Specific antibodies against HPV-18 type E protein and uses thereof

Abstract

The present invention discloses specific antibodies against HPV-18 type E protein and uses thereof. The present invention provides a monoclonal antibody against HPV-18 type E protein. The present invention also provides a nucleic acid molecule encoding the aforementioned monoclonal antibody. The present invention also provides a method for producing the aforementioned monoclonal antibody. The present invention also provides uses of the aforementioned monoclonal antibody in the preparation of related products or drugs. The present invention also provides a recombinant vector carrying the aforementioned nucleic acid molecule. The present invention also provides related cells and conjugates.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to specific antibodies against HPV-18 type E protein and uses thereof. Background Art

[0002] Human papillomavirus (HPV) is a group of spherical, tiny, non-enveloped, circular double-stranded DNA viruses belonging to the genus Papillomavirus in the family Papovaviridae. It is epithelial-tropic and mainly causes proliferative lesions of human skin and mucous membranes. It is divided into three categories: high-risk, medium-risk, and low-risk. Symptoms include common warts, genital warts (condyloma acuminatum), etc. With the rapid increase in the incidence of condyloma acuminatum among sexually transmitted diseases and the increase in cervical cancer, anal cancer, etc., human papillomavirus (HPV) infection has attracted more and more attention.

[0003] Monoclonal antibodies (mAbs) are highly uniform antibodies produced by a single B cell clone that target only a specific antigen epitope. Monoclonal antibodies are very important tools in biomedical research, disease diagnosis, and treatment of certain diseases (such as infectious diseases and cancer). Currently, the common monoclonal antibody preparation technologies mainly include hybridoma, phage antibody library, and single B cell technology. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0005] The present invention provides a monoclonal antibody against HPV-18 type E protein, which comprises a heavy chain variable region CDR1 shown in SEQ ID NO: 1, a heavy chain variable region CDR2 shown in SEQ ID NO: 2, a heavy chain variable region CDR3 shown in SEQ ID NO: 3, a light chain variable region CDR1 shown in SEQ ID NO: 9, a light chain variable region CDR2 shown in LTS, and a light chain variable region CDR3 shown in SEQ ID NO: 10.

[0006] Optionally, the monoclonal antibody further comprises heavy chain variable region framework regions FR1, FR2, FR3, FR4 having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 5, 6, 7, and light chain variable region framework regions FR1, FR2, FR3, FR4 having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 11, 12, 13, 14.

[0007] Optionally, the heavy chain variable region of the monoclonal antibody has a heavy chain variable region with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 8, preferably 95% sequence identity, and the light chain variable region has a light chain variable region with at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 15, preferably 95% sequence identity.

[0008] Optionally, the monoclonal antibody further comprises a heavy chain constant region and a light chain constant region.

[0009] Optionally, the monoclonal antibody is derived from human or non-human mammals.

[0010] Optionally, the monoclonal antibody is humanized.

[0011] The present invention provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody or a functional fragment thereof.

[0012] Optionally, the nucleic acid molecule comprises a nucleotide sequence encoding any one of the amino acid sequences of SEQ ID NOs: 1-15.

[0013] Optionally, the nucleotide sequence encoded by the nucleic acid molecule is subjected to codon degeneracy processing.

[0014] The present invention provides a method for producing the above-mentioned monoclonal antibody, which comprises the following steps:

[0015] a) providing the aforementioned nucleic acid molecule;

[0016] b) transforming a host cell with the substance described in step a);

[0017] c) culturing the host cell obtained in step b) under suitable conditions;

[0018] d) isolating and purifying the aforementioned monoclonal antibody in the host cell culture medium.

[0019] The present invention provides any of the following uses of the aforementioned monoclonal antibody:

[0020] 1) Use in the preparation of products for detecting papillomavirus;

[0021] 2) Use in the preparation of products for testing E protein;

[0022] 3) Use in the preparation of products for diagnosing papillomavirus;

[0023] 4) Use in the preparation of drugs for regulating E protein;

[0024] 5) Use in the preparation of drugs for preventing or treating papillomavirus;

[0025] Optionally, the papillomavirus includes epithelial HPV and mucosal HPV;

[0026] Optionally, the epithelial HPV includes HPV-1, HPV-5, HPV-8, HPV-14, HPV-20, HPV-21, HPV-25, and HPV-47;

[0027] Optionally, the mucosal HPV includes HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-41, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-70, and HPV-72;

[0028] Optionally, the papillomavirus is HPV-18;

[0029] Optionally, the E protein includes HPV virus E protein;

[0030] Optionally, the HPV virus E protein includes HPV virus E1, E2, E3, E4, E5, E6, E7, and E8 proteins.

