Nanometer antibody for detecting respiratory syncytial virus and application thereof

Abstract

The invention relates to a nano antibody for detecting respiratory syncytial virus and application thereof. In particular to a nano antibody specifically combined with respiratory syncytial virus F glycoprotein, which comprises a CDR1 shown as SEQ ID NO: 3, a CDR2 shown as SEQ ID NO: 4 and a CDR3 shown as SEQ ID NO: 5. The nano antibody provided by the invention has the characteristics of strong specificity, high sensitivity and high stability, and has wide application prospects and important economic and social meanings in the research aspects of development of RSV infection detection reagents, targeted drug delivery, neutralizing antibody treatment and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nanobody for detecting respiratory syncytial virus and its application. Background Technology

[0002] Human respiratory syncytial virus (RSV) is the most common pathogen causing acute respiratory infections in infants and young children, and is also recognized as an important pathogen in certain high-risk groups, such as the elderly, adults with chronic diseases, and immunocompromised individuals. Almost all children are infected at least once before the age of two, and are reinfected throughout their lives due to weakened immunity over time. Globally, an estimated 33.1 million cases of acute lower respiratory infection (ALRI) are caused by RSV, resulting in approximately 120,000 deaths in children under the age of five each year. Although RSV poses a moderate threat to healthy adults, it is a leading pathogen in the elderly and those at high risk of chronic cardiopulmonary diseases. Currently, there is a lack of effective preventative and therapeutic vaccines and antibodies for RSV, as well as a lack of highly efficient, sensitive, convenient, and economical detection antibodies and related kits. Therefore, given the continuous mutation of the virus, developing RSV detection nanobodies with different targets and high specificity, and establishing convenient, rapid, and effective detection methods, would be more conducive to the early identification of respiratory syncytial virus infection and is an important measure to reduce RSV transmission.

[0003] RSV belongs to the genus Pneumovirus in the family Paramyxoviridae. It is an enveloped negative-sense RNA virus. Its genome is 15.2 kb in length, containing 10 genes encoding 11 proteins. G and F proteins are two major glycoproteins on the viral surface, playing crucial roles in mediating viral entry into cells. The G protein primarily functions as an adsorption protein, interacting with one or more molecules on the host cell surface to facilitate viral particle binding to target cells, but it is not essential for viral infection. The F protein's main function is to mediate the fusion of the viral envelope and cell membrane, and it is the only protein responsible for membrane fusion, essential for viral entry and spread through syncytial formation. Furthermore, both F and G proteins are important antigens, stimulating a protective immune response, serving as primary targets for neutralizing antibodies, and are also important targets for vaccine design and small-molecule inhibitors. Based on the differences in the G protein, RSV strains are divided into two different subtypes (RSV-A and RSV-B). The two subtypes share only 50% homology in the amino acids of the G protein. The F protein is relatively conserved, with only 10% sequence difference. Therefore, the F protein is considered the most ideal target for both vaccine and drug development and the establishment of pathogen detection methods.

[0004] Currently, common detection techniques for RSV include the classic plaque assay, the TCID50 assay, RT-qPCR, and immunofluorescence. Among these, the plaque assay and TCID50 assay are time-consuming, requiring RSV to be cultured for about a week to produce sufficiently large, visible plaques, followed by fixation and crystal violet staining to calculate the viral titer. However, in practice, the plaque assay has been found to be unstable, with small plaques, indistinct boundaries, and specific requirements for cell type and culture medium. Errors in plaque identification and manual counting often lead to poor reproducibility. RT-qPCR offers high sensitivity, strong specificity, and a short detection cycle, but for RSV titer detection, it requires RNA extraction from the sample, a cumbersome process that is prone to RNA degradation and contamination, resulting in false positives. Indirect immunofluorescence uses specific antibodies to react with the corresponding antigens in the sample. A fluorescently labeled secondary antibody binds to the specific antibody, and the fluorescence signal is observed under a fluorescence microscope. Quantification is then performed by analyzing the fluorescence intensity. This method is rapid, highly sensitive and specific, and exhibits good experimental reproducibility. However, the antibody cost is high when dealing with large sample sizes, and the method requires further optimization and improvement. Although many methods exist for respiratory virus detection, they all have various shortcomings, hindering their widespread clinical application. Therefore, we urgently need to develop a detection antibody, which is of great significance for the early detection and diagnosis of RSV.

