavirus.

Description

Technical Field

[0001] This invention belongs to the fields of nanobody technology and biomedicine, and relates to nanobodies targeting G4 genotype rotavirus and their applications. Background Technology

[0002] Rotavirus (RV) is a double-stranded, non-enveloped RNA virus. Its structure resembles a wheel with short spokes and a smooth outer edge, with a diameter of 70-100 nm. It consists of 11 dsRNA segments and has a complex structure of three concentric capsids. The RNA segments encode six structural viral proteins (VP1, VP2, VP3, VP4, VP6, and VP7) and six non-structural proteins (NSP1, NSP2, NSP3, NSP4, NSP5, and NSP6). VP4 and VP7 are capsid proteins that dominate the viral adsorption and cell invasion processes. Both VP4 and VP7 proteins can independently induce the rotavirus host to produce neutralizing antibodies. Based on VP4, rotaviruses are classified into different P genotypes; based on VP7, they are classified into different G genotypes. VP6 is the middle layer of the three concentric capsids, with a molecular weight of approximately 45 kDa. It accounts for 51% of the total protein content in rotavirus and is highly conserved and immunogenic. Based on the antigenic differences of the VP6 protein, rotaviruses can be divided into 7 groups, A to G. Among them, group A rotaviruses are the main pathogens causing acute gastroenteritis in infants and young children.

[0003] The proteins in mature viral particles determine host specificity, the enzyme functions necessary for cell entry and the production of viral transcripts, and contain epitopes that generate immune responses. They are a major cause of acute gastroenteritis and diarrhea-related deaths in infants and young children worldwide.

[0004] However, as of now, there is no effective drug available to treat rotavirus infection. Therefore, researching a rapid and accurate method for early detection of rotavirus is of profound significance for the prevention and control of this virus.

[0005] In the treatment of rotavirus infection, enzyme-linked immunosorbent assay (ELISA) is often used clinically as an important method for detecting rotavirus antigens in feces. Various rotavirus type-specific antibodies can be used as coatings for antigen capture, and a double-antibody sandwich ELISA method is employed for detection. However, traditional monoclonal antibodies have the following drawbacks: long development and production cycles, high preparation costs, high immunogenicity, difficulty in being cleared in vivo, and weak penetration ability. Nanobodies derived from animals such as alpacas retain the affinity and specificity of traditional monoclonal antibodies and also have the following advantages: high expression levels in prokaryotic or eukaryotic cells, short prokaryotic cell growth cycle, low preparation costs, weak immunogenicity in humans, small molecular weight, strong penetration ability (even penetrating the blood-brain barrier), high specificity, and high affinity. However, the preparation of anti-rotavirus nanobodies has not yet been reported, therefore, more effective methods need to be sought.

[0006] In summary, nanobodies have great potential in the fields of detection, diagnosis and drug development. Nanobodies targeting rotavirus obtained through screening will complement and replace traditional monoclonal antibodies, and will have broad application prospects in the biomedical field. Summary of the Invention

[0007] To address the aforementioned problems in rotavirus (RV) detection, in a first aspect, according to a G4 genotype-targeting nanobody in some embodiments of this application, the complementarity-determining region (CDR) of the nanobody includes CDR1, CDR2, and CDR3 sequences:

[0008] (I): (1) The amino acid sequence of CDR1 is shown in SEQ ID NO.11; (2) The amino acid sequence of CDR2 is shown in SEQ ID NO.21; and (3) The amino acid sequence of CDR3 is shown in SEQ ID NO.31; or (II): Amino acid sequences obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequences of (1), (2) and (3) of (I), and having the same function as the amino acid sequence of (I).

[0009] According to some embodiments of the present application, the nanobody targeting G4 genotype rotavirus, wherein: (II) is an amino acid sequence obtained by substituting one or more amino acids of the amino acid sequence described in (I) (1), (2), (3), including (II-1): the amino acid sequence of CDR1 is as shown in SEQ ID NO.12, the amino acid sequence of CDR2 is as shown in SEQ ID NO.22, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.32; or (II-2): the amino acid sequence of CDR1 is as shown in SEQ ID NO.13, the amino acid sequence of CDR2 is as shown in SEQ ID NO.23, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.33; or (II-3): the amino acid sequence of CDR1 is as shown in SEQ ID NO.14, the amino acid sequence of CDR2 is as shown in SEQ ID NO.24, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.34; or (II-4): the amino acid sequence of CDR1 is as shown in SEQ ID NO.15, the amino acid sequence of CDR2 is as shown in SEQ ID NO.15, and the amino acid sequence of CDR2 is as shown in SEQ ID NO.12. As shown in NO.25, the amino acid sequence of CDR3 is as shown in SEQ ID NO.35; or (II-5): the amino acid sequence of CDR1 is as shown in SEQ ID NO.16, the amino acid sequence of CDR2 is as shown in SEQ ID NO.26, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.36; or (II-6): the amino acid sequence of CDR1 is as shown in SEQ ID NO.17, the amino acid sequence of CDR2 is as shown in SEQ ID NO.27, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.37; or (II-7): the amino acid sequence of CDR1 is as shown in SEQ ID NO.18, the amino acid sequence of CDR2 is as shown in SEQ ID NO.28, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.38; or (II-8): the amino acid sequence of CDR1 is as shown in SEQ ID NO.19, the amino acid sequence of CDR2 is as shown in SEQ ID NO.29, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.39; or (II-9): the amino acid sequence of CDR1 is as shown in SEQ ID NO. As shown in NO.20, the amino acid sequence of CDR2 is shown in SEQ ID NO.30, and the amino acid sequence of CDR3 is shown in SEQ ID NO.40.

[0010] According to some embodiments of the present application, the nanobody targeting G4 genotype rotavirus includes: (III) the framework region FR of the nanobody includes FR1, FR2, FR3 and FR4 sequences, wherein: (1) the amino acid sequence of FR1 is as shown in SEQ ID NO. 41, (2) the amino acid sequence of FR2 is as shown in SEQ ID NO. 50, (3) the amino acid sequence of FR3 is as shown in SEQ ID NO. 58, and (4) the amino acid sequence of FR4 is as shown in SEQ ID NO. 68; or (IV) an amino acid sequence having more than 50% homology with the amino acid sequences described in (1), (2), (3) and (4) of (III).