[0031] Optionally, the drug includes various pharmaceutically acceptable carriers in the art. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dosage and concentration used, and may specifically include but are not limited to: buffers, such as acetate, Tris, phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as blood albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; tonicity modifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactants such as polysorbates; salt-forming counterions such as sodium; metal complexes (such as Zn-protein complexes); and / or nonionic surfactants such as Or polyethylene glycol (PEG). Pharmaceutical preparations for in vivo administration are generally sterile, and methods for achieving sterility of pharmaceutical preparations should be known to those skilled in the art, for example, it can be achieved by filtering with a sterile filter membrane or the like. Those skilled in the art can also select a suitable pharmaceutically acceptable carrier according to the dosage form required for the pharmaceutical composition to prepare it into different dosage forms, for example, the pharmaceutical composition of the present invention can be in various dosage forms including but not limited to tablets, injections, lyophilized agents, etc.

[0032] The present invention provides a recombinant vector, which comprises the nucleic acid molecule described above;

[0033] Optionally, the recombinant vector comprises a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector or an artificial chromosome;

[0034] Optionally, the recombinant vector is a plasmid.

[0035] The present invention provides a cell, wherein the cell comprises the aforementioned monoclonal antibody, the aforementioned nucleic acid molecule or the aforementioned recombinant vector.

[0036] Optionally, the cell comprises a prokaryotic cell or a eukaryotic cell.

[0037] Optionally, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasmas, chlamydia, and rickettsia.

[0038] Optionally, the bacteria include Gram-positive bacteria and Gram-negative bacteria.

[0039] Optionally, the eukaryotic cells include mammalian cells, insect cells, plant cells other than cyanobacteria, fungal cells, and yeast cells.

[0040] More optionally, the mammalian cells include fibroblasts, lymphocytes, epithelial cells, and myeloblasts.

[0041] The present invention provides a conjugate, which comprises the above-mentioned monoclonal antibody and a substance conjugated to the antibody.

[0042] Optionally, the substance does not affect the function of the antibody itself after being conjugated to the antibody.

[0043] Optionally, the substance includes a fluorescent dye, a chemiluminescent compound, a radioactive isotope, an enzyme, or biotin.

[0044] Optionally, the enzyme includes horseradish peroxidase, alkaline phosphatase, β-galactosidase and acetylcholinesterase.

[0045] Exemplary fluorescent dyes include, but are not limited to, xanthene dyes, fluorescein dyes, rhodamine dyes, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE or J), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine 6G (R6G5 or G5), 6-carboxyrhodamine 6G (R6G6 or G6), and rhodamine 110; cyanine dyes such as Cy3, Cy5, and Cy7 dyes; Alexa dyes such as Alexa-fluor-555; coumarins, diethylaminocoumarins, umbelliferone; benzylamine dyes such as Hoechst 33258; phenanthridine dyes, such as Texas red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, BODIPY dyes, quinoline dyes, pyrene, fluoresceinchlorotriazinyl, R110, Eosin, JOE, R6G, tetramethylrhodamine, lissamine, ROX and naphthofluorescein.

[0046] Optionally, the method for preparing the conjugate should be known to those skilled in the art. For example, the monoclonal antibody can be connected to the functional molecule directly or through a spacer of appropriate length. The connection method can be chemical cross-linking or genetic engineering fusion expression to obtain the conjugate.

[0047] For therapeutic purposes, it may be appropriate to combine with therapeutic effector groups such as radioactive groups, i.e., groups derived from radioisotopes or radionuclides (e.g. 3 H. 14 C. 15 N. 33 P. 35 S. 90 Y. 99 Tc, 111 ln, 123 1. 125 1. 131 1. 201 TI, 213 Bi), a toxin or a cytotoxic group such as a cytostatic group or a group consisting of or including it.

[0048] The monoclonal antibody of the present invention can be coupled with a labeling group (labeled polypeptide), which can then be used, for example, for diagnostic purposes. Suitable labeling groups can be selected from radioisotopes (such as those mentioned above) or groups containing radioisotopes, radionuclides, fluorescent groups (such as fluorescent proteins such as GFP, RFP, etc., dyes, rhodamine, fluorescein and derivatives thereof such as FITC, cyanine dyes), enzyme groups (such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase), chemiluminescent groups, biotin groups, metal particles (such as gold particles), magnetic particles (such as having a core containing magnetite (Fe3O4) and / or hematite (Fe2O3)), predetermined polypeptide groups, etc.

