An alpaca-derived nanobody screened by phage display technology that binds to HPV16-L1 protein and its applications

Abstract

The present invention belongs to the technical fields of molecular virology and immunology, and provides a camelid-derived nanobody screened by phage display technology that binds to the HPV16-L1 protein, with a heavy chain variable region VHH, comprising the following CDRs and FRs: CDR1 shown in SEQ ID NO:1; CDR2 shown in SEQ ID NO:2; CDR3 shown in SEQ ID NO:3; FR1 shown in SEQ ID NO:4; FR2 shown in SEQ ID NO:5; FR3 shown in SEQ ID NO:6; FR4 shown in SEQ ID NO:7. It provides potential new nanobody drugs for the clinical prevention, treatment and detection of human papillomavirus HPV16. It has a small molecular weight, low immunogenicity, high solubility, strong stability, can reach the infection site directly, and takes effect faster, providing a potential treatment strategy for virus infection.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular virology and immunology, and particularly relates to a camelid nanobody that binds to the HPV16-L1 protein screened by phage display technology and its applications. Background Art

[0002] Human papillomavirus (HPV) is a group of small, non-enveloped DNA viruses that infect epithelial tissues. Human papillomavirus type 16 (HPV16) belongs to the mucosal carcinogenic type of HPV and mainly infects the mucocutaneous tissues of the urogenital tract, perianal region, and oropharynx. The persistent infection of HPV16 is closely related to the incidence of cervical cancer, and the detection rate of HPV16 in cervical cancer worldwide reaches 50-60%. Cervical cancer is the third most common malignant tumor in women worldwide, with an annual incidence of approximately 527,000, posing a serious threat to the lives and health of women.

[0003] The HPV genome can be divided into three regions: the long control region (LCR), the early open reading frame (EORF), and the late open reading frame (LORF). The coding gene of the L1 protein is located in the LORF and is the major capsid protein of HPV, mainly inducing type-specific neutralizing antibodies and protective responses. Currently, the three HPV vaccines on the market are all assembled into virus-like particles (VLPs) based on non-infectious recombinant specific L1 capsid proteins and purified in the form of VLPs as immunogens. Among them, the nine-valent HPV vaccine has the potential to prevent up to 90% of cervical cancer cases. Therefore, the HPV L1 protein, as a potential vaccine component, has received extensive attention in HPV vaccine research.

[0004] Nanobodies are a type of antibody that naturally lacks a light chain found in the peripheral blood of camels. Such antibodies only contain 1 heavy chain variable region (VHH) and 2 conventional CH2 and CH3 regions, but they are not as prone to sticking to each other or even aggregating into clumps as artificially engineered single-chain antibody fragments. More importantly, the VHH structure cloned and expressed alone has structural stability equivalent to that of the original heavy chain antibody and antigen-binding activity, and is the smallest known unit capable of binding to the target antigen. VHH is highly soluble and stable, and its single-domain property and strict monomeric characteristics make it easy to clone, and it can be quickly screened from an immune nanobody library using phage display technology to screen for antibodies. These advantages have gradually made nanobodies a hot spot in the development of a new generation of therapeutic antibodies. Summary of the Invention

[0005] The object of the present invention is to provide a camelid nanobody screened by phage display technology and binding to HPV16-L1 protein. Further, the present invention provides a camelid nanobody or its antigen-binding fragment binding to human papillomavirus HPV16-L1 protein, a polynucleotide encoding the same, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a preparation method thereof, a transformed cell, and a pharmaceutical composition containing the above.