[0011] According to the G4 genotype-targeting nanobodies in some embodiments of this application, (IV) has amino acid sequences with more than 50% homology as described in (1), (2), (3), and (4) of (III), including (IV-1): the amino acid sequence of FR1 as shown in SEQ ID NO.42, the amino acid sequence of FR2 as shown in SEQ ID NO.51, the amino acid sequence of FR3 as shown in SEQ ID NO.59, and the amino acid sequence of FR4 as shown in SEQ ID NO.69; or (IV-2): the amino acid sequence of FR1 as shown in SEQ ID NO.43, the amino acid sequence of FR2 as shown in SEQ ID NO.52, the amino acid sequence of FR3 as shown in SEQ ID NO.60, and the amino acid sequence of FR4 as shown in SEQ ID NO.70; or (IV-3): the amino acid sequence of FR1 as shown in SEQ ID NO.44, the amino acid sequence of FR2 as shown in SEQ ID NO.53, the amino acid sequence of FR3 as shown in SEQ ID NO.61, and the amino acid sequence of FR4 as shown in SEQ ID NO. As shown in NO. 70; or (IV-4): the amino acid sequence of FR1 is as shown in SEQ ID NO. 45, the amino acid sequence of FR2 is as shown in SEQ ID NO. 54, the amino acid sequence of FR3 is as shown in SEQ ID NO. 62, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 70; or (IV-5): the amino acid sequence of FR1 is as shown in SEQ ID NO. 46, the amino acid sequence of FR2 is as shown in SEQ ID NO. 52, the amino acid sequence of FR3 is as shown in SEQ ID NO. 63, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 70; or (IV-6): the amino acid sequence of FR1 is as shown in SEQ ID NO. 47, the amino acid sequence of FR2 is as shown in SEQ ID NO. 55, the amino acid sequence of FR3 is as shown in SEQ ID NO. 64, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 71; or (IV-7): the amino acid sequence of FR1 is as shown in SEQ ID NO. 48, the amino acid sequence of FR2 is as shown in SEQ ID NO. 56, and the amino acid sequence of FR3 is as shown in SEQ ID NO. 70. As shown in NO. 65, the amino acid sequence of FR4 is as shown in SEQ ID NO. 72; or (IV-8): the amino acid sequence of FR1 is as shown in SEQ ID NO. 49, the amino acid sequence of FR2 is as shown in SEQ ID NO. 57, the amino acid sequence of FR3 is as shown in SEQ ID NO. 66, and the amino acid sequence of FR4 is as shown in SEQ ID NO. 70; or (IV-9): the amino acid sequence of FR1 is as shown in SEQ ID NO. 49, and the amino acid sequence of FR2 is as shown in SEQ ID NO.As shown in Figure 52, the amino acid sequence of FR3 is shown in SEQ ID NO. 67, and the amino acid sequence of FR4 is shown in SEQ ID NO. 73.

[0012] According to some embodiments of the present application, the nanobody targeting G4 genotype rotavirus has (V) an amino acid sequence as shown in SEQ ID NO.1; or (VI) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids as the amino acid sequence of (V), and having the same function as the amino acid sequence of (I).

[0013] According to some embodiments of the present application, the nanobody targeting G4 genotype rotavirus, wherein the amino acid sequence of the nanobody of (VI) is as shown in any of SEQ ID NO.2 ~ SEQ ID NO.10.

[0014] In a second aspect, according to some embodiments of this application, a method for preparing nanobodies targeting rotavirus includes the method of screening a phage-displayed nanobody library using rotavirus as an antigen.

[0015] In a third-party context, according to the preparation method in some embodiments of this application, the whole rotavirus is a live attenuated rotavirus vaccine including at least one of G1 genotype rotavirus, G2 genotype rotavirus, G3 genotype rotavirus, G4 genotype rotavirus, G9 genotype rotavirus, and P1A8 genotype rotavirus.

[0016] In a fourth aspect, according to the methods in some embodiments of this application, the phage display nanobody library includes at least one of a fully synthetic library, a semi-synthetic library, an immune library, a non-immune library, or a natural library.

[0017] In a fifth aspect, the polypeptides in some embodiments of this application include any of the nanobodies described herein.

[0018] In a sixth aspect, the nucleic acid molecule encoding the nanobody according to any one of the embodiments of this application.

[0019] In a seventh aspect, the expression vector according to some embodiments of this application includes the nucleic acid molecule.

[0020] In an eighth aspect, the host cells of the expression vector described herein are transformed or transfected according to some embodiments of this application.

[0021] In a ninth aspect, the conjugates or conjugates according to some embodiments of this application include nanobodies described in any one of the chemically labeled or biologically labeled methods.

[0022] In a tenth aspect, the adsorbents according to some embodiments of this application include any of the nanobodies; or the polypeptides; or the nucleic acid molecules; or the expression vectors; or the host cells; or the conjugates; or the couplings, and the carriers.

[0023] In an eleventh aspect, the kit according to some embodiments of this application is characterized in that it comprises any of the nanobodies; or the polypeptides; or the nucleic acid molecules; or the expression vectors; or the host cells; or the conjugates; or the conjugates; or the adsorbents; and an adjuvant acceptable for detection.

[0024] In a twelfth aspect, the apparatus according to some embodiments of this application is used to capture, adsorb, and / or detect G4 genotype rotavirus, including any of the nanobodies described herein; or the polypeptides; or the nucleic acid molecules; or the expression vectors; or the host cells; or the conjugates; or the coupling agents; or the adsorbents; or the kits described herein.

[0025] In a thirteenth aspect, the use of the nanobody, or the polypeptide, or the nucleic acid molecule, or the expression vector, or the host cell, or the conjugate, or the coupling compound, or the adsorbent, or the kit described in any of the embodiments of this application in the preparation of a specific agent for capturing, adsorbing, and / or detecting G4 genotype rotavirus.

[0026] The use of the nanobody, or the polypeptide, or the nucleic acid molecule, or the expression vector, or the host cell, or the conjugate, or the coupling compound, or the adsorbent, or the kit described in any of the embodiments of this application in the preparation of enriched and / or purified cell preparations for specific capture, adsorption, and / or detection of G4 genotype rotavirus.

[0027] The beneficial effects of the present invention are as follows: The nanobody of the present invention is a nanobody with a novel amino acid sequence that targets G4 genotype rotavirus. This nanobody has high affinity and activity, and can specifically recognize and bind to G4 genotype rotavirus. It can be used for detection and purification of G4 genotype rotavirus. Attached Figure Description

[0028] Figure 1This is a plate plot showing the enrichment of the NiNb1 library (G4 genotype rotavirus and non-immune nanobody phage library, NiNb) in Example 1 of this invention. The results show that the enrichment reached 70-fold after three rounds of biological panning. In the figure, A represents the enrichment rate of the first round of screening; B represents the enrichment rate of the second round of screening; and C represents the enrichment rate of the third round of screening.

[0029] Figure 2 This is to verify the binding ability of G4 genotype rotavirus to positive clones screened from the NiNb1 library in Example 2 of this invention.

[0030] Figure 3 To verify the broad-spectrum activity of the nanobodies in Example 4 of this invention, A represents the verification of the binding ability of RVNb-1 nanobodies with rotaviruses of genotypes G1, G2, G3, G4, G9, and P1A8; B represents the verification of the binding ability of RVNb-2 nanobodies with rotaviruses of genotypes G1, G2, G3, G4, G9, and P1A8; C represents the verification of the binding ability of RVNb-3 nanobodies with rotaviruses of genotypes G1, G2, G3, G4, G9, and P1A8; D represents the verification of the binding ability of RVNb-4 nanobodies with rotaviruses of genotypes G1, G2, G3, G4, G9, and P1A8; and E represents the verification of the binding ability of RVNb-5 nanobodies with rotaviruses of genotypes G1, G2, G3, G4, G9, and P1A8. Rotavirus binding ability verification; F: Validation of the binding ability of RVNb-6 nanobody with rotavirus genotypes G1, G2, G3, G4, G9, and P1A8; G: Validation of the binding ability of RVNb-7 nanobody with rotavirus genotypes G1, G2, G3, G4, G9, and P1A8; H: Validation of the binding ability of RVNb-8 nanobody with rotavirus genotypes G1, G2, G3, G4, G9, and P1A8; I: Validation of the binding ability of RVNb-9 nanobody with rotavirus genotypes G1, G2, G3, G4, G9, and P1A8; J: Validation of the binding ability of RVNb-10 nanobody with rotavirus genotypes G1, G2, G3, G4, G9, and P1A8.

