阅读说明：本技术 一种抗人乳头瘤病毒31型的单克隆中和抗体及其应用 (Monoclonal neutralizing antibody against human papillomavirus 31 and application thereof ) 是由 张海江 张尧 王学红 杨增敏 李亚坤 王艳 郑明卉 伍树明 陈晓 刘永江 于 2021-03-09 设计创作，主要内容包括：本发明公开一种抗人乳头瘤病毒31型的单克隆中和抗体,其是人乳头瘤病毒的15价特异性抗体,可用于制备人乳头瘤病毒疫苗、检测或诊断人乳头瘤病毒,以及在制备防治人乳头瘤病毒引起疾病的药物中的应用。(The invention discloses a monoclonal neutralizing antibody of anti-human papillomavirus 31 type, which is a 15-valent specific antibody of human papillomavirus, can be used for preparing human papillomavirus vaccines, detecting or diagnosing the human papillomavirus and can be applied to preparing medicines for preventing and treating diseases caused by the human papillomavirus.)

1. A monoclonal neutralizing antibody against human papillomavirus type 31, characterized by the fact that in its heavy chain: the CDR1 sequence is: GYGVN; the CDR2 sequence is: MIWGDGSTDYNSAL, respectively; the CDR3 sequence is: GGSSYYYAMDY, respectively; and in its light chain, the CDR1 sequence is: RASENIYSYLA; the CDR2 sequence is: NAKTLAE; the CDR3 sequence is: QHHYGTPLT are provided.

2. The monoclonal neutralizing antibody of claim 1, wherein the heavy chain variable region amino acid sequence is as set forth in SEQ ID NO: 12 is shown in the specification; the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO: shown at 17.

3. The monoclonal neutralizing antibody according to claim 1, which is produced by monoclonal antibody cell line HPV 3119C 2, which has been deposited at CGMCC No.21417 at 29.12.2020.

4. A polynucleotide encoding the monoclonal neutralizing antibody of any one of claims 1 to 3.

5. A nucleic acid expression vector comprising a polynucleotide encoding the polynucleotide of claim 4.

6. A recombinant host comprising the nucleic acid expression vector of claim 5.

7. The monoclonal neutralizing antibody cell strain is characterized by being preserved in China general microbiological culture Collection center (CGMCC) at 29 th 12 th 2020 with the preservation number of CGMCC No. 21417.

8. A method of producing a monoclonal neutralizing antibody according to any one of claims 1 to 3, comprising:

culturing the recombinant host of claim 6 to induce expression of said monoclonal neutralizing antibody;

or culturing the monoclonal neutralizing antibody cell strain of claim 7 to obtain the monoclonal antibody.

9. Use of a monoclonal neutralizing antibody according to any one of claims 1 to 3 in the detection of HPV31 vaccine.

10. The use according to claim 9, wherein the test is a test for process-like levels during HPV31 vaccine development or manufacture, identification of type, detection of antigen levels, or in vitro potency.

11. The use of claim 10, wherein the detection is performed by a double antibody sandwich ELISA.

12. Use of the monoclonal neutralizing antibody according to any one of claims 1 to 3 for the preparation of a medicament for the prevention and treatment of diseases caused by human papillomavirus.

13. The use according to claim 12, wherein the disease is a disease caused by human papillomaviruses of the HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68 types.

14. The use of claim 13, wherein the disease is cervical cancer, vulvar cancer, vaginal cancer, anal cancer, rectal cancer, oral cancer, tonsil cancer, penile cancer, genital warts, flat warts, or verruca vulgaris.

15. A human papillomavirus type 31 detection or diagnostic kit comprising a monoclonal neutralizing antibody according to any of claims 1 to 3.

16. The kit of claim 15, wherein the detection is performed by ELISA double antibody sandwich method and the monoclonal neutralizing antibody of any one of claims 1 to 3 is used as a coating antibody.

The technical field is as follows:

the invention belongs to the technical field of biology and medicine, and particularly relates to a monoclonal neutralizing antibody for resisting human papilloma virus 31 and application thereof.