[0049] The immunoconjugate can also be a chimeric antigen receptor (CAR), which can be expressed in immune cells. The "immune cells" and "immune effector cells" are used interchangeably, including: T lymphocytes, NK cells or NKT cells, etc., preferably, NK cells and T lymphocytes. The chimeric antigen receptor generally comprises an extracellular binding region, a transmembrane region and an intracellular signaling region connected in sequence, wherein the extracellular binding region comprises an antibody or fusion protein of the present invention. The design of the transmembrane region and the intracellular signaling region based on CAR technology is well known in the art: for example, the transmembrane region adopts the transmembrane region of molecules such as CD8 and CD28, and the intracellular signaling region adopts the intracellular signaling region of the immunoreceptor tyrosine activation motif (ITAM) CD3ζ chain or FcεRIγ tyrosine activation continuation and co-stimulatory signal molecules CD28, CD27, CD137, CD134, MyD88, CD40, etc.

[0050] As used herein, the terms "fluorescent label", "fluorescent dye" and "fluorophore" refer to a portion that absorbs light energy at a defined excitation wavelength and emits light energy at a different wavelength. Examples of fluorescent labels include, but are not limited to, AlexaFluor dyes (AlexaFluor 350, AlexaFluor 488, AlexaFluor 532, Alexa Fluor 546, Alexa Fluor568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, and AlexaFluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPYFL, BODIPY R6G, BODIPYTMR, BODIPYTR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665), Carboxyrhodamine 6G, Carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Coumarin DM-NERF, Eosin, Erythrosine, Fluorescein, FAM, Hydroxycoumarin, IR dyes (IRD40, IRD 700, IRD 800), JOE, Lissamine Rhodamine B, Marina Blue, Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodamine Green, 2',4',5',7'-Tetrabromosulfone-fluorescein, Tetramethylrhodamine (TMR), Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X.

[0051] The present invention illustratively described herein can be properly implemented in the absence of any one or more elements, one or more restrictions not specifically disclosed herein. Thus, for example, the terms "comprise", "include", "contain", etc. should be understood broadly and without limitation. In addition, the terms and expressions used herein have been used as descriptive terms rather than limiting, and there is no intention to use these terms and expressions to exclude any equivalents or portions thereof of the features shown and described, but it should be recognized that various modifications can be made within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and changes embodied in the present invention disclosed herein can be adopted by those skilled in the art, and these modifications and changes are considered to be within the scope of the present invention.

[0052] As used herein, the term "antibody" or "immunoglobulin" is used as a general term herein, including full-length antibodies, single-chain antibodies, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments). In addition, the term "sequence" used herein (e.g., in the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

[0053] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0054] As used herein, the term "humanized antibody" refers to a molecule having an antigen binding site substantially derived from an immunoglobulin of a non-human species, wherein the rest of the immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain, or may comprise only a complementarity determining region (CDR) transplanted to an appropriate framework region in a variable domain. The antigen binding site may be wild type, or modified by one or more amino acid substitutions, such as modification to be closer to a human immunoglobulin. Certain forms of humanized antibodies retain all CDR sequences. Other forms have one or more CDRs that have been altered relative to the original antibody.

[0055] As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is the electrophoresis diagram of monoclonal antibody 7G4;

[0057] Figure 2 It is the HPLC chart of monoclonal antibody 7G4;

[0058] Figure 3 It is the active binding diagram of monoclonal antibody 7G4. DETAILED DESCRIPTION

[0059] Other aspects and advantages of the present invention will be disclosed in the following experimental section, which should be considered illustrative and not limiting the scope of the present application.

[0060] Example 1

[0061] 1. Recombinant expression of immunogens

[0062] The human papillomavirus type 18 E protein sequence was synthesized and constructed into the pEM5.1 vector; the transfection plasmid was extracted; the HEK293 cells were transfected and the cells were cultured for 7 days; the supernatant was harvested, purified by Ni column, and the recombinant human papillomavirus type 18 E protein was obtained after concentration and replacement of buffer.

[0063] The recombinant human papillomavirus type 18 E protein sequence was obtained from Uniprot.