[0005] Studies have found that camels and cartilaginous fish (such as sharks) possess naturally occurring heavy-chain antibodies (HCAbs). Compared to traditional antibodies, nanobodies offer numerous advantages: for example, nanobodies possess the smallest natural antigen-binding domain, with a molecular weight of approximately 15 kDa, one-tenth the size of traditional antibodies. This small size allows them to bind to narrow epitopes that are typically inaccessible to traditional antibodies. Therefore, nanobodies exhibit better specificity and affinity than traditional antibodies. Furthermore, nanobodies offer advantages such as high stability, high expression levels, low production costs, and ease of engineering, which are unmatched by traditional monoclonal antibodies. Since the first preparation of nanobodies from alpacas in 1994, nanobodies have demonstrated enormous application potential in the biomedical field. Currently, several nanobodies and their engineered antibodies are in clinical trials and may be used in the detection, prevention, and treatment of diseases such as tumors, autoimmune diseases, and viral infections. Summary of the Invention

[0006] Based on the aforementioned research background, there is an urgent need to address the shortcomings of current RSV detection antibodies on the market. This invention aims to develop a nanobody that specifically binds to the F glycoprotein on the surface of respiratory syncytial virus (RSV), establishing a simple, rapid, and cost-effective RSV detection method. This method facilitates early identification of RSV infection and is an important measure to reduce RSV transmission.

[0007] In order to solve the above problems, the inventors conducted in-depth research and successfully screened a nanobody that specifically binds to the F protein on the surface of respiratory syncytial virus (RSV).

[0008] Specifically, the present invention relates to the following aspects:

[0009] On one hand, the present invention relates to a nanobody comprising CDR1 as shown in SEQ ID NO:3, CDR2 as shown in SEQ ID NO:4, and CDR3 as shown in SEQ ID NO:5.

[0010] The amino acid sequence of the above-mentioned nanobody is shown in SEQ ID NO:1.

[0011] Among them, the above-mentioned nanobody specifically binds to the spatial conformational epitope and / or linear epitope of the fusion F glycoprotein on the surface of respiratory syncytial virus.

[0012] On the other hand, the present invention also relates to polynucleotides that encode the aforementioned nanobodies.

[0013] Its sequence is shown in SEQ ID NO:2.

[0014] On the other hand, the present invention also relates to expression vectors comprising the aforementioned polynucleotides.

[0015] On the other hand, the present invention also relates to host cells that contain the above-described expression vector.

[0016] On the other hand, the present invention also relates to the use of the above-mentioned nanobodies in the preparation of diagnostic agents or kits for detecting RSV infection.

[0017] On the other hand, the present invention also relates to a method for detecting respiratory syncytial virus (RSV) in a sample, comprising adding the above-described nanobody to the sample.

[0018] The embodiments of the present invention also relate to a method for preparing the above-mentioned nanobodies, which includes the following steps:

[0019] (1) Camels were immunized four times with 0.5 mg / time of high-purity RSV post-F protein, and the amino acid sequence of RSV post-F protein is shown in SEQ ID NO:6;

[0020] (2) Blood was collected and PBMCs were isolated, and V was amplified by PCR. H Fragment H, wherein the primers used for PCR are shown in SEQ ID NO:7 and SEQ ID NO:8;

[0021] (3) The amplified V H H is cloned into the phage particle to form a recombinant plasmid;

[0022] (4) The recombinant plasmid was introduced into E. coli TG1 competent cells by electroporation to construct V H H's phage library, wherein, if necessary, a further step of freezing the phage library bacteria at -80°C can be added;

[0023] (5) Using phage display technology to display V H The H fragment is expressed onto the capsid protein on the surface of the phage, and KM13 is added to help the phage amplify the V fragment. H H's bacteriophage;

[0024] (6) Two rounds of biopane screening were performed to screen the nanobodies in the library;

[0025] (7) By obtaining nanobodies that specifically target post-F protein.