Background art:

human Papillomaviruses (HPV) are non-enveloped, double-small DNA viruses that mainly invade human epithelial tissues, thereby inducing various benign and malignant proliferative lesions. Certain types of human papillomaviruses are associated with epithelial malignancies such as cervical cancer, laryngeal cancer, bladder cancer, esophageal cancer, and the like. High risk HPV infection is associated with the development of a variety of malignancies. The epidemic range of HPV infection is wide, the caused lethal malignant tumor has serious harm to human health, and the development of safe and effective preventive or therapeutic vaccine has great significance. The vaccine currently on the market, the most expensive vaccine is the mershadong 9-valent vaccine (Gardasil 9), consisting of HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58. Research on HPV vaccines shows that the HPV L1 protein expressed in vitro can be self-assembled into virus-like particles (VLPs), the structure of the VLPs is highly similar to that of natural HPV, most of neutralizing epitopes of the natural virus are reserved, and high-titer neutralizing antibodies can be induced.

In the development process of HPV vaccines, at least four aspects of determination, namely, detection of sample content in the process, type identification experiment, detection of antigen content and detection of in vitro efficacy, need to be carried out. The method can adopt a double-antibody sandwich ELISA. In vitro potency assay requires at least one neutralizing antibody for ELISA assay, therefore, monoclonal antibodies are important tools in vaccine development, especially antibodies with specificity and neutralizing activity have irreplaceable effects. In addition, antibody drugs developed using neutralizing antibodies can also be used for the therapeutic action of diseases associated with papillomavirus.

In the vaccine development process, in vitro potency detection, detection by a double antibody sandwich ELISA method, the requirement of the used antibody, besides one of the paired antibodies being a neutralizing antibody, also has to be specific, because the multivalent vaccine, which is a mixture of multiple HPVs, first has to be able to specifically detect the target protein for each HPV in vitro potency determination. The most multivalent form currently on the market is the 9-valent form, and some reports have already developed clinical 14-valent vaccines, but no documents report higher-valent, e.g., 15-valent, specific antibodies. In addition to the requirement for neutralizing activity and specificity, the epitope recognized by the antibody is preferably the dominant epitope for neutralizing activity as an antibody for in vitro potency assay, which is of greater significance to represent vaccine viability.

The invention content is as follows:

in response to the needs and deficiencies of the prior art, the present inventors have developed a monoclonal neutralizing antibody against human papillomavirus type 31, which is a 15-valent specific antibody and has a neutralizing epitope, and thus have come up with the present invention.

The present inventors provide a monoclonal neutralizing antibody against human papillomavirus type 31, in which in the heavy chain: the CDR1 sequence is: DYAMH; the CDR2 sequence is: VMSTYYDDANYNQKFKG, respectively; the CDR3 sequence is: GPLYGNYVDYSMDY, respectively; and in its light chain, the CDR1 sequence is: KSSQSLLDSDGKTYLN, respectively; the CDR2 sequence is: LVSKLDS; the CDR3 sequence is: QGTHFPRT;

or in its heavy chain: the CDR1 sequence is: GYGVN; the CDR2 sequence is: MIWGDGSTDYNSAL, respectively; the CDR3 sequence is: GGSSYYYAMDY, respectively; and in its light chain, the CDR1 sequence is: RASENIYSYLA; the CDR2 sequence is: NAKTLAE; the CDR3 sequence is: QHHYGTPLT are provided.

Preferably, the monoclonal neutralizing antibody has the full-length amino acid sequence of the heavy chain as shown in SEQ ID NO: 2 is shown in the specification; the full-length amino acid sequence of the light chain is shown as SEQ ID NO: 7 is shown in the specification; or the full-length amino acid sequence of the heavy chain is shown as SEQ ID NO: 12 is shown in the specification; the full-length amino acid sequence of the light chain is shown as SEQ ID NO: shown at 17.

More preferably, the monoclonal neutralizing antibody is produced by monoclonal antibody cell strains HPV 3119B 4 and HPV 3119C 2, wherein the monoclonal antibody cell strains HPV 3119B 4 and HPV 3119C 2 have been deposited at China general microbiological culture Collection center (CGMCC) at 29.12.2020 with the deposition numbers of CGMCC No.21416 and CGMCC No.21417, respectively.

The invention also provides polynucleotides encoding the monoclonal neutralizing antibodies.

Further provided is a nucleic acid expression vector comprising a polynucleotide encoding the same.

Recombinant hosts containing the nucleic acid expression vectors are also provided.

The invention also provides a monoclonal neutralizing antibody cell strain HPV 3119B 4 with the preservation number of CGMCC No. 21416; the monoclonal neutralizing antibody cell strain HPV 3119C 2 has preservation number of CGMCC No. 21417.

The invention also provides a preparation method of the monoclonal neutralizing antibody, which comprises the following steps:

culturing said recombinant host to induce expression of said monoclonal neutralizing antibody; or culturing the monoclonal neutralizing antibody cell strain to obtain the monoclonal antibody.