[0064] The sequence information is as follows:

[0065] (SEQ ID NO:16)

[0066] 2. Immunity

[0067] Immunization of mice, SP2 / 0 fusion, screening, subcloning, the specific steps are:

[0068] The first immunization was performed with Freund's complete adjuvant, 100 μg per mouse, intraperitoneally, with a total dose of 0.5 ml / mouse. The second immunization was performed 3 weeks later. The second immunization was performed with Freund's incomplete adjuvant, with a dose of 50 μg / 0.5 ml / mouse. The third immunization was performed 2 weeks later. Cell fusion was prepared 10 days after the third injection.

[0069] Take feeder cells and press 10 5 / well, plate 10 one day before fusion 5 / 100μl / well; Mouse immune spleen cells were taken and fused with the prepared myeloma cells using the fusion agent PEG, and then plated into a 96-cell culture plate that had been added with feeder cells, 100μl / well.

[0070] Positive wells were screened by ELISA detection method, and recombinant human papillomavirus type 18 E protein was plated overnight; the plate was washed, and skimmed milk powder was added for blocking at 37°C for 1 hour; the plate was washed, and 100 μl of 96-well culture supernatant was added, and the plate was incubated at 37°C for 1 hour; the plate was washed, and HRP-labeled goat anti-mouse secondary antibody was added, and the plate was incubated at 37°C for 30 minutes; the plate was washed, and the color developing solution was added, and the color was developed for 10 minutes, and the stop solution was added to read the OD450 value; the high-expression cell lines were screened for subcloning.

[0071] 3. Sequence Retrieval

[0072] Collect cells, extract RNA, reverse transcribe, design primers, PCR, transform, select clones, send for sequencing, human papillomavirus type 18 E protein antibody, targeting E protein. Clone number 7G4, the specific sequence is shown in Table 1.

[0073] Table 1

[0074]

[0075]

[0076]

[0077] 4. Antibody Expression

[0078] Monoclonal Antibody Expression and Purification:

[0079] (1) The screened sequences are chemically synthesized and cloned into a eukaryotic expression vector.

[0080] (2) Amplify and extract the plasmid.

[0081] (3) The antibody encoding plasmid was transiently transfected into mammalian HEK293 cells.

[0082] (4) Collect the supernatant and purify the monoclonal antibody using affinity chromatography.

[0083] HEK293 cells were transiently transfected with PEI, and the expression level of monoclonal antibody 7G4 was 203 mg / L.

[0084] 5. Physical and chemical properties

[0085] 5.1 Gel electrophoresis detection

[0086] A. Sample preparation

[0087] Take 20 μL of sample and mix it evenly with 5 μL of 5× reducing buffer, heat it at 95°C for 5 min, and cool it down; take 20 μL of sample and mix it evenly with 5 μL of 5× non-reducing buffer.

[0088] B. Electrophoresis

[0089] Prepare gel, add appropriate amount of electrophoresis buffer, add sample, and perform electrophoresis.

[0090] C. Dyeing and bleaching

[0091] After electrophoresis, place the gel in an appropriate amount of Coomassie Brilliant Blue staining solution and stain at room temperature for 1 hour or longer; pour out the staining solution, add an appropriate amount of Coomassie Brilliant Blue staining decolorizing solution, and decolorize at room temperature for 4-24 hours. After decolorization, use ddH 2 O, refer to the marker protein, compare with the unstained gel, cut out the gel of the desired protein component, collect it, and then separate the protein to be purified from the gel.

[0092] 5.2 HPLC detection of monoclonal antibody purity

[0093] A. Mobile phase preparation

[0094] Add potassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate and potassium chloride to about 900 mL of purified water, stir to dissolve, dilute to 1 L, measure with a pH meter to determine that the pH is between 6.2±0.1. Filter through a 0.22 μm filter membrane and store at room temperature.

[0095] B. Sample Preparation

[0096] System suitability sample: The standard was diluted to 2 mg / mL with mobile phase;

[0097] Test sample: Dilute the sample to be tested to 2 mg / mL with mobile phase.

[0098] The detection was carried out under conventional chromatographic conditions.

[0099] The results are as follows Figure 1 and Figure 2 As shown, the results showed that the purity of the monoclonal antibody 7G4 against papillomavirus type 18 E protein was greater than 95% by electrophoresis and liquid chromatography detection.

[0100] 6. Binding activity

[0101] Binding activity assay of monoclonal antibodies:

[0102] A. Coating: Dilute papillomavirus type 18 E protein to 2 μg / ml with coating solution, mix well, add to 96-well coating plate, 100 μl / well, seal with film, and incubate at 4°C overnight.

[0103] B. Wash the plate three times in a plate washer. No liquid should remain on the plate during the last wash. Pat the liquid on the surface of the plate dry with absorbent paper.