[0026] More specifically, the present invention relates to the following aspects of research:

[0027] Research Content 1: Constructing a phage-displaying nanobody library, performing biopanning and nanobody purification;

[0028] (1) First, RSV post-F protein was prepared as an antigen and used to immunize camels. After four immunizations, peripheral blood lymphocytes (PBMCs) were isolated from camel blood. RNA was extracted from PBMCs and reverse transcribed into cDNA. V was then amplified using specific primers. H The H fragment was constructed onto a phage particle and transformed into V in E. coli. H H gene library.

[0029] (2) The post-F protein was coated on an ELISA plate, and the nanobodies were displayed on the surface of the phage using phage display technology. The nanobodies that specifically target the post-F protein were obtained from the nanobodies library through multiple rounds of biological panning.

[0030] (3) The obtained positive monoclonal bacteria were sequenced, and the nanobody sequence was constructed into a eukaryotic expression vector, transfected into eukaryotic cells for expression and purification.

[0031] Research Content 2: Detection of the binding ability of candidate nanobodies;

[0032] Antibodies can recognize both conformational epitopes, which are spatial structures formed by the folding of non-connected amino acid residues in a sequence, and linear epitopes, which are structures formed by covalent bonds between connected amino acid residues in a sequence.

[0033] (1) Spatial conformation epitope binding: ELISA is a commonly used method to characterize the binding activity of antibody spatial epitopes. 50 The lower the value, the stronger the binding between the antibody and the antigen. The binding activity of candidate nanobodies was evaluated by ELISA.

[0034] (2) Linear epitope binding: Western blotting is a commonly used method for characterizing the binding of antibody linear epitopes. After denaturation, proteins are separated by molecular weight using gel electrophoresis, and then specific antibodies are used to recognize the target protein. This technique can be used to further determine whether candidate detection nanobodies can recognize the linear epitope of the F glycoprotein on the surface of respiratory syncytial virus.

[0035] Research Content 3: Establishment and optimization of an indirect immunofluorescence assay for RSV detection.

[0036] To accurately and intuitively detect RSV, we established an indirect immunofluorescence detection method for RSV based on the detection antibody F-E2 screened in this invention. Cells were pre-seeded in well plates and co-cultured with the virus. Finally, the detection antibody F-E2 was added to react with the corresponding antigen in the sample, and the viral infection status was analyzed and observed using fluorescence microscopy.

[0037] The term "spatial conformational epitope" refers to an epitope formed by a discontinuous sequence of amino acid residues that depends on the spatial conformation of the antigen. In protein antigens, it refers to an antigenic determinant cluster, typically composed of 3 to 15 elements, formed by the spatial conformation of discontinuous amino acid residues. In nucleic acid and carbohydrate antigens, it refers to an antigenic determinant cluster formed by the spatial conformation of discontinuous nucleotide or monosaccharide residues, respectively.

[0038] The term "linear epitope" refers to a continuous amino acid sequence extending from a specific region of a protein, which interacts with antibodies based on its primary structure.

[0039] The term "RSV post-F protein" refers to the RSV virus surface fusion F glycoprotein, which, after infecting cells, undergoes changes in its secondary structure to form a more stable post subtype, whose amino acid sequence remains unchanged from the nucleotide sequence encoding it.

[0040] The term "PBMC" refers to peripheral mononuclear cells, which include lymphocytes and mononuclear cells. In the embodiments of this invention, it refers to camel lymphocytes.

[0041] The term "phage particle" refers to a plasmid containing the replication origin of filamentous bacteriophages.