[0006] The nanobody obtained in the present invention is a nanobody with high binding activity to HPV16-L1 protein, with a small molecular weight of about 12 KD. It can be made into various dosage forms such as sprays, injections, oral medications, etc., and can reach the infection site directly, with a faster onset of action, providing a potential treatment strategy for virus infections.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A camelid nanobody screened by phage display technology and binding to HPV16-L1 protein, having a heavy chain variable region VHH, and the VHH contains the following complementarity-determining regions CDR and framework regions FR within the antibody variable region:

[0009] CDR1 with the amino acid sequence shown in SEQ ID NO:1, namely GIIFSTTVL;

[0010] CDR2 with the amino acid sequence shown in SEQ ID NO:2, namely TITRGGTT;

[0011] CDR3 with the amino acid sequence shown in SEQ ID NO:3, namely NVGDAVAWGY;

[0012] FR1 with the amino acid sequence shown in SEQ ID NO:4, namely ESGGGLVPGGSLRLSCVAS;

[0013] FR2 with the amino acid sequence shown in SEQ ID NO:5, namely GWYRQAPGKQRESVA;

[0014] FR3 with the amino acid sequence shown in SEQ ID NO:6, namely NYADSVKGRFTISRDNAKN TVYLQMN SLKLEDTATYYC;

[0015] FR4 with the amino acid sequence shown in SEQ ID NO:7, namely WGQGTHVIVSS;

[0016] FR1-4 and CDR1-3 are arranged in sequence in an interleaved manner. The amino acid sequence of the heavy chain variable region VHH is as shown in SEQ ID NO:8, wherein: positions 1-19 are the framework region FR1, positions 20-28 are CDR1, positions 29-43 are the framework region FR2, positions 44-51 are CDR2, positions 52-89 are the framework region FR3, positions 90-99 are CDR3, and positions 100-110 are the framework region FR4.

[0017] The amino acid sequence of the HPV16-L1 protein is as shown in SEQ ID NO:10.

[0018] A nucleotide encoding the alpaca-derived nanobody or its antigen-binding fragment as described above, and the sequence of the nucleotide is as shown in SEQ ID NO:9.

[0019] A nucleic acid construct comprising the above nucleotide.

[0020] An expression vector comprising the above nucleic acid construct.

[0021] A transformed cell comprising the above nucleotide, nucleic acid construct or expression vector.

[0022] A pharmaceutical composition comprising an alpaca-derived nanobody or its antigen-binding fragment that specifically binds to human papillomavirus HPV as described above, the nucleotide, nucleic acid construct or expression vector as described above, or a transformed cell, and a pharmaceutically acceptable carrier and / or excipient.

[0023] The pharmaceutical composition is a spray, suppository or transmucosal preparation.

[0024] Furthermore, the spray is an aerosol, nebulizer and powder inhaler; the suppository is a solid preparation or semi-solid preparation; the transmucosal preparation is a transdermal agent, ointment, plaster, topical liquid preparation, injectable or pushable preparation.

[0025] Use of the alpaca-derived nanobody that binds to the HPV16-L1 protein screened by phage display technology as described above, or a nucleotide, nucleic acid construct or expression vector, or a transformed cell in the preparation of a drug for preventing, treating and / or detecting human papillomavirus infection, wherein the human papillomavirus is the HPV16-L1 original strain and / or the HPV16-L1 variant strain.

[0026] The dosage of the active ingredient of the pharmaceutical composition of the present invention varies depending on the administration object, object site, symptoms, administration method, etc. It can be determined according to the doctor's judgment by considering the type of dosage form, administration method, age and weight of the patient, symptoms of the patient, etc.

[0027] The present invention is directed to the development of nanobody drugs against HPV16. Specific nanobodies were screened from a llama phage library using the HPV16-L1 protein. With the HPV16-L1 protein as a "bait", nanobodies that specifically bind to HPV16-L1 with high affinity were screened. The nanobody obtained in the present invention is subsequently named NB1 nanobody. The NB1 nanobody of the present invention can bind to HPV16-L1 with high efficiency and specificity. The present invention provides potential new nanobody drugs for the clinical prevention, treatment, and detection of human papillomavirus HPV16. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] One or more embodiments are illustrated by way of example in the accompanying drawings, and these exemplary illustrations do not constitute a limitation on the embodiments. The specific term "exemplary" used herein means "serving as an example, embodiment, or illustration". Any embodiment described as "exemplary" herein need not be construed as superior to or better than other embodiments.

