Monoclonal Antibody Against E6 Oncoprotein of HPV16, Preparation Method and Application Thereof

Abstract

The present invention relates to a monoclonal antibody against HPV16 E6 oncoprotein, its preparation method and application. The present invention screens and obtains a monoclonal antibody that specifically binds to the HPV16 E6 recombinant antigen. Applying the monoclonal antibody to a biotin-avidin amplified ELISA system can specifically detect the E6 oncoprotein of HPV16, with a sensitivity of 100 pg / mL, no cross-reaction with the oncoproteins of other high-risk HPVs, and showing high specificity and sensitivity in the detection of cervical exfoliated cell samples from normal populations, CIN3 and cervical cancer populations. It can be applied to the early screening of cervical cancer in the future; it is faster and more accurate than traditional exfoliative cytology examinations, and at the same time can avoid the risks of false positives and misdiagnosis caused by the too high sensitivity of nucleic acid detection, and has high application value.

Description

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody against HPV16 E6 oncoprotein, a preparation method thereof, and an application thereof. Background Art

[0002] Human papillomavirus (HPV) belongs to the papillomavirus DNA virus. HPV can infect epidermal cells through skin damage and spread to each other. In addition to causing cervical cancer in women, HPV infection is also highly associated with anal and genital tumors in the general population. Generally, persistent infection with high-risk human papillomavirus is considered to be the primary cause of cervical cancer and precancerous lesions. It is reported that approximately 600,000 new cases of cervical cancer are diagnosed globally each year, and approximately 340,000 people die from the disease. The economic burden caused by HPV-related diseases in China is approximately 18 billion yuan per year, of which cervical cancer is approximately 7 billion yuan, bringing a huge health cost to individuals and society as a whole.

[0003] The papillomavirus genome is divided into the early region (E), which contains six open reading frames (ORFs) (E1, E2, E4, E5, E6, and E7) that are immediately expressed after the initial infection of host cells, and the late region (L), which mainly includes the major capsid protein L1 and the minor capsid protein L2. Among them, studies on E6 have found that it interacts with many other proteins in cells. Its main function is to mediate the degradation of the tumor suppressor protein p53, thereby reducing the cell's ability to respond to DNA damage. E6 has also been shown to target other cellular proteins, thus altering metabolic pathways. For example, it targets NFX1-91, which inhibits telomerase production. When NFX1-91 is degraded by E6, the level of telomerase increases, inactivating the main mechanism that inhibits cell growth. In addition, E6 interacts with the cell transcription factors E2F1 / DP1, the PDZ domain, and MAGUK (membrane-associated guanylate kinase family) proteins. In summary, the E6 protein blocks normal protein activities in various ways, enabling cells to grow and reproduce at an increased rate characteristic of cancer. The role played by the expression of E6 in HPV-related cancers is also considered to be an attractive target for therapeutic HPV vaccines for cervical cancer tumors. Summary of the Invention

[0004] To solve the above problems, the present invention provides a monoclonal antibody against HPV16 E6 oncoprotein, its preparation method and application. The present invention screens and obtains a monoclonal antibody that specifically binds to the HPV16 E6 recombinant antigen. Applying the monoclonal antibody to the biotin-avidin amplified ELISA system can specifically detect the E6 oncoprotein of HPV16, with a sensitivity of 100 pg / mL, no cross-reactivity with the oncoproteins of other high-risk HPVs, and showing high specificity and sensitivity in the detection of cervical exfoliated cell samples from normal populations, CIN3, and cervical cancer patients. It can be applied to the early screening of cervical cancer in the future; it is faster and more accurate than traditional exfoliative cytology examinations, and at the same time can avoid the risk of false positives and misdiagnosis caused by the high sensitivity of nucleic acid detection, and has high application value.

[0005] The technical solution of the present invention is as follows:

[0006] A monoclonal antibody against HPV16 E6 oncoprotein, the antibody comprising a VH domain and a VL domain;

[0007] The VH domain comprises CDR-H1 of the amino acid sequence shown in SEQ ID No.1, CDR-H2 of the amino acid sequence shown in SEQ ID No.2, and CDR-H3 of the amino acid sequence shown in SEQ ID No.3;

[0008] The VL domain comprises CDR-L1 of the amino acid sequence shown in SEQ ID No.4, CDR-L2 with the amino acid sequence YAS, and CDR-L3 of the amino acid sequence shown in SEQ ID No.5.

[0009] The VH domain has the nucleotide sequence shown in SEQ ID No.6 and the amino acid sequence shown in SEQ ID No.7.

[0010] The VL domain has the nucleotide sequence shown in SEQ ID No.8 and the amino acid sequence shown in SEQ ID No.9.