[0042] The term "phage library" refers to the introduction of a specific target gene into a microorganism and its storage within that microorganism. In this invention, it specifically refers to the introduction of the aforementioned phage particles into *E. coli* TG1 competent cells, ensuring that all cells contain V... H The H fragment gene can be preserved in E. coli for a long time for subsequent screening.

[0043] The term "bacteriophage library" refers to a microorganism that has preserved the target gene. In this invention, it specifically refers to a microorganism that has preserved the entire V gene. H The H fragment gene in E. coli TG1.

[0044] The term "phage display technology" refers to inserting the gene sequence of a foreign protein into an appropriate position in the gene region of a bacteriophage that encodes the capsid protein, so that the foreign protein can be expressed on the capsid protein on the surface of the bacteriophage as the bacteriophage proliferates.

[0045] The term "EC" 50 "" refers to the half-maximal effect concentration, in this invention EC 50 The lower the value, the higher the antibody-antigen binding activity.

[0046] Advantages and positive effects:

[0047] Unlike traditional monoclonal antibodies, nanobodies, such as those derived from camelids (V), are different. H The H fragment contains only the variable domain of the antibody heavy chain. It is the smallest natural antigen-binding domain, with a molecular weight of approximately 15 kDa, which is 1 / 10 the size of a traditional antibody. Its small size allows it to bind to narrow epitopes that are typically inaccessible to full-length antibodies, and it can also penetrate the blood-brain barrier to reach regions inaccessible to traditional antibodies. Nanobodies exhibit high specificity, high affinity, and stability, and are easy to store and transport over long distances. Their simple structure makes them easy to modify, and their preparation process is faster and less costly than that of traditional monoclonal antibodies. The nanobody (F-E2) provided by this invention, compared to commercially available traditional monoclonal antibodies, features high specificity, high accuracy, cost-effectiveness, and ease of operation. It is suitable for research in pathogen detection, diagnostic kit development, targeted drug delivery, and neutralizing antibody therapy. Furthermore, its high stability, high sensitivity, and strong specificity make it particularly advantageous in detection applications. Attached Figure Description

[0048] Figure 1 The image shows the purification results of RSV F (post-F) protein.

[0049] Figure 2 The image shows the results of PCR amplification of the nanobody gene sequence using specific primers.

[0050] Figure 3 The results show the calculated library size of the RSV F protein nanobody library for plate detection.

[0051] Figure 4 The results of monoclonal phage ELISA for various nanobodies (Nbs) are shown in the figure.

[0052] Figure 5 The figure shows the protein purification results of seven candidate nanobodies.

[0053] Figure 6 To validate the binding activity of seven candidate nanobodies to RSV post-F protein using ELISA.

[0054] Figure 7 The image shows the results of indirect immunofluorescence staining screening of seven candidate nanobodies.

[0055] Figure 8 The image shows a comparison of the effects of RSV plaque assay (left) and F-E2-based indirect immunofluorescence assay (right).

[0056] Figure 9 To detect the neutralizing effect of Niservimab monoclonal antibody against RSV virus using indirect immunofluorescence (left figure); the fluorescence intensity was quantitatively and statistically analyzed using ImageJ software; finally, the inhibition curve was fitted using GraphPad, and the half-inhibition concentration (IC50) was calculated. 50 Value (right image)

[0057] Figure 10 A Western blotting diagram showing the binding of nanobody F-E2 to RSV F protein. Detailed Implementation

[0058] This invention is conducted through the following process: 1. Expression and purification of antigen proteins; 2. Immunization of animals; 3. Establishment of phage libraries; 4. Screening using phage display technology; 5. Characterization of nanobodies; 6. Establishment and optimization of a methodology for detecting RSV using indirect immunofluorescence.

[0059] We obtained PBMCs by immunizing camels with antigens, collecting blood after four rounds of immunization, and then separating the PBMCs. Nanobodies (V) were then amplified using specific primers. H The H) sequence was then cloned into phage particles, and a phage library was constructed using electroporation. Specific nanobody sequences were then screened using phage ELISA, and the nanobodies were expressed via a eukaryotic system. The binding of the nanobodies to the F protein was then identified using ELISA and Western blotting. Finally, the staining effect of the nanodetector antibody was verified using indirect immunofluorescence. Therefore, the nanobody F-E2 provided by this invention can be further developed and prepared as a detection antibody reagent for RSV detection.