[0029] Figure 1 It is a schematic diagram of the SDS-PAGE identification result of HPV16-L1 in Example 1 of the present invention;

[0030] Figure 2 It is a result diagram of the plate detection of the library capacity of the HPV16-VHH nanolibrary in Example 3 of the present invention;

[0031] Figure 3 It is a result diagram of the library abundance of the HPV16-VHH nanolibrary detected by plate in Example 3 of the present invention;

[0032] Figure 4 It is a result diagram of the insertion rate of the HPV16-VHH nanolibrary detected by PCR in Example 3 of the present invention;

[0033] Figure 5 It is a schematic diagram of the SDS-PAGE identification result of the prokaryotic expression of the NB1 nanobody in Example 5 of the present invention;

[0034] Figure 6 It is a schematic diagram of the SDS-PAGE and molecular sieve chromatography identification results of the refolding of the inclusion body of the prokaryotic expression of the NB1 nanobody in Example 5 of the present invention;

[0035] Figure 7 It is a schematic diagram of the SDS-PAGE and molecular sieve chromatography identification results of the eukaryotic expression of the NB1 nanobody in Example 5 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative work fall within the scope of protection of the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. All materials cited herein and the materials they cite will be incorporated by reference.

[0038] Equivalent technologies of the specific embodiments described that can be understood by those skilled in the art through routine experiments will be included in this application.

[0039] In addition, to better illustrate the present invention, numerous specific details are given in the following specific embodiments.

[0040] Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the instruments and equipment used in the following embodiments are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following embodiments are all obtained from conventional biochemical reagent stores.

[0041] Definition: "Nanobody", that is, "heavy chain single domain antibody", this type of antibody only contains one heavy chain variable region (VHH), and compared with other antibodies, the light chain is naturally absent.

[0042] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding refers to determining the binding reaction of a protein such as the nanobody of the present invention with the HPV16-L1 protein in a heterogeneous population of proteins and / or other biological reagents. Therefore, under the specified conditions, a specific ligand / antigen binds to a specific receptor / antibody and does not bind to other proteins present in the sample in a significant amount.

[0043] The reagents used in the following embodiments of the present invention, such as enzymes, culture media, antibiotics, and milk, etc., and some commonly used biological materials, such as competent cells, vectors, helper phages, cells to be transformed, etc., are all commercially available products. Some synthetic biological materials, such as primers, sequences, etc., which need to be artificially synthesized, are all commissioned to be synthesized by Genewiz Biotechnology Co., Ltd. The HPV16-L1 protein of the present invention was obtained in the inventor's laboratory (see Example 1).

[0044] Example 1: Expression and Purification of the Antigen HPV16-L1 Protein

[0045] At the 3' end of the HPV16-L1 protein coding sequence (L1, GenBank accession number No. AACO9292, 1879 - 2385, as shown in SEQ ID NO:10), the coding sequence of a 6-histidine tag (hexa-His-tag) and the translation termination codon TGA were ligated. Through the restriction enzyme sites EcoRI and XhoI, it was constructed into the pCAGGS vector and transfected into 293F cells. After 9 days of culture, the supernatant was collected by centrifugation, filtered through a 0.22 μm filter membrane, and purified by nickel ion affinity chromatography. The identification result of the purified protein by SDS-PAGE is as Figure 1 shown. The size of the HPV16-L1 protein is about 56 KD, and a relatively pure target protein was obtained.