[0011] A preparation method of the monoclonal antibody as described above, the specific operation is as follows:

[0012] S1) Immunize female BALB / c mice with the purified recombinant antigen;

[0013] S2) Take all spleen cells of the immunized mice and fuse them with SP2 / 0 myeloma cells in the logarithmic growth phase, screen and culture to obtain hybridoma cells;

[0014] S3) Expand the culture of the obtained hybridoma cells, intraperitoneally inject female BALB / c mice, collect ascites, purify after centrifugation to obtain the monoclonal antibody.

[0015] In step (S1), an adjuvant is also added to the purified recombinant antigen.

[0016] The volume ratio of the purified recombinant antigen to the adjuvant is 1:1 。

[0017] The adjuvant is any one of Freund's complete adjuvant, MF59 aqueous adjuvant, and aluminum hydroxide adjuvant. An ELISA detection system is constructed based on the monoclonal antibody described above.

[0018] The ELISA detection system is a biotin-avidin amplified ELISA system.

[0019] Application of the ELISA detection system described above in detecting HPV16 E6 oncoprotein.

[0020] The beneficial effects of the present invention are as follows:

[0021] The present invention screens and obtains a monoclonal antibody that specifically binds to the HPV16 E6 recombinant antigen. Applying the monoclonal antibody to a biotin-avidin amplified ELISA system can specifically detect the E6 oncoprotein of HPV16, with a sensitivity of 100 pg / mL, no cross-reaction with the oncoproteins of other high-risk HPVs, and showing high specificity and sensitivity in the detection of cervical exfoliated cell samples from normal populations, CIN3, and cervical cancer patients. In the future, it can be applied to the early screening of cervical cancer; it is faster and more accurate than traditional exfoliative cytology examinations, and at the same time can avoid the risks of false positives and misdiagnosis caused by the high sensitivity of nucleic acid detection, and has high application value. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0023] Figure 1 Shown is the ELISA identification result diagram of the HPV16 E6 recombinant protein and the commercial monoclonal antibody of the present invention;

[0024] Figure 2 Shown is the sensitivity result of the biotin-avidin amplified ELISA system based on 14D10 of the present invention for detecting recombinant antigen;

[0025] Figure 3 Shown is the binding activity of the HPV16 E6 monoclonal antibody (14D10) of the present invention;

[0026] Figure 4 Shown is the Western Blot identification result map of the anti-HPV16 E6 monoclonal antibody (14D10) and the TC-1 cell line expressing the HPV16 E6 oncoprotein;

[0027] Figure 5 Shown is the identification result map of 13 purified anti-HPV16 E6 monoclonal antibodies (1 μg / mL) screened by the indirect ELISA method. Detailed implementation manners

[0028] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other implementation manners obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope protected by the present invention.

[0029] The above technical solutions will be described in detail below in combination with specific embodiments.

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels.

[0031] Example 1 Preparation of recombinant antigen

[0032] This example provides a recombinant antigen of the HPV16 type E6 oncoprotein, whose amino acid sequence is shown in SEQ ID No.10, and the nucleotide sequence of the gene encoding the recombinant antigen is shown in SEQ ID No.11.

[0033] SEQ ID No.10 is as follows:

[0034] MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVY.

[0035] SEQ ID No.11 is as follows:

[0036] ATGCACCAAAAGAGAACTGCAATGTTTCAGGACCCACAGGAGCGACCCAGAAAGTTACCACAGTTATGCACAGAGCTGCAAACAACTATACATGATATAATATTAGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTGTAT.

[0037] This embodiment provides a method for preparing a recombinant antigen of HPV16 E6 oncoprotein, comprising the following steps:

[0038] (1) Plasmid construction:

[0039] Select a partial fragment (1 - 147 nt) of the HPV16 E6 gene, as shown in SEQ ID No. 2. After optimization, it is synthesized by Anhui General Biotech and cloned into the pET32a expression vector to obtain the recombinant plasmid Pet32a - HPV16 / E6 expressing HPV16 E6.

[0040] (2) Induced expression:

[0041] Transform the recombinant plasmid Pet32a - HPV16 / E6 into BL21(DE3) competent cells. Spread the transformed bacteria on an LB agar plate containing 100 μg / mL ampicillin and culture overnight at 37°C. Pick a single colony and inoculate it into 5 mL of LB medium containing 100 μg / mL ampicillin, and culture overnight at 37°C with shaking at 220 rpm. Inoculate it into LB medium containing 100 μg / mL ampicillin at a ratio of 1% of the total volume of the medium, and culture with shaking at 37°C and 220 rpm for about 3 hours until the OD600 is 0.6 - 0.9. Add IPTG with a final concentration of 0.1 mM, and induce at 30°C and 200 rpm for 4 hours, then collect the bacterial cells.