[0060] The present invention will now be described in detail with reference to the accompanying drawings (unless otherwise specified, the methods used in the following embodiments are conventional methods, and the reagents used are commercially available unless otherwise specified):

[0061] Example 1: Expression and purification of antigen protein post-F

[0062] Based on the protein sequence described in the literature, we designed and synthesized a PTT5-his plasmid vector (Addgene, #52326) for the RSV post-F protein (SEQ ID NO:6), and transformed it into a eukaryotic expression system HEK 293F cells (Thermo Fisher Scientific, R70007) using standard methods. The cells were cultured at 37°C, 120 rpm, and 5% CO2 for 4 days, after which the cell supernatant was collected. The supernatant was filtered through a 0.22 μm filter, and the filtrate was passed through a nickel column to adsorb the target protein. An elution buffer containing a high concentration (400 mM) of imidazole was prepared, and the protein specifically bound to the nickel column was eluted and recovered using a protein purification instrument (Union-Biotech Co., Ltd., UEV 25D). The obtained proteins were used as samples for SDS-PAGE electrophoresis.

[0063] The result is as follows Figure 1 As shown, a band containing a single target protein (i.e., post-F protein) was obtained. This was used as an antigen immunoassay reagent for immunizing camels.

[0064] Example 2: Immunization, Library Construction and Screening

[0065] Camels were immunized four times with the RSV post-F protein obtained in Example 1, with each immunization occurring 30 days apart. The antigen protein was administered at 0.5 mg per immunization. Finally, serum ELISA was performed, and the antibody titer reached over 10,000. Blood was collected and PBMCs were isolated. Total RNA was extracted, reverse transcribed into cDNA, and then processed using specific primers (upstream and downstream primer sequences are shown in SEQ ID NO:7 and SEQ ID NO:8) via conventional PCR methods (see [reference]). Instructions for use of Max DNA Polymerase (#R045A) to amplify V H H fragments, such as Figure 2 The results are shown.

[0066] V HThe H fragment was cloned into the NotI and NocI restriction sites of pR2 phagemid (BioVector, 67739) to form a recombinant vector. This recombinant vector was then transformed into *E. coli* TG1 competent cells (HonorGene, HG-VSW0286) via electroporation to establish a cell line containing V... H Phage libraries containing the H fragment. For example... Figure 3 The results show that the final library size is 1.35 × 10⁻⁶. 8 The prepared phage library was then stored at -80°C.

[0067] The screening process was completed using phage display technology (wherein, the phage display technology is a conventional technique known in the prior art, for example, see the literature: Jaroszewicz, Weronika et al. “Phage display and other peptide display technologies.” FEMS microbiology reviews vol.46,2(2022):fuab052.). The specific steps are as follows: Frozen library bacteria were taken, amplified, and then KM13 helper phage (New England Biolabs, N0315S) was added to help the phage continuously amplify, thereby enriching the nanobodies. Through two rounds of biopanning, antigen-specific nanobodies were screened out. After identification by monoclonal phage ELISA, the positive clones of phage were subjected to gene sequencing to obtain nanobodies V. H H sequence.

[0068] like Figure 4 The results showed that among the 96 positive clones screened by the post-F protein, there were 86 different nanobody sequences. Based on the grouping of the sequences, we initially selected 7 candidate antibodies, namely antibody F-E2 (i.e., the antibody of this invention), F-G11, F-A1, F-A5, F-H1, F-G8 and F-A11.