The invention further provides the application of the monoclonal neutralizing antibody in detection of the HPV31 vaccine, and further the detection refers to detection of process sample content in the process of successfully developing or preparing the HPV31 vaccine, identification of type, detection of antigen content, or detection of in vitro efficacy, preferably detection by double antibody sandwich ELISA.

The invention also provides application of any one of the monoclonal neutralizing antibodies in preparing a medicament for preventing and treating diseases caused by human papilloma virus, further the diseases are further used for the diseases caused by the human papilloma virus of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and HPV68 types, and further the diseases are cervical cancer, vulvar cancer, vaginal cancer, anal cancer, rectal cancer, oral cancer, tonsil cancer, penile cancer, genital wart, flat wart or common wart.

In addition, the invention further provides a human papillomavirus type 31 detection or diagnosis kit, which is characterized by comprising any one of the monoclonal neutralizing antibodies, preferably an ELISA double antibody sandwich method for detection, and using the other monoclonal neutralizing antibody as a coating antibody.

The HPV 3119B 4 and HPV 3119C 2 monoclonal antibodies obtained by the invention have particularly obvious advantages, good sensitivity and specificity, neutralizing activity, especially 15-valent specific antibodies, and antigen dominant epitopes. However, the HPV31 antibody is only a 9-valent specific antibody at present, and no research data on whether the antibody is a dominant epitope or not exists. Therefore, the two antibodies of the invention have great application value in clinical diagnosis of HPV31 and treatment or prevention of HPV virus infection.

The monoclonal antibody cell strain HPV 3119B 4 has been deposited in China general microbiological culture Collection center (No. 3, Xilu 1. Beijing, Chaoyang, the republic of Beijing) at 29.12.2020 with the deposit number of CGMCC No. 21416. The monoclonal antibody cell strain HPV 3119C 2 has been deposited in China general microbiological culture Collection center (No. 3, Xilu 1, Beijing, Chaoyang, North Cheng) at 29.12.2020 and has a collection number of CGMCC No. 21417.

Drawings

FIG. 1 shows the blocking experiment results of monoclonal antibody and rabbit polyclonal antibody.

Detailed Description

The following examples and figures of the present invention are merely illustrative of specific embodiments for carrying out the invention and these should not be construed as limiting the invention and any changes which may be made without departing from the principles and spirit of the invention are within the scope of the invention.

The experimental techniques and experimental methods used in this example are conventional techniques unless otherwise specified. The materials, reagents and the like used in the present examples are all available from normal commercial sources unless otherwise specified.

Example one, hybridoma cell line development and antibody purification of monoclonal antibodies:

1. animal immunization

1) Basic immunity: the HPV 31L 1VLP antigen and Freund's complete adjuvant are mixed in equal volume and emulsified thoroughly, and injected subcutaneously in several portions to immunize 6 mice, each Balb/c mouse has 10ug per injection.

2) And (3) boosting immunity: the boosting immunity adopts emulsion of antigen and Freund's incomplete adjuvant. 3 days before cell fusion, a physiological saline solution containing 15ug of antigen was intraperitoneally injected. The results of the experiment are shown in table 1.

TABLE 1 immune mouse serum assay (OD450)

As can be seen from the above table, the highest serum titer was observed in mouse No. 5, which was selected for splenectomy and fusion with SP20 cells. Namely, the mouse No. 5 with the strongest signal is selected for fusion and subsequent preparation of hybridoma cells.

2. Preparation of hybridoma cells and establishment of cell lines

Spleen cells from mice were harvested conventionally and fused with SP2/0 cells at a 10:1 ratio with 500g/L PEG 4000. Selectively culturing in HAT semi-solid culture solution (IMDM culture solution containing 1-3% methylcellulose, 1 × HAT and 10-20% fetal calf serum) for 10-20 days, and selecting 1000 clones to 96-well plate for liquid DMEM culture. And continuously culturing for 5-10 days, and when the cells grow up, sucking half of the culture medium to perform primary screening and cloning by an indirect ELISA method.

The indirect ELISA method was performed as follows: plates were packed with 200 ng/well HPV 31L 1VLP, immune mouse serum 1:2000 as a positive control, medium supernatant without clonal growth as a negative control, 1:2000 HRP-goat anti-mouse IgG 100. mu.l per well, and finally OD450 nm was determined. If the OD450 value is more than 2 times larger than the negative control, the positive clone can be primarily determined.