[0046] Example 2: Alpaca Immunization

[0047] 1. Immunization procedure: Select adult male alpacas in good physical condition. Before immunization, collect 10 ml of coagulant-promoting blood from the experimental alpacas, separate the serum, and store it for later use. 100 μg of the HPV16-L1 protein prepared in Example 1 was diluted to a final volume of 1 ml with PBS and used to immunize the experimental alpacas by subcutaneous multi-point injection. The first immunization was emulsified with complete Freund's adjuvant. After that, immunization was carried out every two weeks with incomplete Freund's adjuvant. After the 3rd immunization, collect the coagulant-promoting blood of the alpaca, separate the serum, and detect the immunization effect by ELISA. 12 days after the 4th immunization, collect anticoagulated venous blood from the jugular vein for the isolation of peripheral blood lymphocytes (PBMC).

[0048] . Immunization identification: Add 100 μL of the diluted HPV16-L1 protein to a specific ELISA coating plate, set blank wells (only add coating solution) and negative control wells (add non-immunized serum), and let it stand overnight at 4 °C; discard the coating solution from the overnight-coated ELISA plate, wash it with PBS, add 5% calf serum, and incubate at 37 °C for 40 min. After the blocking is completed, wash the coating plate and blot dry the washing solution with filter paper; let the collected coagulant-promoting blood stand at room temperature for two hours to allow natural separation of serum, collect the immune serum with a sterile collection tube, and gradient-dilute the non-immune serum and immune serum with sample diluent. The gradient-diluted serum was used as the primary antibody for the reaction. HRP-labeled llama was used as the secondary antibody. After the reaction, add the TMB-hydrogen peroxide urea solution chromogenic solution, develop color at 37 °C in the dark for 15 min, and then add the termination solution to terminate the reaction. Measure the absorbance value with an ELISA reader at a wavelength of 450 nm within 15 min. The results are shown in Table 1. The non-immune serum does not contain antibodies against HPV16-L1, while the positive reaction in the immune whole serum is very strong and still shows a positive reaction after dilution 10 -2 times.

[0049] Table 1 Neutralization effect of alpaca immune serum against HPV16-L1 antigen

[0050]

[0051] Example 3: Construction of Antibody Library

[0052] 1. Library construction: Dilute the collected anticoagulated venous blood with 1× PBS at a ratio of 1:1. Add 15 ml of lymphocyte separation solution to the bottom of a 50 ml centrifuge tube, slowly add 15 ml of diluted anticoagulated blood along the tube wall, centrifuge at 2000 rpm and 20 °C for 20 minutes, and aspirate the liquid of the PBMC layer with a pipette; wash with 1× PBS and centrifuge at 3000 g to collect the precipitate to obtain peripheral blood mononuclear cells (PBMCs), and after counting, aliquot into small tubes for standby.

[0053] Add the isolated PBMCs to TRIzol, and extract total RNA according to the steps in the instruction manual. Using the extracted total RNA as a template, synthesize cDNA with the long-chain cDNA reverse transcription kit developed by TaKaRa Reagent Company. Using cDNA as a template, perform the first-round PCR experiment with specific primers CALL001 and CALL002 (the primer sequences are shown in Table 2), where Call001-F corresponds to the Leader region of the alpaca antibody, and Call002-R corresponds to the second constant region (CH2) of the alpaca antibody. The PCR reaction products mainly contain two kinds of amplified products with sizes of 900 bp and 700 bp. Cut and recover the band with a size of 700 bp.

[0054] Table 2 Primers for the First-round PCR Reaction

[0055]

[0056] Table 3 Reaction System for the First-round PCR

[0057]

[0058] Table 4 Reaction Conditions for the First-round PCR

[0059]

[0060] Connect the first-round PCR gel recovery product to the pMD19-T simple vector, the reaction system is shown in Table 5, transfer the ligation product into DH5α competent cells, pick monoclonal colonies and determine their base sequences. Analyze the sequencing results with Vector NTI software and design the second-round PCR primers VHH2-F and VHH2-R for library construction.