[0069] Example 3: Characterization of the binding activity and fluorescence screening of candidate nanobodies

[0070] To explore the functions of the seven candidate nanobodies, we first constructed the nucleotide sequences of all seven candidate nanobodies into the PTT5-Fc vector (Addgene, #52326), transfected it into HEK 293F cells (Thermo Fisher Scientific, #R70007) for eukaryotic expression, and cultured them on a shaker at 37°C, 5% CO2, and 120 rpm for 4 days. The cell supernatant was then collected. The supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was passed through a Protein A column with 0.1 M acetic acid elution buffer. The target protein was eluted using a protein purification instrument (Union-Biotech Co., Ltd; UEV 25D). The concentrated protein was then subjected to SDS-PAGE electrophoresis, and the results are shown below. Figure 5 As shown, seven candidate nanobodies with high purity were obtained. The Fc fragment was used solely for labeling the nanobodies; its size and binding relationship did not affect the binding of the nanobodies to the antigen.

[0071] Subsequently, to explore the binding activity of the seven candidate nanobodies, we first used post-F as the antigen. One day in advance, we plated the purified post-F protein onto an ELISA plate at a concentration of 1 μg / mL, 100 μL / well. We then diluted the nanobodies starting from 30 nM, making seven 1:3 dilutions, and incubated them with the antigen for 1 hour. We then diluted HRP-Rabbit Anti-Human IgG-Fc (Sino Biotech, #10702-T16-H) as the secondary antibody and incubated it at room temperature for 1 hour. The colorimetric values ​​were then read.

[0072] like Figure 6 The results show that the EC values ​​of F-A5, F-E2, and F-G8 are... 50 The values ​​range from 0.04 to 0.06 nM, and compared with the remaining two antibodies F-A1 and F-A11, they have a higher affinity for RSV F.

[0073] To further determine whether these nanobodies could bind to the native F protein on the membrane, we conducted an immunofluorescence screening experiment. The study found that the mpe8 antibody (Wuhan Bailubo Biotechnology Co., Ltd., RVV02814) could be used to detect RSV (Reference: Xun, Guangjin et al. “Potent Human Single-Domain Antibodies Specific for a Novel Prefusion Epitope of Respiratory Syncytial Virus FGlycoprotein.” Journal of Virology vol. 95, 18(2021):e0048521). Therefore, we selected the mpe8 antibody as the positive screening antibody.

[0074] like Figure 7 The results showed that, compared with the positive antibody mpe8, the staining fluorescence of antibody F-E2 was clear, and it could specifically recognize the natural F protein.

[0075] Example 4: Verification and Advantages of F-E2 Detection Performance

[0076] To compare the advantages and disadvantages of the classic plaque assay and indirect immunofluorescence assay for RSV, we conducted both experiments using the same virus. Vero cells (biocode, #C5168) were seeded one day in advance in 12-well and 96-well plates to ensure a confluence of approximately 70% by the next day. Using serum-free DMEM medium, RSV virus (National Virus Resource Center [NVRC]) was diluted 1:10, with five dilutions, resulting in a total virus dilution factor of 10. -1 10 -2 10 -3 10 -4 10 -5 After co-incubating the virus and cells for 1 hour, for the classic plaque assay, after removing the virus solution, add 1 mL of DMEM maintenance medium (Thermofisher, #11995065) containing 0.9% sodium carboxymethyl cellulose (Sigma, M0512) per well, and place in a cell culture incubator for continued culture. When plaque formation is observed, fix the cells with 4% paraformaldehyde (Wuhan Sewell Biotechnology Co., Ltd., G1101-500ML), add 1 mL of crystal violet staining solution, stain at room temperature for 15 min, gently rinse the wells with running water, and observe the plaques formed by cell shedding after rinsing.

[0077] For the indirect immunofluorescence assay, cells were divided into 4 groups (n=4) and subjected to the following conditions. After approximately 72 hours of culture, the cells showed obvious cytopathic effects. The cell plates were recovered and fixed with 4% paraformaldehyde (Wuhan Sewell Biotechnology Co., Ltd., G1101-500ML), and then sealed overnight at 4°C with 5% skim milk (Biosharp, BS102 500g). The detection antibody F-E2 was added to react with the RSV antigen, and finally, the secondary antibody FITC-anti-human IgG-Fc (Proteintech, #SA00003-12) was added. After staining with DAPI (Sigma Aldrich, 28718-90-3), the fluorescence signal was observed under a fluorescence microscope.