[0031] Figure 4The images show SDS-PAGE images of the 10 nanobodies in Example 5 of this invention after separation and purification by nickel affinity chromatography. In these images, A represents SDS-PAGE of RVNb-1 expression and purification; B represents SDS-PAGE of RVNb-2 expression and purification; C represents SDS-PAGE of RVNb-3 expression and purification; D represents SDS-PAGE of RVNb-4 expression and purification; E represents SDS-PAGE of RVNb-5 expression and purification; F represents SDS-PAGE of RVNb-6 expression and purification; G represents SDS-PAGE of RVNb-7 expression and purification; H represents SDS-PAGE of RVNb-8 expression and purification; I represents SDS-PAGE of RVNb-9 expression and purification; J represents SDS-PAGE of RVNb-10 expression and purification; M represents protein molecular weight markers; NE represents whole-cell extract before induction; IE represents whole-cell extract after IPTG induction; SS represents the lysed supernatant; and SP represents the lysed precipitate.

[0032] Figure 5 The images show SDS-PAGE images of the eight nanobodies RVNb-1, RVNb-3, RVNb-5 to RVNb-10 in Example 5 of this invention after purification by size exclusion chromatography. In the images, A is the SDS-PAGE of RVNb-1 and RVNb-3 purified by size exclusion chromatography; B is the SDS-PAGE of RVNb-8 purified by size exclusion chromatography; C is the SDS-PAGE of RVNb-9 purified by size exclusion chromatography; D is the SDS-PAGE of RVNb-5 and RVNb-6 purified by size exclusion chromatography; and E is the SDS-PAGE of RVNb-7 and RVNb-10 purified by size exclusion chromatography.

[0033] Figure 6 This invention relates to the affinity determination of eight nanobodies in Example 6 of this invention, where A represents the RVNb-1 dilution ratio and the corresponding OD. 450 Curve; B represents the RVNb-3 dilution ratio and corresponding OD. 450 Curve; C represents the RVNb-5 dilution ratio and corresponding OD. 450 Curve; D represents the RVNb-6 dilution ratio and corresponding OD. 450 Curve; E represents the RVNb-7 dilution ratio and corresponding OD. 450 Curve; F represents the RVNb-8 dilution ratio and corresponding OD. 450 Curve; G represents the RVNb-9 dilution ratio and corresponding OD. 450 Curve; H represents the RVNb-10 dilution ratio and corresponding OD. 450 curve.

[0034] Figure 7 A standard curve was constructed for the rotavirus ELISA quantification method in Example 8 of this invention.

[0035] Figure 8 This is a flowchart of the A-anti-rotavirus nanobody screening method of the present invention. Detailed Implementation

[0036] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0037] (1) Unless otherwise specified, the term “sequence” as used herein (such as in terms like “antibody sequence,” “variable region sequence,” “VHH sequence,” or “protein sequence”) should generally be understood to include the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding said amino acid sequence, unless the context requires a narrower interpretation.

[0038] (2) Unless otherwise specified, all methods, steps, techniques and operations not specifically described are known and well-known to those skilled in the art. For example, references are still made to the general background art cited above and other references cited therein.

[0039] (3) The term "specificity" in this invention refers to the ability of a specific antigen-binding molecule (such as the nanobody or polypeptide of this invention) to bind to different types of antigens or antigenic determinants. The specificity of an antigen-binding molecule can be determined based on its affinity and / or activity. Affinity is expressed as the dissociation equilibrium constant (K0) between the antigen and the antigen-binding molecule. D K is a measure of the binding strength between antigens and antigen-binding molecules. D The smaller the KD value, the stronger the binding strength between the antigen and the antigen-binding molecule; conversely, the larger the KD value, the weaker the binding strength between the antigen and the antigen-binding molecule.

[0040] (4) The term "specific binding" in this invention refers to binding selectivity for antigens, which can be distinguished from unwanted or non-specific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance (SPR)). Furthermore, those skilled in the art should understand that affinity parameters measured using different methods can vary considerably, even by two to three orders of magnitude.

[0041] (5) The term “antibody” in this invention covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single-chain molecules and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0042] The preparation method of "nanobody" is not limited in its broadest sense to specific biological resources or specific preparation methods. For example, the nanobody of the present invention can be obtained by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; (2) expressing a nucleotide sequence encoding the naturally occurring VHH domain; (3) preparing a protein, polypeptide, or other amino acid sequence using synthetic or semi-synthetic techniques; (4) preparing a nucleic acid encoding the nanobody using nucleic acid synthesis techniques, and then expressing the nucleic acid obtained in this way; and / or (5) by any of the foregoing combinations.

[0043] According to a non-limiting embodiment of the present invention, the above-mentioned polypeptide is substantially composed of nanobodies. "Substantially composed of" means that the amino acid sequence of the polypeptide of the present invention is identical or corresponds to the amino acid sequence of the nanobodies, wherein a limited number of amino acid residues, such as 1 to 10 amino acid residues, and preferably 1 to 6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, are added to the amino terminus (N-terminus) and / or carboxyl terminus (C-terminus) of the nanobodies or polypeptides.

[0044] The aforementioned amino acid residues may not alter the biological properties of the nanobody, and may even impart other functionalities to the nanobody. For example, the amino acid residues may:

[0045] 'a' is a purification tag, i.e., an amino acid sequence or residue that facilitates the purification of the nanobody, for example, by using affinity techniques targeting the sequence or residue for purification. Some preferred, but not limited, examples of such residues are multiple sets of His-tags (His6 or His8), GST-tags, MBP-tags, Myc-tags, Strep-tags, Flag-tags, HA-tags, V5-tags, S-tags, and E-tags;

[0046] b is a soluble tag, which is a tag that increases the solubility of nanobodies, such as SUMO;

[0047] c is an N-terminal amino acid residue, such as Met, Ala, Gln or MetAlaGln, AlaGln, which can be expressed in a heterologous host cell or in a host organism.

[0048] d is a C-terminal Cys residue, which can, for example, react with -SH on a ligand or with the Au surface;

[0049] e is a hinge to provide a link or spacer between the nanobody and other groups, such as a combination of GlySer, an IgG hinge, an IgA hinge, or other synthetic hinges.

[0050] f is provided in a known manner with one or more amino acid residues that have functional groups and / or have been functionalized, for example, as is known in the art, amino acid residues such as lysine or cysteine ​​allow PEG groups to attach.