In the primary screening of 1000 clones, 12 clones with OD values greater than 2-fold negative value were selected by indirect ELISA.

TABLE 2 Primary screening of Positive clones (Note: the reading in the Table is OD450)

	19B4	19C2	19D6	19D11	20A11	20B11	20F9	20F10	20E10	21E5	22G8	24D1	Negative of
														OD450	2.326	2.571	0.563	0.641	0.959	1.257	1.06	0.858	2.064	2.302	1.865	1.954	0.058

And (3) selecting the primary screening positive clone, transferring to a 24-pore plate for continuous culture, culturing for 5-10 days, and after the cells grow up, sucking the cell culture solution of each pore to perform indirect ELISA (enzyme-linked immunosorbent assay) method, re-screening and cloning, and finally retaining 6 strains of positive clones. The experimental results are shown in table 3 below.

TABLE 3 summary of the rescreened cloning results (Note: the reading in the table is OD450)

	19B4	19C2	19D6	19D11	20A11	20B11	20F9	20F10	20E10	21E5	22G8	24D1	Negative of
														OD450	2.158	2.321	0.088	0.063	0.046	0.092	0.084	0.082	1.908	2.588	1.981	2.316	0.061

Positive clones were screened initially, and some were converted to negative after rescreening. In this example, 6 strains, namely, 19B4, 19C2, 20E10, 21E5, 22G8 and 24D1 positive clones were selected, and subjected to continuous cell culture, passaging, cryopreservation, and the like, and then subjected to experiments such as ascites preparation and antibody purification.

3. Ascites production and antibody purification

Adult BALB/c mice were selected and intraperitoneally inoculated with 0.5ml of pristane per mouse. The hybridoma cells were inoculated intraperitoneally 7-10 days later, at 1X 10 cells/mouse⁶-2×10⁶And (4) respectively. After 5 days, when the abdomen is obviously enlarged and the hand touches the abdomen, the skin is tense, and the ascites can be collected by using a 16-gauge needle.

TABLE 4 Abdominal water statistics table

The collected ascites volume was at a reasonable level for each clone. The ascites fluid was centrifuged (13000r/min for 30 minutes), the cell fraction and other precipitates were removed, and the supernatant was collected. Purifying with Protein G-Sepharose CL-4B, wherein the upper column liquid is 20mM PBS buffer solution, and the column chromatography eluent is: glycine buffer, ph2.7,20mM, yielded a monoclonal antibody against HPV31 VLP.

TABLE 5 antibody purification amounts

As is clear from Table 5 above, the antibody of 21E5 was significantly lower in amount than other antibody strains after purification, and the results of other antibodies showed that the amount of antibody purified 2ml ascites was 4.9mg or more.

4. Identification of the specificity of antibodies

The specificity of the clones was assessed by ELISA indirect method using type 15 VLP protein-coated plates. The indirect ELISA method comprises the following steps: the VLP protein was diluted to 2. mu.g/ml. Add 100. mu.l/well to 96-well microplate and coat overnight at 4 ℃. And (3) sealing: and (5) drying the coated ELISA plate. Add 300. mu.l/well blocking solution (2% BSA) to the plate and allow to stand at room temperature for 1-2 hours. Sample dilution: and (3) respectively diluting the antibody samples to 0.3 mu g/ml by using sample diluent, uniformly mixing, adding 100 mu l/hole into an enzyme label plate, and standing at room temperature for 1 h. Adding a secondary antibody: and (3) drying the enzyme label plate after sealing, adding 100 mul/hole of HRP-labeled goat-anti-mouse secondary antibody into the enzyme label plate at the concentration of 1:4000, and standing for 1h at room temperature. Color development: washing the plate for 5 times with 300 mul per hole, and drying by spin. The bottom was wiped with toilet paper. The developing solution was added at 100. mu.l/well, and the mixture was developed in the dark at room temperature for 10 minutes. And (4) terminating: the reaction was terminated by adding 100. mu.l/well of a stop solution. Reading: the microplate was placed in the microplate reader and OD450 read and data analyzed. The results are given in the table below.

TABLE 6 results of specificity evaluation (OD450)

The target type specificity is judged according to the following steps: the target type detection is positive, the other types are negative, or the reading value of the target type OD450 is more than 5 times larger than that of the other types OD 450. Thus, as can be seen from table 6, 19B4, 19C2, 21E5,24D1 are 15-valent specific antibodies, and 20E10 and 22G8 are 15-valent non-specific antibodies.