[0078] like Figure 8 The results show that the traditional plaque assay has certain limitations. The plaques are small and the boundaries are unclear, making it difficult to accurately quantify them. However, the indirect immunofluorescence assay based on F-E2 can not only perform qualitative analysis intuitively, but also detect and analyze the fluorescence intensity using ImageJ software. The viral titer can be quantitatively detected based on the fluorescence intensity, and the results can be used to objectively, quickly and accurately calculate the viral titer.

[0079] Example 5: Establishment and application of RSV neutralization immunofluorescence method

[0080] To further explore whether the RSV neutralization immunofluorescence method based on F-E2 is suitable for detecting antibody neutralization titers, we selected Nirsevimab (Pujian Biotechnology Co., Ltd., DVV02802), a newly marketed RSV prophylactic antibody, as a positive antibody. Nirsevimab is a fully human monoclonal antibody with strong RSV neutralizing activity; its neutralizing activity against clinical isolates is approximately 50 times higher than that of Palvizumab. Cells were divided into 3 groups (n=3). Nirsevimab monoclonal antibody was set in 8 gradients, starting from 1 μg / ml and diluted 3-fold. After incubating Nirsevimab monoclonal antibody with virus, it was added to cultured cells. Finally, RSV viral titer was determined by immunofluorescence assay (refer to Xun G, Song X, Hu J, et al. Potent Human Single-Domain Antibodies Specific for a Novel Prefusion Epitope of Respiratory Syncytial Virus F Glycoprotein. J Virol. 2021; 95(18):e0048521.).

[0081] like Figure 9 The results showed that F-E2 could be used to detect the neutralizing effect of Nirsevimab on RSV A subtypes, as verified by fluorescence imaging. Statistical analysis of fluorescence intensities at different concentration gradients was performed using ImageJ software. Figure 9 (Left), the suppression curve was fitted using GraphPad ( Figure 9 (Right) and calculate the half-inhibition concentration (IC50). 50 The result can be used to calculate the IC50 value of Nirsevimab against RSV virus. 50 The concentration was 37.03 ng / ml, further demonstrating the accuracy of F-E2 in the neutralization assay for RSV.

[0082] Example 5: Linear epitope detection of F-E2 nanobodies

[0083] In the above embodiments, we identified the binding of F-E2 to the conformational epitope of the antigen using an ELISA experiment. Therefore, we wanted to further detect whether F-E2 binds to the linear epitope of the antigen using a Western blotting experiment. First, we collected the supernatant of the virus (RSVA2 strain) and heated it in a metal bath at 100°C for 10 minutes to inactivate the protein and open its secondary structure. We then used polypropylene gel electrophoresis to separate the protein that had become linear due to inactivation. The electrophoretically separated components were then transferred from the gel to a PVDF membrane (Merck Millipore, #IPVH00010). Next, we added the detection antibody F-E2-Fc (dilution ratio 1:500) to the PVDF membrane and incubated it at room temperature for 1 hour. Then, we added the secondary antibody Rabbit Anti-Human IgG-Fc Secondary Antibody (HRP) (Sino Biotech Ltd., #10702-T16-H-100, dilution ratio 1:8000) and incubated it at room temperature on a shaker at 80 rpm for 1 hour. Antibodies specifically bind to target antigen epitopes attached to the membrane. After incubation with secondary antibodies to amplify the binding signal, developing solution is added, and the mixture is exposed under a chemiluminescence imaging device to qualitatively analyze the antigen-antibody binding.

[0084] like Figure 10 The results showed that F-E2-Fc specifically binds to the linear epitope of the F glycoprotein on the surface of respiratory syncytial virus, meaning that F-E2 has excellent binding ability to both the spatial conformational epitope and the linear epitope of the post-F protein.