[0051] The nanobodies, peptides, and nucleic acids encoding the present invention can be prepared in known ways, as will be clear to those skilled in the art from the further description herein. A particularly useful method for preparing the nanobodies, peptides, and nucleic acids generally includes the following steps:

[0052] (1) Expressing nucleic acid encoding the nanobody or polypeptide of the present invention in a suitable host cell or host organism or in another suitable expression system, optionally followed by the expression of the present invention.

[0053] (2) Isolate and / or purify the nanobodies or polypeptides of the present invention thus obtained.

[0054] Alternatively, other methods can be used, including the following steps:

[0055] (3) Cultivate and / or maintain the host of the present invention under certain conditions, so that the host of the present invention expresses and / or produces a nanobody and / or polypeptide of the present invention; optionally, proceed thereafter;

[0056] (4) Isolate and / or purify the nanobodies or polypeptides of the present invention thus obtained.

[0057] The nucleic acid of the present invention can be in the form of single-stranded or double-stranded DNA or RNA, and is preferably in the form of double-stranded DNA. For example, the nucleic acid sequence of the present invention can be genomic DNA, cDNA or synthetic DNA (such as DNA having codons particularly suitable for expression in the host cell or host organism to which it is intended, i.e., codon optimized).

[0058] The nucleic acids of the present invention can be prepared or obtained by methods that are essentially known, based on the amino acid sequence information of the nanobodies or polypeptides of the present invention given herein, and / or can be isolated from suitable natural sources. For example, site-directed mutagenesis of the nucleic acid sequence of a naturally occurring VHH domain can be used to provide the nucleic acids of the present invention encoding said analogs.

[0059] The nucleic acids of the present invention can also be present in and / or as part of a genetic construct, as is well known to those skilled in the art. Such genetic constructs typically include at least one nucleic acid of the present invention, which can be in vector form, such as a plasmid, YAC, viral vector, or transposon. In particular, the vector can be an expression vector, i.e., a vector that can provide in vitro and in vivo expression (e.g., in a suitable host cell, host organism, and / or expression system).

[0060] The nucleic acids and / or genetic constructs of the present invention can be used to transform host cells or host organisms, i.e., for expressing and / or producing the nanobodies or peptides of the present invention. Suitable hosts or host cells are well known to those skilled in the art, and may be, for example, any suitable fungus, prokaryotic or eukaryotic cell or organelle or organism, as well as all other hosts or host cells known essentially for expressing and producing antibodies and antibody fragments (including, but not limited to, single-domain antibodies and ScFv fragments), are well known to those skilled in the art.

[0061] For production, the nanobodies and peptides of the present invention can be produced in the milk of transgenic mammals, such as rabbits, cows, goats or sheep, or in plants or parts of plants, including but not limited to their leaves, flowers, fruits, roots or seeds.

[0062] As mentioned above, one advantage of using nanobodies is that the peptides based on them can be expressed and prepared in prokaryotic systems, and suitable prokaryotic expression systems, vectors, and host cells are well known to those skilled in the art. However, it should be noted that this invention is not limited, in its broadest sense, to expression in bacterial systems.

[0063] Preferably, in this invention, the nanobodies or peptides are produced in bacterial cells, particularly in bacterial cells suitable for large-scale drug production, as described above.

[0064] When expressing the nanobodies or peptides of the present invention used to produce them in cells, the nanobodies or peptides of the present invention can be generated intracellularly (e.g., in the cytoplasm or periplasmic space), then isolated from the host cell, and optionally further purified; or they can be generated extracellularly (i.e., secretory expression), then isolated from the culture medium, and optionally further purified. Some preferred but non-limiting vectors for use with these host cells include vectors for expression in mammalian cells—pMANneo (Clonetech), pUCTtag (ATCC37460), and pMClneo (Stratagene); vectors for expression in bacterial cells—pET vector (Novagen) and pQE vector (Qiagen); expression vectors for yeast or other fungal cells—pYES2 (Invitrogen) and Pichia pastoris expression vector (Invitrogen); expression vectors for insect cells—pBlueBacⅡ (Invitrogen) and other baculovirus vectors; and so on.

[0065] The corresponding techniques for transforming the host or host cells of the present invention are well known to those skilled in the art.

[0066] The amino acid sequence of the present invention can then be isolated from the host cell / host organism and / or from the culture medium in which the host cell or host organism is cultured, using essentially known protein separation and / or purification techniques, such as (preparative) chromatography and / or electrophoresis, differential precipitation, affinity techniques (e.g., using a specific / cleavable amino acid sequence fused with the amino acid sequence of the present invention) and / or preparative immunological techniques (i.e., using antibodies against the amino acid sequence to be isolated).

[0067] The adsorbent of the present invention can be used to specifically recognize G4 genotype rotavirus.

[0068] The nanobodies, peptides, or adsorbents of this invention can be used to purify and detect G4 genotype rotavirus.

[0069] In a first aspect, the present invention provides a nanobody targeting G4 genotype rotavirus, wherein the variable region of the amino acid sequence of the nanobody includes a complementarity-determining region (CDR) and a frame region (FR), and the CDR includes complementarity-determining regions CDR1, CDR2, and CDR3, wherein CDR1 is a sequence SEQ ID NO.11 to SEQ ID NO.20, CDR2 is a sequence SEQ ID NO.21 to SEQ ID NO.30, and CDR3 is a sequence SEQ ID NO.31 to SEQ ID NO.40.

[0070] Preferably, the amino acid sequence of the nanobody includes: SEQ ID NO.1 to SEQ ID NO.10.

[0071] Secondly, the present invention provides a method for preparing nanobodies targeting rotavirus, wherein the method involves using rotavirus as an antigen and screening a phage-displayed nanobody library.

[0072] Thirdly, the present invention provides a polypeptide obtained by modifying the amino acids at the N-terminus and / or C-terminus of the above-mentioned nanobody.

[0073] Preferably, the N-terminal and / or C-terminal amino acid modification of the nanobody includes:

[0074] Method 1: Tagging the N-terminal and / or C-terminal amino acids of the nanobody;

[0075] Method 2: After tagging the N-terminal and / or C-terminal amino acids of the nanobody, the tag is further connected to protect the amino acids via a hinge;

[0076] Preferably, the tag includes at least one of His-tag, GST-tag, Myc-tag, SUMO-tag, Strep-tag, and Flag-tag; the hinge includes at least one of GS hinge, IgG hinge, IgA hinge, and PEG; and the protected amino acid includes Ala, Gln, Glu, Met, or any combination of two or more of the aforementioned amino acids.

[0077] Fourthly, the present invention provides a polypeptide obtained by multivalent synthesis of the above-mentioned nanobody.

[0078] Fifthly, the present invention provides a nucleic acid that encodes the aforementioned nanobody or the aforementioned polypeptide.

[0079] In a sixth aspect, the present invention provides an expression vector comprising the expression frame of the nucleic acid described above.

[0080] In a seventh aspect, the present invention provides a host cell containing the expression vector described above.