5. Detection of neutralizing Activity of antibodies

The HPV31 type pseudovirus was mixed with 50. mu.l each of the cloned cell culture supernatants, and added to the pre-cultured 293FT cells after 1 hour. After further culturing for about 72 hours, the neutralizing activity of the clones was judged by detecting the pseudoviral infection of the cells in each well of each plate using an I3 multifunctional plate reader.

The Positive Control (PC) for neutralizing activity replaced the cloned cell supernatant with the same volume of cell culture medium and the rest of the experimental procedure was the same as the other experimental groups. The smaller the detection value of the experimental group to which the supernatant of the clone cells was added, the higher the neutralizing activity of the clone. In the antibody neutralization test, if the reading value of the neutralization test is not more than 50% of the mean value of the PC control group, the clone is judged to have the neutralization activity, otherwise, the clone is judged to have the non-neutralization activity. The results of the experiment are shown in Table 7.

Table 7 neutralization evaluation results

Table shows the value of green fluorescence excited after infection of 293FT cells with pseudovirus. The results show that, except for 20E10,22G8 is a non-neutralizing antibody, 19B4, 19C2, 21E5,24D1 is a neutralizing antibody.

6. Epitope detection of antibodies

The 4 selected neutralizing antibodies were analyzed for whether the antigen-recognizing epitope of the neutralizing antibodies was a neutralizing dominant epitope using an ELISA competition experiment.

Plates were coated with HPV31 VLPs, and monoclonal antibodies, diluted in a gradient, were competed with rabbit polyclonal enzyme-labeled antibodies, and a non-competitive control without added monoclonal antibodies was set. And calculating competitive inhibition rate, and taking the inhibition rate of more than 50% as an inhibition standard. If the monoclonal antibody can block the rabbit polyclonal antibody from being combined by more than 50%, the monoclonal antibody is shown to recognize the neutralizing dominant epitope.

The inhibition was calculated as (1-experimental OD/control OD) × 100%.

Calculating the inhibition rate and drawing an inhibition curve among different monoclonal antibodies. The results of the experiment are shown in fig. 1 and table 8 below.

TABLE 8 summary of blocking experiments between monoclonal antibody and rabbit polyclonal antibody

The results showed that mabs 19B4 and 19C2 recognized the epitope dominant and 21E5 and 24D1 recognized the epitope non-dominant of VLPs.

In conclusion, the antibodies of 19C2 and 19B4 have high purification yield, specificity, neutralization activity and antigen dominant epitope recognition, are the best choice of the antibodies used in the vaccine development process and are the targets for further research. 21E5 and 24D1 are also neutralizing active and specific antibodies, but both recognize non-dominant epitopes of the antigen and are not optimal. The 20E10 and 22G8 antibodies are not specific and have no neutralizing activity, so that the antibodies are not suitable for in vitro efficacy research of vaccines. Wherein the preservation number of the single-cell strain of the 19B4 antibody is CGMCC No: 21416; the preservation number of the single-cell strain of the 19C2 antibody is CGMCC No: 21417.

EXAMPLE two, identification of 19B4 and 19C2 antibodies

1. Conformational epitope identification of antibodies

The VLP proteins of each type are subjected to alkali denaturation and heat denaturation to destroy the secondary or tertiary structure, and the primary structure is preserved. Then reacting with the monoclonal antibody, and detecting the monoclonal antibody by adopting indirect ELISA. Through the experiment, the identification condition of the antibody to various VLPs can be identified, and whether the antibody is a conformational epitope recognition antibody can also be identified. If the OD450 value of the protein after denaturation and the monoclonal antibody reaction is obviously reduced, the monoclonal antibody is proved to be a conformational epitope recognition antibody, and if the OD450 value of the protein after denaturation and the monoclonal antibody reaction is not obviously changed, the monoclonal antibody is proved to be a linear epitope recognition antibody.

The experimental steps are as follows: VLP denatured protein treatment: 0.2M sodium carbonate, 0.01M DTT, pH 10.6 was incubated at room temperature for 30 minutes and then boiled for 5 minutes. Denatured and undenatured VLP proteins were coated separately and tested by indirect ELISA and finally OD450 readings were taken. The results are shown in Table 9.