[0061] Table 5 Ligation System

[0062]

[0063] The 700 bp DNA fragment after the first-round PCR recovery was used as a template, and the second-round PCR was performed using primers VHH2-F and VHH2-R to amplify the nanobody (VHHs) sequence. The primer sequences are shown in Table 6, and the PCR reaction conditions are shown in Table 7. The VHHs sequence with a size of about 400 bp was recovered and purified.

[0064] Table 6 Primers for the second-round PCR reaction

[0065]

[0066] Table 7 Second-round PCR reaction conditions

[0067]

[0068] Using the double digestion method, the VHHs fragment was ligated into the plasmid pMES4 through the restriction enzyme sites PstⅠ and BstEⅡ. The purified cloning vector and electrocompetent E. coli TG1 cells were mixed, and the cloning vector was transformed into electrocompetent E. coli TG1 cells using an electroporator. The incubated bacterial solution was centrifuged to discard the supernatant, and the precipitate was resuspended in 8 ml of fresh LB medium. 100 μl of the bacterial solution was taken for gradient dilution and then spread on solid culture plates to determine the library capacity, with two plates for each gradient. The remaining bacterial solution was all spread on the selective medium containing AMP. After standing overnight for culture, all the colonies were collected in LB medium, centrifuged and the supernatant was discarded. The cells were resuspended in LB liquid medium containing 15% glycerol, and the tubes were sealed to obtain the prepared primary antibody library. 100 μl of the primary library bacteria was taken to determine the library abundance.

[0069] 2. Phage library capacity and abundance

[0070] A. Method for determining the library capacity: The electrotransformed bacterial solution was gradient diluted, with dilution factors from 10 -1 to 10 -8 ; 100 μl of each dilution was spread on solid culture plates and cultured overnight at 25 °C; the next day, the colonies on the gradient plates were counted to calculate the library capacity, and the results are shown in Figure 2 . There were 163 monoclonal colonies on the 10 -6 dilution plate, and 800 × 163 × 10 6 = 13.04 × 10 10 colonies.

[0071] B. Method for determining the library abundance: The primary library bacterial solution was gradient diluted, with dilution factors from 10 -4 to 10 -10; Take 100 μl of the bacterial solution for each dilution and spread it on a solid culture plate, and culture it overnight at 25 °C; the next day, count the colonies on the gradient plate and calculate the library abundance. The results are as Figure 3 shown, the abundance of the library is 291×10×10 7 =2.91 × 10 10 CFU / ml.

[0072] C. Method for determining the insertion rate and insertion diversity of the library VHH fragment: After the library storage capacity is determined, randomly pick 34 monoclonal colonies from the solid culture plate with the determined storage capacity, culture them overnight with shaking at 37 °C, and collect part of the bacterial solution for PCR identification. Analyze the PCR identification results and sequencing results. The fragment lengths of 34 bacterial solution PCR samples are all consistent with the VHH fragment, and the calculated insertion rate of the library VHH fragment is 100%.

[0073] Example 4: Screening specific nanobodies by phage display technology

[0074] Take the E. coli TG1 transfected with the recombinant plasmid in Example 3, add the VCSM13 helper phage at a multiplicity of infection of about 20, culture overnight, centrifuge to take the supernatant, add PEG6000 / NaCl at a volume ratio of 1:4, mix, place at 4 °C for at least 1 hour and then centrifuge for 30 min, collect the precipitate and resuspend it with PBS, which is the collected phage particles, and measure the phage titer.

[0075] Mix 1×10 10 of the above-collected phages with an equal volume of 5% (w / v) skim milk, add them to a 96-well plate coated with HPV16-L1 antigen, incubate at room temperature for 1 hour, elute the specific phages with 0.2 M glycine, and neutralize the eluted phages with Tris-HCl (pH 9.1). Then infect E. coli TG1 cells with the phages and amplify the phages.