[0081] Eighthly, the present invention provides the use of the nanobodies and / or the peptides in detection and / or purification.

[0082] Preferably, the nanobody and / or the polypeptide are used in the preparation of G4 genotype rotavirus adsorbents, G4 genotype rotavirus purification kits, and G4 genotype rotavirus detection kits.

[0083] Example

[0084] The following examples illustrate specific embodiments of the present invention. However, the embodiments of the present invention are not limited to these examples, and any selections and modifications can be made within the scope of the technical effects to be achieved by the present invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the field, or according to the product instructions. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional reagent products that can be obtained commercially.

[0085] Example 1:

[0086] Screening of nanobodies

[0087] Phage screening technology has requirements for the concentration and purity of the coated antigen. Among the six rotaviruses G1, G2, G3, G4, G9, and P1A8, only the concentrated rotavirus of genotype G4 has high purity and meets the screening requirements. Therefore, the concentrated rotavirus of genotype G4 was selected for phage library screening.

[0088] First, G4 genotype rotavirus was diluted to 10 μg / mL with PBS. 1 mL of this solution was added to an immunoassay tube. 1 mL of Vero cell lysis buffer from African green monkey kidney cells was added to the negative control tube, and the tubes were incubated overnight at 4°C. 2 mL of the phage library (number: NiNb1) was added to a final concentration of 0.1% blocking agent and 100 μL of Vero cell lysis buffer, and incubated at 4°C for 30 min. The antigen dilution was aspirated from the tubes, and the tubes were washed three times with PBST, blotted dry, and 3 mL of 3% skim milk blocking buffer (purchased from Sangon Biotech Co., Ltd.) was added per tube. The tubes were incubated at room temperature for 2 h. The blocking agent was aspirated from the tubes, and the tubes were washed six times with PBST, blotted dry. 1 mL of the phage library was added per tube, and the tubes were incubated at room temperature for 30 min. The tubes were washed 10 times with PBST, and the phages were eluted with elution buffer (10 mM pH 1.5 Gly-HCl). The tubes were incubated at room temperature for 30 min, and the elution buffer was amplified for the next round of screening. Once the enrichment level meets the requirements, ELISA can be used to verify specificity. For example... Figure 1 As shown in Figure A, the enrichment rate was approximately 10-fold in the first round of screening; in Figure B, it was approximately 20-fold in the second round; and in Figure C, it was approximately 70-fold in the third round of biological screening. This indicates that after three rounds of biological screening, phages exhibiting specific binding to G4 genotype rotavirus nanobodies were effectively enriched, and the phages eluted in the third round of biological screening could be used to screen for binders.

[0089] Example 2:

[0090] Specific detection.

[0091] After three rounds of screening, the phages obtained from the screening eluent were used to infect TG1 *E. coli* and plated on LB Amp solid medium plates. Sixteen single clones were picked and added to 1 mL of 2XTY medium and cultured at 37°C and 180 rpm for 4–6 h. Then, 200 μL of 2XTY medium containing 10 mM IPTG was added to induce nanobody expression; this is the periplasmic space extract containing nanobodies. G4 genotype rotavirus was diluted to 10 μg / mL with PBS, and 100 μL was added to 96-well plates. The negative wells were coated with Vero cell lysis buffer and incubated at 4°C for 12 h. The antigen dilution was aspirated from the wells, the plates were washed three times with PBST, dried, and 300 μL of 3% skim milk blocking buffer was added to each well. The plates were incubated at room temperature for 2 h. The blocking agent was aspirated from the tubes, the plates were washed six times with PBST, and dried. Add 100 μL of pericytic space extract to each well and incubate at room temperature for 1 h. Wash the plate 5 times with 300 μL PBST and drain. Add 100 μL of mouse anti-His6 IgG diluted 1:2000 with PBS (purchased from Sangon Biotech Co., Ltd.) to each well and incubate at 37°C on a horizontal shaker for 1 h. Wash the plate 5 times with 300 μL PBST and drain. Add 100 μL of HRP-conjugated goat anti-mouse IgG diluted 1:1000 with PBS (purchased from Sangon Biotech Co., Ltd.) to each well and incubate at 37°C on a horizontal shaker for 1 h. Wash the plate 5 times with 300 μL PBST and drain. Add 100 μL of TMB working solution to each well and incubate at 37°C in the dark for 30 min. Stop the reaction by adding 50 μL of 2 M H2SO4 to each well. Measure the OD using a microplate reader. 450 Absorbance at nm, OD of test well and control well 450 The ratio can reflect specificity. Figure 2 It can be seen that among the 16 conjugates obtained by screening G4 genotype rotavirus concentrate and NiNb1, 12 conjugate wells showed OD values ​​that were significantly different from those of the control wells. 450 The ratios were all greater than 8, indicating that the 12 conjugates screened had high affinity and specificity for G4 genotype rotavirus, meeting the screening requirements.

[0092] Example 3:

[0093] Nanobody sequencing analysis.

[0094] Twelve highly specific phage clones were selected and inoculated into 20 mL LB liquid medium (100 mg / mL Amp). The cultures were incubated at 37°C and 180 rpm for 12–16 h. The bacterial cultures were then sequenced externally to obtain the nanobody sequence information. The nanobody nomenclature and sequence number information are shown in Tables 1-2.

[0095] Table 1. Comparison of Nanobody Serial Numbers and CDR Sequence Information

[0096]

[0097] Table 2 Comparison of Nanobody Sequence Numbers and FR Sequence Information - 1

[0098]

[0099] Table 2 Comparison of Nanobody Sequence Numbers and FR Sequence Information - 2

[0100]

[0101] SEQ ID NO.1: QLQESGGGLVQPGDSLRLSCAASG-RTSGSGAM-AWFRQAPGKEREFVAYI-TWSGGDT-WYADAAKGRFTIARDNAKNTAYLQMNNLKPEDTAVYYCA-ANPFEQLQFTPASYR-DWGQGTQVTVSS

[0102] SEQ ID NO.2: QLQESGGGLVQAGDSLRLSCSSSSD-PSLNSYSL-GWFRQAPGVERAGEREFVAGV-SWSGGSAE-YGDSAKGRFTVSRDNAKNTLYLQMNDLRPDDAGNYWCA-AAYLRPVSPYDMD-YWGRGTQVTVSS

[0103] SEQ ID NO.3: QLQESGGGLVQAGGSLRLSCAASG-RTSSSYAM-GWFRQAPGKEREFVAAI-TWPSDSP-YYSNSIKGRFTIAVDNAKNTLYLQMNSLKPDDTAVYYCA-AQRSLRETVTVADTYD-YWGQGTQVTVSS

[0104] SEQ ID NO.4: QLQESGGGLVQAGGSLRLSCAVSG-FDSDYGLDYYAI-GWFRQAPGKEREAISCI-INRSGST-DYADSVKGRFTISRDTTKNTVYLQMNSLKPEDTAVYYCT-IKMEYSGNSCPPLWQYD-YWGQGTQVTVSS