TABLE 9 conformational epitope detection

The results show that both clones 19B4 and 19C2 recognized non-denatured HPV31 VLPs with OD450 values greater than 2, as significant positive results, while those recognizing denatured HPV31 VLPs with values less than 2-fold negative values, as negative results. It shows that 19B4 and 19C2 monoclonal antibodies are both conformation recognition monoclonal antibodies. Except positive for the non-denatured HPV31 VLP, both mAbs reacted negatively with other non-denatured VLPs of type 8, indicating that both 19B4 and 19C2 are specific, conformational epitope mAbs.

2. Antibody subtype identification

The IgG subclasses of the antibodies produced by the above hybridoma cells were identified using antibodies against various IgG subclasses of mice by an indirect ELISA method. The results are shown in Table 10 below.

Subtype identification of the antibodies in tables 1019B 4 and 19C2

From the above results, it was found that both 19B4 and 19C2 are kappa IgG1 type antibodies.

EXAMPLE III preparation of HPV31 VLP detection reagent Using 19B4 purified antibody

The ELISA double antibody sandwich method is used for carrying out the mutual pairing experiment of the antibodies. In this example, the ELISA detection method was determined using clone 19B4 as a coating antibody and HRP-labeled clone 19C2 as a detection antibody. The results are shown in Table 11.

TABLE 11 preliminary pairing results for the kit

The results of the preliminary pairing experiments show that the pairing combination signals of 19C2-HRP detection are higher when the 19B4 is used for coating, and the pairing combination signals are more suitable for ELISA detection. Therefore, ELISA detection is further carried out by the pair.

The detection method comprises the following steps: the coated antibody 19B4 was diluted to 10. mu.g/mL with 0.05mol/L carbonate buffer solution of pH 9.6, 100. mu.L was added to each well of the ELISA plate, coated overnight at 4 ℃, the coating solution was decanted, washed 2 times with PBST, patted dry, 200. mu.L of 3% Bovine Serum Albumin (BSA) was added to each well, sealed in a 37 ℃ incubator for 2 hours, washed 1 time with PBS, added with 10% sucrose aqueous solution, protected at room temperature for 1 hour, patted dry, dried, filled in aluminum foil bags, vacuumed, and stored at 4 ℃.

The 19C2 antibody was labeled with horseradish peroxidase to give 19C2-HRP and stored. Adding 100 mu L/hole of VLP sample into the ELISA plate, incubating for 1.5 hours at 37 ℃, washing the plate, adding 100 mu L/hole of 19C2-HRP (0.5ug/ml), incubating for 1 hour at 37 ℃, washing, drying, adding color developing agent for color development, incubating for 10min at 37 ℃, adding 50 mu L/hole of stop solution, and reading by an ELISA reader at the wavelength of 450 nm.

And (3) specific test of the kit: sample detection of HPV15 type VLPs was performed using the established method described above, with VLP denatured protein treatment: 0.2M sodium carbonate, 0.01M DTT, pH 10.6 was incubated at room temperature for 30 minutes and then boiled for 5 minutes. The 96-well plate detects 100ul of 10ug/ml VLP per well. The experimental results are shown in table 12 below. The results show that the kit detects non-denatured HPV31 VLPs with good signal, does not recognize denatured HPV31 VLPs, and has no crossover with other HPV VLPs of type 8.

TABLE 12 evaluation results of HPV31 VLP specificity by ELISA method (OD450)

Therefore, the kit of the embodiment can be used for specifically detecting HPV31 VLP with biological activity, and even if the protein is not degraded in vaccine development, the change of OD value can be reflected in time by ELISA method only because of the quality change caused by conformation change, thereby reflecting the change of vaccine quality. Therefore, the HPV31 vaccine can be widely applied to HPV31 vaccine development.

EXAMPLE four determination of the variable region sequences of clones 19B4 and 19C2

The obtained 19B4 and 19C2 monoclonal cells were separately extracted for mRNA, reverse transcribed into cDNA, subjected to high fidelity PCR amplification using variable region universal primers, the PCR product fragments were inserted into T-vectors for DNA sequencing, and the obtained sequences were translated into the amino acid sequence of proteins. The obtained sequences were aligned and showed no identical sequence, indicating that the obtained sequences were specific. The relevant sequences of the antibodies of the two clones were as follows:

the full-length nucleotide sequence of the 19B4 heavy chain is as follows (366 bp):