[0085] In summary, this invention screened a detection nanobody, F-E2, using phage display technology. ELISA and Western blotting experiments confirmed that this nanobody specifically binds to the F glycoprotein on the surface of respiratory syncytial virus (RSV). Finally, indirect immunofluorescence experiments verified that the F-E2 nanobody exhibits good antigen-binding and secondary antibody-binding capabilities. Therefore, the F-E2 nanobody provided by this invention can be further developed into detection antibody reagents and / or detection kits for RSV detection. Sequence Listing:

[0086] SEQ ID NO:1: F-E2 amino acid sequence

[0087] QVQLVESGGGSVQPGGSLRLSCAAPGYIYSSGCCMGWFRQAPGKE

[0088] REGVAVRYIGGGNTYYFDLVKGRFTISQDSAKNTLYLQMNSLRPE

[0089] DTAMYYCAADVDPVRCSLMGEPWQYNYRGQGTQVTVSS

[0090] SEQ ID NO:2: Encoding the F-E2 nucleotide sequence

[0091] CAGGTGCAGCTCGTGGAGGTCTGGGGGAGGCTCGGTGCAGCCTG

[0092] GAGGGTCTCTGAGACTCTCCTGTGCAGCCCCTGGATACATCTAC

[0093] AGTAGCGGGTGCATGGGTTGGTTCCGCCAGGCTCCAGGAAAAG

[0094] AGCGCGAGGGGGTCGCAGTTAGATATATTGGTGGTGGTAACACA

[0095] TACTATTTCGACCTCGTGAAGGGCCGATTCACCATCTCCCAAGA

[0096] CAGCGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGA

[0097] CCTGAGGACACTGCCATGTACTACTGTGCGGCAGACGTCGACC

[0098] CCGTACGGTGCTTCTCAATGGGCGAGCCGTGGCAGTATAACTAC

[0099] AGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0100] SEQ ID NO:3: F-E2 CDR1 amino acid sequence

[0101] SGCMG

[0102] SEQ ID NO:4: F-E2 CDR2 amino acid sequence

[0103] VRYIGGGNTYYFDLVKG

[0104] SEQ ID NO:5: F-E2 CDR3 amino acid sequence

[0105] DVDPVRCSLMGEPWQYNY

[0106] SEQ ID NO:6: RSV post-F protein amino acid sequence

[0107] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSAL

[0108] RTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTEL

[0109] QLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRA

[0110] IASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGS NICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLFPSDEFDASISQVNEKINQSLAFIRKSDELL

[0111] SEQ ID NO:7: Upstream primer

[0112] GCTGCACAGCCTGCTATGGCACAGKTGCAGCTCGTGGAGTCTGGGGG

[0113] SEQ ID NO:8: Downstream primer

[0114] GAGTTTTTGTTCGGCTGCTGCTGAGGAGACGGTGACCTGGGTCCCC

[0115] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanobody comprising CDR1 as shown in SEQ ID NO:3, CDR2 as shown in SEQ ID NO:4, and CDR3 as shown in SEQ ID NO:

5.

2. The nanobody according to claim 1, wherein, The amino acid sequence of the nanobody is shown in SEQ ID NO:

1.

3. The nanobody according to claim 1 or 2, wherein, The nanobody specifically binds to the spatial conformational epitope and / or linear epitope of the respiratory syncytial virus surface fusion F glycoprotein.

4. A polynucleotide encoding a nanobody according to any one of claims 1-3.

5. The polynucleotide according to claim 4, the sequence of which is shown in SEQ ID NO:

2.

6. An expression vector comprising the polynucleotide of claim 4 or 5.

7. A host cell comprising the expression vector of claim 6.

8. The use of the nanobody according to any one of claims 1-3 in the preparation of a diagnostic agent or kit for detecting respiratory syncytial virus (RSV) infection.

9. A method for detecting respiratory syncytial virus (RSV) in a sample, comprising adding a nanobody according to any one of claims 1-3 to the sample, the method being used for non-disease diagnostic purposes.

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