[0105] SEQ ID NO.5:QLQESGGGLVQAGGSVRLSCTSSK-STFSNYDM-AWFRQAPGKDREFVAFI-RSSGEAT-WYGDSMKGRVTISLDNAKNTLYLYMNRLTPEDTAMYFCA-ARRRGFDLVGTRPDQYD-YWGQGSQVTVSS

[0106] SEQ ID NO.6:QLQESGGGLVQPGGSLRLSCVVSG-RTYSDMAM-GWFRQAPGKEREFVAAI-RWTSDSP-WYGEPAKGRFTIARDNAKDTLYLHLNFLKPEDTAVYYCA-AWRGEFASGTIPPD-YWGQGTQVTVSS

[0107] SEQ ID NO.7:QLQESGGGLVQAGDSLRLSCAASG-DSLSGFNM-GWFRQPPGRERWEREFVASI-TWTEGFT-DYADDVKGRFTISRDASKEMVYLQMNSLKPEDTAAYFCA-AKWTETYTLFRRWADSS-YWGEGTQVTVSS

[0108] SEQ ID NO.8:QLQESGGGLVQTGGSLKLSCAASG-RTMSAYRM-AWFRQAPGKGREFVAAF-GDGP-YYGASAMGRFTIARDNARNTVYLHMYNLKPEDTAVYYCA-AGISDSDIRRPDNYD-SWGQGTQVTVSS

[0109] SEQ ID NO.9:QLQESGGGSVQAGGSLRLSCAASG-RTFSSYGM-GWFRQAPGKEREFVAFI-NQNGDSP-YYTDSVKGRFTIAKDNAKNTLYLQMNSLKPEDTAVYYCA-IHNSAGLVTGARTYD-YWGQGTQVTVSS

[0110] SEQ ID NO.10:QLQESGGGSVQAGGSLRLSCAASG-LGSGDYVV-GWFRQAPGKEREFVAAI-SWPSNSPF-YSNSVVKGRFTIALDNTKNTAYLHMYRLEAEDTAVYYCA-AQRSQRPIVTSASTYD-VWGQGTQVTVSS

[0111] Example 4:

[0112] Broad spectrum verification.

[0113] To determine whether these nanobodies bind only to the G4 genotype rotavirus or have broad-spectrum binding, hydrophobic coatings were applied to rotaviruses of different genotypes (G1, G2, G3, G4, G9, and P1A8). Multiple nanobodies binding to the G4 genotype rotavirus were incubated to verify broad-spectrum binding. The G1, G2, G3, G4, G9, and P1A8 genotype rotaviruses were diluted to 10 μg / mL with PBS, and 100 μL was added to each well of a 96-well plate. The negative wells were coated with Vero cell lysis buffer and incubated at 4°C for 12 h. The antigen dilution buffer was aspirated from the wells, the plates were washed three times with PBST, blotted dry, and 300 μL of 3% skim milk blocking buffer was added to each well. The plates were incubated at room temperature for 2 h. The blocking buffer was aspirated from the tubes, the plates were washed six times with PBST, and blotted dry. 100 μL of 10 different periplasmic space extracts were added to each well and incubated at room temperature for 1 h. The plates were washed five times with 300 μL of PBST and drained. Add 100 μL of mouse anti-His6 IgG diluted 1:2000 with PBS to each well and incubate at 37°C on a horizontal shaker for 1 h. Wash the plate 5 times with 300 μL PBST and drain. Add 100 μL of HRP-conjugated goat anti-mouse IgG diluted 1:1000 with PBS to each well and incubate at 37°C on a horizontal shaker for 1 h. Wash the plate 5 times with 300 μL PBST and drain. Add 100 μL of TMB working solution to each well and incubate at 37°C in the dark for 30 min. Stop the reaction by adding 50 μL of 2 M H2SO4 to each well. Measure the OD using a microplate reader. 450 Absorbance at nm, OD of test well and control well 450 The ratio can reflect specificity. Figure 3 It can be seen that AJ represents the validation of the binding ability of RVNb-1 to RVNb-10 nanobodies with rotaviruses of genotypes G1, G2, G3, G4, G9, and P1A8, respectively. The results show that the OD values ​​of these 10 nanobodies with G1, G2, G3, G9, and P1A8 rotaviruses in the experimental and control wells are... 450 The ratios were all less than 3, indicating that the above 10 nanobodies had low specificity for other rotavirus genotypes. Furthermore, the OD values ​​of the 10 nanobodies and the G4 rotavirus in the test wells and control wells were... 450 A ratio greater than 8 indicates that the above 10 nanobodies have the ability to bind to the G4 genotype rotavirus.

[0114] Example 5:

[0115] Expression and purification of nanobodies.

[0116] 1) Eight His tags were added to the C-terminus of the amino acid sequence of the nanobody, and the above nanobody was expressed using Escherichia coli shuffle T7 strain. Uniformly sized single colonies were picked and inoculated into 20 mL LB liquid medium (20 μL 100 mg / mL Amp), and activated overnight by shaking at 37℃ and 180 rpm for 12-16 h.

[0117] 2) Transfer 10 mL of the overnight culture to 1 L of TB medium (1 mL 100 mg / mL Amp) and incubate at 37℃ and 180 rpm on a shaker until OD reaches 100 mg / mL. 600 =0.6~0.8 (approximately 4~6 hours).

[0118] 3) Add IPTG inducer to a final concentration of 0.5 mM, and induce expression overnight at 30℃ and 180 rpm. Centrifuge the obtained bacterial culture at 4℃ and 3200 g for 30 min, discard the supernatant, and collect the bacterial cells.

[0119] 4) Resuspend the bacterial cells in 5 times the weight of the wet bacterial cells in pre-cooled loading buffer (20 mM Na2HPO4, 20 mM imidazole, 500 mM NaCl).

[0120] 5) The bacterial cells were disrupted using a high-pressure homogenizer at 700 MPa for 30 min to expose the target nanobody in *E. coli*. The supernatant was filtered through a 0.45 μm filter and then purified using an affinity chromatography column (GE Healthcare, US) with Ni Sepharose High Performance packing material. The purified nanobody was then subjected to SDS-PAGE electrophoresis to determine its purity. Figure 4 As shown, AJ represents SDS-PAGE of RVNb-1 to RVNb-10 nanobodies purified by Ni affinity chromatography. The results indicate that, except for RVNb-2 and RVNb-4, which could not be effectively recombinantly expressed in E. coli SHuffle T7 host bacteria, RVNb-1, RVNb-3, and RVNb-5 to RVNb-10 could all be effectively recombinantly expressed in E. coli SHuffle T7 host bacteria. However, these eight nanobodies still contained contaminating proteins with significant differences in molecular weight.

[0121] 6) The above 8 nanobodies were further purified by size exclusion chromatography, and SDS-PAGE electrophoresis was used to determine the purity. Figure 5As shown, A is an SDS-PAGE of RVNb-1 and RVNb-3 purified by size exclusion chromatography; B is an SDS-PAGE of RVNb-8 purified by size exclusion chromatography; C is an SDS-PAGE of RVNb-9 purified by size exclusion chromatography; D is an SDS-PAGE of RVNb-5 and RVNb-6 purified by size exclusion chromatography; and E is an SDS-PAGE of RVNb-7 and RVNb-10 purified by size exclusion chromatography. The results indicate that the purity of the eight nanobodies can reach 95%, and the protein concentration was determined using the BCA method.