GTGAAGCTGCAGCAGTCAGGGGCTGAGCTGGTGAGGCCTGGGGTCTCAGTGAAGATTTCCTGCAAGGGTTCTGGCTACACATTCACTGATTATGCTATGCACTGGGTGAAGCAGAGTCATGCAAAGAGTCTAGAGTGGATTGGAGTTATGAGTACTTACTATGATGATGCTAATTACAACCAGAAGTTCAAGGGCAAGGCCACAATGACTGTAGACAAATCCTCCAGAACAGCCTATATGGAACTTGCCAGACTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGGGGACCCCTGTATGGTAACTACGTCGACTATTCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA。

the full length of the amino acid sequence encoded by the 19B4 heavy chain is as follows (122 aa):

VQLQESGAELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHAKSLEWIGVMSTYYDDANYNQKFKGKATMTVDKSSRTAYMELARLTSEDSAIYYCARGPLYGNYVDYSMDYWGQGTTVTVSS。

by performing identification according to a conventional method, it can be known that, in the heavy chain: the CDR1 sequence is: DYAMH; the CDR2 sequence is: VMSTYYDDANYNQKFKG, respectively; the CDR3 sequence is: GPLYGNYVDYSMDY are provided.

Wherein, the full-length nucleotide sequence of the 19B4 light chain is as follows (336 bp):

GATATCCAGCTGACCCAGTCTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGCAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCGGACGTTCGGAGCAGGTACCAAGCTGGAGATCAAA。

the full length of the amino acid sequence encoded by the 19B4 light chain was as follows (122 aa):

DIQLTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLQISRVEAEDLGVYYCWQGTHFPRTFGAGTKLEIK

by performing identification according to a conventional method, it can be known that, in the light chain: the CDR1 sequence is: KSSQSLLDSDGKTYLN, respectively; the CDR2 sequence is: LVSKLDS; the CDR3 sequence is: QGTHFPRT.

The full-length nucleotide sequence of the 19C2 heavy chain is as follows (354 bp):

GTGCAGCTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACCGGCTATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATGATATGGGGTGATGGAAGCACAGACTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGGTACTACTGTGCCATCGGCGGTAGTAGCTATTACTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA。

the full-length amino acid sequence of the 19C2 heavy chain is as follows (118 aa):

VQLQESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSISKDNSKSQVFLK MNSLQTDDTARYYCAIGGSSYYYAMDYWGQGTTVTVSS。

by performing identification according to a conventional method, it can be known that, in the heavy chain: the CDR1 sequence is: GYGVN; the CDR2 sequence is: MIWGDGSTDYNSAL, respectively; the CDR3 sequence is: GGSSYYYAMDY are provided.

The full-length nucleotide sequence of the 19C2l light chain is as follows (318 bp):

GACATTCAGCTGACCCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGATC。

the full-length nucleotide sequence of the 19C2 heavy chain is as follows (106 aa):

DIQLTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQP EDFGSYYCQHHYGTPLTFGAGTKLEI。

by performing identification according to a conventional method, it can be known that, in the light chain: the CDR1 sequence is: RASENIYSYLA; the CDR2 sequence is: NAKTLAE; the CDR3 sequence is: QHHYGTPLT are provided.

Using the sequences identified above, various genetically engineered antibodies, such as chimeric antibodies, humanized antibodies, single chain antibodies, diabodies, and the like, can be prepared by known antibody engineering techniques, while retaining the biological properties of the monoclonal antibody from which it is derived.

Sequence listing

<110> Beijing Kangle guard Biotechnology Ltd

<120> monoclonal neutralizing antibody against human papillomavirus type 31 and application thereof