[0104] C. Blocking: Add 5% milk powder (0.5 g milk powder dissolved in 10 mL DPBS), 300 μL / well, incubate at 37°C for 1 h, and wash the plate 3 times according to step B.

[0105] D. Dilute the antibody in a gradient manner, 100 μL / well, react at 37°C for 1 h, and wash the plate 3 times according to step B.

[0106] E. Add secondary antibody: dilute with DPBS at 1:2000, add to 96-well plate, 100 μL / well, react at 37℃ for 1 hour, and wash the plate 3 times according to step B.

[0107] F. Color development: Add TMB, 100 μL / well, and develop color for 10 min at room temperature in the dark.

[0108] G. Termination: Add 2N H 2 SO 4 , 100 μL / well.

[0109] H. Measure OD450 with an enzyme-labeled instrument within 10 minutes.

[0110] Coat VP1 protein, add antibody, add secondary antibody, develop color, read, and analyze data.

[0111] The results are as follows Figure 3 As shown, the results showed that the EC50 of the papillomavirus type 18 E protein monoclonal antibody 7G4 was 0.139 ng / ml.

Claims

1. A monoclonal antibody against HPV-18 type E protein, characterized in that: The monoclonal antibody comprises a heavy chain variable region CDR1 as shown in SEQ ID NO: 1, a heavy chain variable region CDR2 as shown in SEQ ID NO: 2, a heavy chain variable region CDR3 as shown in SEQ ID NO: 3, a light chain variable region CDR1 as shown in SEQ ID NO: 9, a light chain variable region CDR2 as shown in an amino acid sequence abbreviation such as LTS, and a light chain variable region CDR3 as shown in SEQ ID NO:

10.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody also comprises heavy chain variable region framework regions FR1, FR2, FR3, FR4 having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 5, 6, and 7, and light chain variable region framework regions FR1, FR2, FR3, FR4 having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 11, 12, 13, and 14.

3. The monoclonal antibody according to claim 1 or 2, characterized in that The heavy chain variable region of the monoclonal antibody has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:8, and the light chain variable region has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

15.

4. The monoclonal antibody according to claim 1 or 2, characterized in that The monoclonal antibody further comprises a heavy chain constant region and a light chain constant region.

5. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 4.

6. A method for producing the monoclonal antibody according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: a) providing the nucleic acid molecule according to claim 5; b) transforming a host cell with the nucleic acid molecule described in step a); c) culturing the host cell obtained in step b) under suitable conditions; d) isolating and purifying the monoclonal antibody according to any one of claims 1 to 4 in the host cell culture medium.

7. Use of the monoclonal antibody according to any one of claims 1 to 4 in the preparation of a product for detecting E protein.

8. The use according to claim 7, wherein the E protein is HPV-18 papillomavirus E protein.

9. A recombinant vector comprising the nucleic acid molecule according to claim 5.

10. The recombinant vector of claim 9, wherein the recombinant vector comprises a DNA vector. The recombinant vector of claim 9 , wherein the recombinant vector comprises a transcriptional viral vector.

12. The recombinant vector of claim 9, wherein the recombinant vector comprises a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retrovirus vector, an mRNA vector, a transposon-based vector or an artificial chromosome. The recombinant vector according to claim 9 , which is a plasmid.

14. A cell comprising the monoclonal antibody according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5 or the recombinant vector according to any one of claims 9 to 13.

15. The cell of claim 14, comprising a prokaryotic cell or a eukaryotic cell.

16. The cell of claim 15, wherein the prokaryotic cell comprises bacteria, mycoplasma, chlamydia, rickettsia.

17. The cell of claim 16, wherein the bacteria comprises actinomycetes and cyanobacteria.

18. The cell of claim 16, wherein the bacteria comprises Gram-positive bacteria and Gram-negative bacteria.

19. The cell of claim 15, wherein the eukaryotic cell comprises a mammalian cell, an insect cell, or a fungal cell.

20. The cell of claim 19, wherein the fungal cell comprises a yeast cell.

21. The cell of claim 19, wherein the mammalian cell comprises a fibroblast, a lymphocyte, an epithelial cell, or a myeloblast.

22. A conjugate comprising the monoclonal antibody according to any one of claims 1 to 4 and a fluorescent dye, a chemiluminescent compound, a radioactive isotope, an enzyme, or biotin conjugated to the antibody.

23. The conjugate of claim 22, wherein the enzyme comprises horseradish peroxidase, alkaline phosphatase, β-galactosidase and acetylcholinesterase.

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