[0122] Example 6:

[0123] Affinity determination of nanobodies.

[0124] G4 genotype rotavirus was diluted to 10 μg / mL with PBS. 100 μL was added to each well of a 96-well plate. The negative wells were coated with Vero cell lysis buffer and incubated at 4°C for 12 h. The antigen dilution buffer was aspirated from the wells, the plates were washed three times with PBST, blotted dry, and 300 μL of 3% skim milk blocking buffer was added to each well. The plates were incubated at room temperature for 2 h. The blocking buffer was aspirated from the tubes, the plates were washed six times with PBST, and blotted dry. Eight nanobodies with a purity >95% were serially diluted 1:20. 100 μL of nanobody dilution buffer was added to each well, and the plates were incubated at 37°C for 1 h. The plates were washed five times with 300 μL of PBST, each time with a horizontal shaker at 170 rpm for 1 min. 100 μL of mouse anti-His6 IgG diluted 1:2000 with PBS was added to each well, and the plates were incubated at 37°C for 1 h. The plates were washed five times with 300 μL of PBST, each time with a horizontal shaker at 170 rpm for 1 min. Add 100 μL of PBS (1:1000 dilution) of HRP-conjugated goat anti-mouse IgG to each well and incubate at 37°C for 1 h. Wash the plate 5 times with 300 μL PBST, shaking horizontally at 170 rpm for 1 min each time. Add 100 μL of TMB working solution to each well and incubate at 37°C in the dark for 10–30 min. Stop the reaction by adding 50 μL of 2 M H₂SO₄. Measure the OD using a microplate reader. 450 The absorbance value at nm, the measured OD 450 Plot a curve showing the absorbance at nm and the corresponding nanobody dilution ratio. For example... Figure 6 As shown, AH represents the dilution ratios of RVNb-1, RVNb-3, RVNb-5—RVNb-10 nanobody nanoparticles and their corresponding OD values. 450 The curve, the OD when the curve tends to flatten. 450 The curve represents the 100% saturation state, and the antibody concentration corresponding to 50% saturation is the affinity of the nanobody. The kinetic parameters, as shown in Table 3, were calculated using equations. Except for RVNb-8, the other seven nanobodies all exhibited high affinity for the G4 genotype rotavirus. DThe range is 1-100 (nM).

[0125] Table 3. Affinity of nanobodies to G4 genotype rotavirus

[0126]

[0127] Example 7:

[0128] Antibody labeling

[0129] Antibody labeling techniques well-known to those in the art include enzyme labeling, biotin labeling, fluorescein labeling, colloidal gold labeling, and radioisotope labeling. Each labeling technique has its specific application scenarios and advantages. Choosing the appropriate labeling technique can improve the sensitivity and specificity of the experiment.

[0130] The invention employs a commonly used enzyme-labeled antibody labeling method, as follows:

[0131] Weigh 3 mg of horseradish peroxidase (HRP) and add 0.5 mL of 0.1 mol / L NaIO4 solution. Mix well and incubate at room temperature in the dark for 30 min. Add 0.5 mL of 0.16 mol / L ethylene glycol and incubate at room temperature for 30 min. Mix the activated enzyme solution with an equimolar amount of the RVNb-10 nanobody to be conjugated (dissolved in PBS), transfer to an ultrafiltration tube, and replace the buffer with 0.05 mol / L pH 9.5 carbonate buffer. Add 0.1 mL of 5 mg / mL NaBH4 solution, mix well, and incubate at 4°C for 3 h. After ultrafiltration with PBS at 4°C, centrifuge at 18000 g for 10 min to remove the precipitate. The supernatant (approximately 1–1.5 mL) is the enzyme-labeled nanobody, denoted as HRP-RVNb-10.

[0132] Example 8:

[0133] Construction of quantitative detection method.

[0134] The nanobody RVNb-10 was diluted to 10 μg / mL with PBS, and 100 μL was added to a 96-well plate and incubated overnight at 4°C with shaking. 300 μL of 1×PBST was added to each well to wash 5 times. 250 μL of 3% skim milk powder was added to each well, and the plate was blocked at room temperature with horizontal shaking for 2 hours. 300 μL of 1×PBST was added to each well to wash 5 times. The G4 genotype rotavirus concentrate was diluted with PBS to final concentrations of 1000, 800, 600, 500, 400, 200, and 100 ng / mL, and 100 μL of the G4 genotype rotavirus concentrate dilution was added to each well. 100 μL of Vero cell lysis buffer was added to the negative control wells, and the plate was incubated at room temperature with horizontal shaking for 1 hour. 300 μL of 1×PBST was added to each well to wash 5 times, shaking horizontally for 4 minutes each time. Enzyme-labeled antibody: Add 100 μL of HRP-RVNb-10 diluted 1:2000 with PBS to each well and incubate on a horizontal shaker at room temperature for 1 h. Wash each well 5 times with 300 μL of 1×PBST, shaking horizontally for 4 min each time. Add 100 μL of TMB working solution to each well and incubate at room temperature in the dark for 30 min. After the reaction, add 50 μL of 2 M H2SO4 to each well and measure the OD using a microplate reader. 450 Absorbance value at nm. For example Figure 7 As shown, the standard curve exhibits good linearity, capable of detecting G4 genotype rotavirus at concentrations ranging from 100 to 1000 ng / mL, with a linear correlation coefficient R0. 2 The value is 0.9954, indicating that this quantitative detection method has good linear correlation.

[0135] Industrial availability

[0136] The nanobody of the present invention is a novel amino acid sequence targeting G4 genotype rotavirus, discovered through screening of a phage library. This nanobody and its polypeptide have high affinity and activity, and can specifically recognize and bind to G4 genotype rotavirus. The nanobody prepared by the present invention can be used for the detection of G4 genotype rotavirus.