[0076] Prepare a 96-well plate coated with HPV16-L1 antigen again, enrich the phages expressing specific nanobodies through the second round of panning, and perform a total of 3 rounds of panning. After each round of panning, randomly select different single colonies from the agar plate with growing colonies, then add the VCSM13 helper phage and culture overnight at 37 °C, centrifuge the next day to take the phage supernatant for ELISA experiment. When OD 450nM >0.2, it is determined as a positive reaction. Take the corresponding clone, use the specific primers MP57 and GⅢ to sequence the plasmid (the primers are shown in Table 8), and obtain the sequence encoding VHHs in the plasmid. Through sequence determination, the core coding sequence of NB1 is obtained.

[0077] Table 8 Sequencing primers

[0078]

[0079] Example 5: Expression of Nanobody

[0080] After the core coding sequence obtained in Example 4, the coding sequence of six histidine tags (hexa-His-tag) and the translation termination codon TGA were ligated, which was the coding sequence of the NB1 nanobody of the present invention; through the restriction enzyme sites EcoRI and XhoI, it was constructed into the pColdII and pCAGGS vectors to obtain the recombinant plasmids NB1-pColdII and NB1-pCAGGS containing the NB1 nanobody coding sequence.

[0081] Prokaryotic expression: The NB1-pColdII recombinant plasmid was transferred into the E. coli BL21 strain. Single colonies were picked and inoculated into LB liquid medium containing Amp. After culturing at 37 °C for 2 hours, IPTG with a final concentration of 0.3 mM was added to induce the expression of the target protein, and the culture was continued overnight at 16 °C. Then, the cell pellet was collected by centrifugation at 4000 rpm for 10 min. The cell pellet was resuspended in PBS and sonicated. After centrifugation at 10000 rpm for 30 min, the supernatant and the pellet were collected respectively. The results were identified by SDS-PAGE as Figure 5 shown. The target protein was mainly expressed in the form of inclusion bodies, and only a small amount was expressed in the supernatant.

[0082] The inclusion bodies in the precipitate were collected and dissolved in 10 mmol / L Tris-HCl (pH 7.0, containing 8 mol / L urea, 1 mmol / L DTT). The insoluble matter was removed by centrifugation. Then, a renaturation buffer (100 mmol / L Tris-HCl pH 8.0, 400 mmol / L arginine, 5 mmol / L reduced glutathione, 0.5 mmol / L oxidized glutathione, 0.1 mmol / L PMSF) was prepared for dilution renaturation. The renatured product was analyzed by nickel ion affinity chromatography and gel filtration chromatography (SuperdexTM 75 Increase Hiload column (GE Healthcare)). The results were as Figure 6 shown. The position of the target peak showed that the obtained target protein existed in the form of a multimer.

[0083] Eukaryotic expression: The recombinant plasmid NB1-pCAGGS was transfected into 293F cells. After 9 days of culture, the supernatant was collected by centrifugation at 5000 rpm for 30 min, filtered through a 0.22 μm filter membrane, and then purified by nickel ion affinity chromatography and gel filtration chromatography (SuperdexTM 75 Increase Hiload column (GE Healthcare)) to obtain a relatively pure target protein. The target peak was determined by SDS-PAGE, and the results are as Figure 7 shown, and the purified NB1 nanobody was obtained.

[0084] Example 6: Antigen-antibody specific binding experiment

[0085] The diluted HPV16-L1 protein was added to a specific ELISA coating plate, and blank wells (without adding any liquid) and negative control wells (only adding coating solution) were set. It was left standing overnight at 4 °C. The coating solution of the overnight-coated ELISA plate was discarded, and after washing with PBS, 5% calf serum was added and incubated at 37 °C for 40 min. After the blocking was completed, the ELISA plate was washed, and the washing solution was blotted dry with filter paper. The purified NB1 was serially diluted as the primary antibody, and HRP-labeled llama was used as the secondary antibody for the reaction. After the reaction, TMB-hydrogen peroxide urea solution chromogenic solution was added, and it was developed at 37 °C in the dark for 15 min, and then the reaction was terminated by adding the termination solution. The absorbance was measured at a wavelength of 450 nm by an enzyme-linked immunosorbent assay (ELISA) reader within 15 min. The reaction results are shown in Table 9. The NB1 nanobody had a positive reaction with the HPV16-L1 antigen protein.