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<400> 1

gtgaagctgc agcagtcagg ggctgagctg gtgaggcctg gggtctcagt gaagatttcc 60

tgcaagggtt ctggctacac attcactgat tatgctatgc actgggtgaa gcagagtcat 120

gcaaagagtc tagagtggat tggagttatg agtacttact atgatgatgc taattacaac 180

cagaagttca agggcaaggc cacaatgact gtagacaaat cctccagaac agcctatatg 240

gaacttgcca gactgacatc tgaggattct gccatctatt actgtgcaag gggacccctg 300

tatggtaact acgtcgacta ttctatggac tactggggcc aagggaccac ggtcaccgtc 360

tcctca 366

<210>2

<211> 122

<212>PRT

<213>19B4 heavy chain full-length amino acid sequence

<400>2

VQLQESGAEL VRPGVSVKIS CKGSGYTFTD YAMHWVKQSH AKSLEWIGVM STYYDDANYN 60

QKFKGKATMT VDKSSRTAYM ELARLTSEDS AIYYCARGPL YGNYVDYSMD YWGQGTTVTV 120

SS 122

<210>3

<211> 5

<212>PRT

<213> CDR1 of the heavy chain of 19B4

<400>3

DYAMH 5

<210>4

<211> 17

<212>PRT

<213> CDR2 of the heavy chain of 19B4

<400>4

VMSTYYDDAN YNQKFKG 17

<210>5

<211> 14

<212>PRT

<213> CDR3 of the heavy chain of 19B4

<400>5

GPLYGNYVDY SMDY 14

<210>6

<211> 336

<212>DNA

<213>19B4 light full-length nucleotide sequence

<400> 6

gatatccagc tgacccagtc tccactcact ttgtcggtta ccattggaca accagcctcc 60

atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata tttgaattgg 120

ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180

tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgcaaatc 240

agcagagtgg aggctgagga tttgggagtt tattattgct ggcaaggtac acattttcct 300

cggacgttcg gagcaggtac caagctggag atcaaa 336

<210>7

<211> 112

<212>PRT

<213>19B4 light chain full-length amino acid sequence

<400>7

DIQLTQSPLT LSVTIGQPAS ISCKSSQSLL DSDGKTYLNW LLQRPGQSPK RLIYLVSKLD 60

SGVPDRFTGS GSGTDFTLQI SRVEAEDLGV YYCWQGTHFP RTFGAGTKLE IK 112

<210>8

<211> 16

<212>PRT

<213> CDR1 of 19B4 light chain

<400>8

KSSQSLLDSD GKTYLN 16

<210>9

<211> 17

<212>PRT

<213> CDR2 of 19B4 light chain

<400>9

VMSTYYDDAN YNQKFKG 17

<210>10

<211> 8

<212>PRT

<213> CDR3 of 19B4 light chain

<400>10

QGTHFPRT 8

<210>11

<211> 354

<212>DNA

<213>19C2 heavy chain full-length nucleotide sequence

<400> 11

gtgcagctgc aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcaca 60

tgcaccgtct cagggttctc attaaccggc tatggtgtaa actgggttcg ccagcctcca 120

ggaaagggtc tggagtggct gggaatgata tggggtgatg gaagcacaga ctataattca 180

gctctcaaat ccagactgag catcagcaag gacaactcca agagccaagt tttcttaaaa 240

atgaacagtc tgcaaactga tgacacagcc aggtactact gtgccatcgg cggtagtagc 300

tattactatg ctatggacta ctggggccaa gggaccacgg tcaccgtctc ctca 354

<210>12

<211> 118

<212>PRT

<213>19C2 heavy chain full-length amino acid sequence

<400>12

VQLQESGPGL VAPSQSLSIT CTVSGFSLTG YGVNWVRQPP GKGLEWLGMI WGDGSTDYNS 60

ALKSRLSISK DNSKSQVFLK MNSLQTDDTA RYYCAIGGSS YYYAMDYWGQ GTTVTVSS 118

<210>13

<211> 5

<212>PRT

<213> CDR1 of the heavy chain of 19C2

<400>13

GYGVN 5

<210>14

<211> 14

<212>PRT

<213> CDR2 of the heavy chain of 19C2

<400>14

MIWGDGSTDY NSAL 14

<210>15

<211> 11

<212>PRT

<213> CDR3 of the heavy chain of 19C2

<400>15

GGSSYYYAMD Y 11

<210>16

<211> 318

<212>DNA

<213>19C2 light full-length nucleotide sequence

<400> 16

gacattcagc tgacccagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60

atcacatgtc gagcaagtga gaatatttac agttatttag catggtatca gcagaaacag 120

ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagaagg tgtgccatca 180

aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 240

gaagattttg ggagttatta ctgtcaacat cattatggta ctccgctcac gttcggtgct 300

gggaccaagc tggagatc 318

<210>17

<211> 106

<212>PRT

<213>19C2 light chain full-length amino acid sequence

<400>17

DIQLTQSPAS LSASVGETVT ITCRASENIY SYLAWYQQKQ GKSPQLLVYN AKTLAEGVPS 60

RFSGSGSGTQ FSLKINSLQP EDFGSYYCQH HYGTPLTFGA GTKLEI 106

<210>18

<211> 11

<212>PRT

<213> CDR1 of the light chain of 19C2

<400>18

RASENIYSYL A 11

<210>19

<211> 7

<212>PRT

<213> CDR2 of the light chain of 19C2

<400>19

NAKTL AE 7

<210>20

<211> 9

<212>PRT

<213> CDR3 of the light chain of 19C2

<400>20

QHHYG TPLT 9