[0137] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A nanobody targeting G4 genotype rotavirus, characterized in that, The complementarity-determining region (CDR) of the nanobody includes CDR1, CDR2, and CDR3 sequences: (I): (1) The amino acid sequence of CDR1 is shown in SEQ ID NO.11, and (2) The amino acid sequence of CDR2 is shown in SEQ ID NO.21; And (3) the amino acid sequence of CDR3 is shown in SEQ ID NO.31; or (II): Amino acid sequences obtained by modifying, substituting, deleting, or adding one or more amino acids as described in (1), (2), and (3) of (I), and which have the same function as the amino acid sequences described in (I), including (Ⅱ-1): The amino acid sequence of CDR1 is shown in SEQ ID NO.12, the amino acid sequence of CDR2 is shown in SEQ ID NO.22, and the amino acid sequence of CDR3 is shown in SEQ ID NO.32; or (Ⅱ-2): The amino acid sequence of CDR1 is shown in SEQ ID NO.13, the amino acid sequence of CDR2 is shown in SEQ ID NO.23, and the amino acid sequence of CDR3 is shown in SEQ ID NO.33; or (Ⅱ-3): The amino acid sequence of CDR1 is shown in SEQ ID NO.14, the amino acid sequence of CDR2 is shown in SEQ ID NO.24, and the amino acid sequence of CDR3 is shown in SEQ ID NO.34; or (Ⅱ-4): The amino acid sequence of CDR1 is shown in SEQ ID NO.15, the amino acid sequence of CDR2 is shown in SEQ ID NO.25, and the amino acid sequence of CDR3 is shown in SEQ ID NO.35; or (Ⅱ-5): The amino acid sequence of CDR1 is shown in SEQ ID NO.16, the amino acid sequence of CDR2 is shown in SEQ ID NO.26, and the amino acid sequence of CDR3 is shown in SEQ ID NO.36; or (Ⅱ-6): The amino acid sequence of CDR1 is shown in SEQ ID NO.17, the amino acid sequence of CDR2 is shown in SEQ ID NO.27, and the amino acid sequence of CDR3 is shown in SEQ ID NO.37; or (Ⅱ-7): The amino acid sequence of CDR1 is shown in SEQ ID NO.18, the amino acid sequence of CDR2 is shown in SEQ ID NO.28, and the amino acid sequence of CDR3 is shown in SEQ ID NO.38; or (Ⅱ-8): The amino acid sequence of CDR1 is shown in SEQ ID NO.19, the amino acid sequence of CDR2 is shown in SEQ ID NO.29, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (Ⅱ-9): The amino acid sequence of CDR1 is shown in SEQ ID NO.20, the amino acid sequence of CDR2 is shown in SEQ ID NO.30, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

40.

2. The nanobody according to claim 1, characterized in that, in: (III) The framework region FR of the nanobody includes FR1, FR2, FR3 and FR4 sequences, wherein: (1) the amino acid sequence of FR1 is shown in SEQ ID NO.41, (2) the amino acid sequence of FR2 is shown in SEQ ID NO.50, (3) the amino acid sequence of FR3 is shown in SEQ ID NO.58, and (4) the amino acid sequence of FR4 is shown in SEQ ID NO.68; or (IV) Amino acid sequences that have more than 50% homology with the amino acid sequences described in (1), (2), (3), and (4) of (III), including (IV-1): The amino acid sequence of FR1 is shown in SEQ ID NO.42, the amino acid sequence of FR2 is shown in SEQ ID NO.51, the amino acid sequence of FR3 is shown in SEQ ID NO.59, and the amino acid sequence of FR4 is shown in SEQ ID NO.69; or (IV-2): The amino acid sequence of FR1 is shown in SEQ ID NO.43, the amino acid sequence of FR2 is shown in SEQ ID NO.52, the amino acid sequence of FR3 is shown in SEQ ID NO.60, and the amino acid sequence of FR4 is shown in SEQ ID NO.70; or (IV-3): The amino acid sequence of FR1 is shown in SEQ ID NO.44, the amino acid sequence of FR2 is shown in SEQ ID NO.53, the amino acid sequence of FR3 is shown in SEQ ID NO.61, and the amino acid sequence of FR4 is shown in SEQ ID NO.70; or (IV-4): The amino acid sequence of FR1 is shown in SEQ ID NO.45, the amino acid sequence of FR2 is shown in SEQ ID NO.54, the amino acid sequence of FR3 is shown in SEQ ID NO.62, and the amino acid sequence of FR4 is shown in SEQ ID NO.70; or (IV-5): The amino acid sequence of FR1 is shown in SEQ ID NO.46, the amino acid sequence of FR2 is shown in SEQ ID NO.52, the amino acid sequence of FR3 is shown in SEQ ID NO.63, and the amino acid sequence of FR4 is shown in SEQ ID NO.70; or (IV-6): The amino acid sequence of FR1 is shown in SEQ ID NO.47, the amino acid sequence of FR2 is shown in SEQ ID NO.55, the amino acid sequence of FR3 is shown in SEQ ID NO.64, and the amino acid sequence of FR4 is shown in SEQ ID NO.71; or (IV-7): The amino acid sequence of FR1 is shown in SEQ ID NO.48, the amino acid sequence of FR2 is shown in SEQ ID NO.56, the amino acid sequence of FR3 is shown in SEQ ID NO.65, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-8): The amino acid sequence of FR1 is shown in SEQ ID NO.49, the amino acid sequence of FR2 is shown in SEQ ID NO.57, the amino acid sequence of FR3 is shown in SEQ ID NO.66, and the amino acid sequence of FR4 is shown in SEQ ID NO.70; or (IV-9): The amino acid sequence of FR1 is shown in SEQ ID NO.49, the amino acid sequence of FR2 is shown in SEQ ID NO.52, the amino acid sequence of FR3 is shown in SEQ ID NO.67, and the amino acid sequence of FR4 is shown in SEQ ID NO.

73.

3. The nanobody according to claim 1 or 2, characterized in that, The nanobody has (V) the amino acid sequence shown in SEQ ID NO. 1; or (VI) An amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids as described in (V), and having the same function as the amino acid sequence described in (I); wherein the amino acid sequence of the nanobody in (VI) is shown in any one of SEQ ID NO.2 ~ SEQ ID NO.

10.

4. A method for preparing nanobodies targeting rotavirus, characterized in that, The method includes screening a phage-displayed nanobody library using rotavirus as an antigen; The whole rotavirus is a live attenuated rotavirus vaccine, including at least one of the following: G1 genotype rotavirus, G2 genotype rotavirus, G3 genotype rotavirus, G4 genotype rotavirus, G9 genotype rotavirus, and P1A8 genotype rotavirus. The phage-displaying nanobody library includes at least one of the following: fully synthetic library, semi-synthetic library, immune library, non-immune library, and natural library.

5. A polypeptide, characterized in that, Includes the nanobody described in any one of claims 1 to 3.

6. A nucleic acid molecule encoding the nanobody of claims 1 to 3.

7. An expression carrier, characterized in that, Includes the nucleic acid molecule as described in claim 6.

8. A host cell that transforms or transfects the expression vector of claim 7.

9. A conjugate, coupling compound, or adsorbent, characterized in that, The conjugate or conjugate includes the nanobodies of claims 1 to 3 that have been chemically or biologically labeled; The adsorbent includes the nanobody as described in any one of claims 1 to 3; or the polypeptide as described in claim 5; Or the nucleic acid molecule as described in claim 6; or the expression vector as described in claim 7; or the host cell as described in claim 8; Or the aforementioned conjugate; or the aforementioned coupling, and the carrier.

10. A reagent kit, characterized in that, Includes the nanobody according to any one of claims 1 to 3; or the polypeptide according to claim 5; Or the nucleic acid molecule as described in claim 6; or the expression vector as described in claim 7; or the host cell as described in claim 8; Or the combination as described in claim 9; or the coupling as described in claim 9; Or the adsorbent as described in claim 9, and an auxiliary agent acceptable in the detection.

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