[0086] Table 9 Detection of the binding of nanobody to HPV16-L1 antigen

[0087]

[0088] The results showed that: in the present invention, specific nanobodies were screened from an alpaca phage library using the HPV16-L1 protein. Using the HPV16-L1 protein as a "bait", nanobodies that specifically bind to HPV16-L1 with high affinity were screened, and they can bind to HPV16-L1 efficiently and specifically. The present invention provides potential new nanobody drugs for the clinical prevention, treatment, and detection of human papillomavirus HPV16.

[0089] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An alpaca-derived nanobody screened by phage display technology and binding to HPV16-L1 protein, having a heavy chain variable region VHH, characterized in that: The VHH comprises the following complementarity-determining regions CDRs and framework regions FRs within the antibody variable region: CDR1 with the amino acid sequence shown in SEQ ID NO:1, i.e., GIIFSTTVL; CDR2 with the amino acid sequence shown in SEQ ID NO:2, i.e., TITRGGTT; CDR3 with the amino acid sequence shown in SEQ ID NO:3, i.e., NVGDAVAWGY; FR1 with the amino acid sequence shown in SEQ ID NO:4, i.e., ESGGGLVPGGSLRLSCVAS; FR2 with the amino acid sequence shown in SEQ ID NO:5, i.e., GWYRQAPGKQRESVA; FR3 with the amino acid sequence shown in SEQ ID NO:6, i.e., NYADSVKGRFTISRDNAKN TVYLQMN SLKLEDTATYYC; FR4 with the amino acid sequence shown in SEQ ID NO:7, i.e., WGQGTHVIVSS; FR1-4 and CDR1-3 are arranged alternately in sequence. The amino acid sequence of the heavy-chain variable region VHH is shown in SEQ ID NO:8, wherein: positions 1-19 are the framework region FR1, positions 20-28 are CDR1, positions 29-43 are the framework region FR2, positions 44-51 are CDR2, positions 52-89 are the framework region FR3, positions 90-99 are CDR3, and positions 100-110 are the framework region FR4.

2. An alpaca-derived nanobody screened by phage display technology and binding to HPV16-L1 protein according to claim 1, characterized in that: The amino acid sequence of the HPV16-L1 protein antigen is shown in SEQ ID NO:

10.

3. A polynucleotide encoding the alpaca-derived nanobody as claimed in claim 1, characterized in that: The sequence of the polynucleotide is shown in SEQ ID NO:

9.

4. A nucleic acid construct comprising the polynucleotide of claim 3.

5. An expression vector comprising the nucleic acid construct of claim 4.

6. A transformed cell comprising the polynucleotide of claim 3, the nucleic acid construct of claim 4, or the expression vector of claim 5.

7. A pharmaceutical composition comprising the alpaca-derived nanobody specifically binding to the HPV16-L1 protein as described in claim 1 or 2, comprising the polynucleotide of claim 3, the nucleic acid construct of claim 4, or the expression vector of claim 5, or comprising the transformed cell of claim 6, and a pharmaceutically acceptable carrier and / or excipient.

8. The pharmaceutical composition according to claim 7, characterized in that: The pharmaceutical composition is a spray, suppository, or transmucosal preparation.

9. The pharmaceutical composition according to claim 8, wherein: The spray is an aerosol, nebulizer, or powder inhaler; the suppository is a solid preparation or semi-solid preparation; the transmucosal preparation is a transdermal agent, ointment, plaster, topical liquid, injectable or pushable preparation.

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