[0042] (3) Purification of the recombinant protein:

[0043] Since the expressed recombinant protein carries a histidine tag, it is purified using the AKTA Start and HisTrapTM HP affinity chromatography columns from GE Healthcare to obtain the purified recombinant protein, which is the recombinant antigen. The amino acid sequence is as shown in SEQ ID No. 1.

[0044] Specifically: Buffer A is 50 mM PB, 300 mM NaCl, pH 8.0, and buffer B is 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. Equilibrate the chromatography column with buffer A. Then centrifuge the fermented bacterial solution at 8000 rpm for 10 min, resuspend the precipitate with buffer A, ultrasonically disrupt it in ice water for 30 min, ultrasonically for 5 s every 5 s, centrifuge at 12000 rpm for 30 min, filter the supernatant through a 0.22 - micron filter, load the sample, wash the chromatography column with buffer A, and finally elute it with a gradient of buffer B. Observe the purification situation by SDS - PAGE protein gel electrophoresis. Select the peak of the target protein, dialyze and change the buffer to buffer A, measure the protein concentration with Nanodrop, aliquot it into 1 ml / tube, and store it at - 20°C.

[0045] (4) Identification of the recombinant protein:

[0046] Coated with purified recombinant HPV16 E6 protein (1 μg / mL), and its reaction with the positive monoclonal antibody was identified by indirect ELISA method. The positive monoclonal antibody was a commercially available monoclonal antibody purchased externally (Santa Cruz, C1P5). First, coat the recombinant protein in a microplate: the coating buffer is carbonate buffer (prepared by dissolving 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate in 1 L of pure water), the coating concentration is 1 μg / mL, 50 μL per well, overnight at 4 °C; block with 1% BSA, 100 μL per well, 2 hours at 37 °C, wash the plate once with washing solution PBST (PBS containing 0.05% Tween-20), and pat dry; dilute the monoclonal antibody in PBS at gradients of 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL, add 50 μL to the microplate coated with antigen, and at the same time use the mouse monoclonal antibody against BK polyomavirus VP1 as a negative control, react at 37 °C for 30 min. Discard the liquid in the wells, wash the plate 4 times with PBST washing solution, pat dry, add 50 μL of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS) per well, react at 37 °C for 30 min, wash the plate 4 times again, pat dry, add 50 μL of TMB chromogenic solution per well and develop color at room temperature for 10 min, finally add 0.5 M sulfuric acid to terminate the reaction, and measure the OD450nm value with an enzyme-linked immunosorbent assay instrument.

[0047] As Figure 1 shown, where "C1P5" represents the commercially available mouse monoclonal antibody against HPV16 E6, and "Ctrl" represents the mouse monoclonal antibody against BK polyomavirus VP1. It can be seen from the figure that the recombinantly expressed HPV16 E6 protein can specifically bind well with the commercially available monoclonal antibody, but does not react with the control monoclonal antibody (mouse monoclonal antibody against BK polyomavirus VP1), indicating the successful expression of the recombinant antigen.

[0048] Example 2

[0049] This example provides another preparation method of the recombinant antigen of HPV16 type E6 oncoprotein. The difference from Example 1 is only that the expression vector is different. The expression vector in this example is pET40b.

[0050] Example 3 Screening of Monoclonal Antibodies

[0051] This example provides a monoclonal antibody obtained by screening based on the recombinant antigen. The specific operation is as follows:

[0052] S1) Mouse Immunization

[0053] Mix 200 μL of the mixture obtained by mixing the purified recombinant protein with an equal volume of Freund's complete adjuvant, and subcutaneously inject it into 6-week-old female BALB / c mice at multiple points. The dose is 30 μg / mouse. Then, at the 2nd and 4th weeks, mix it with an equal volume of MF59 adjuvant at the above dose and immunize it again by intramuscular injection. At the 5th week, collect the mouse serum to detect the antibody titer. Select the mouse with the highest titer and boost the immunity by intraperitoneal injection with 20 μg of recombinant HPV16 E6 protein. Three days later, take the mouse spleen for the preparation of hybridoma cells;

[0054] S2) Screening of hybridoma cell lines

[0055] Fuse all the spleen cells of the immunized mice with SP2 / 0 myeloma cells in the logarithmic growth phase, and place them in HAT medium for screening culture. When the fused cells grow to 1 / 2 of the bottom of the well, screen and obtain the clones positive for HPV16 E6 protein by indirect ELISA method. Since the immunogen is from prokaryotic expression and contains His tag, it is necessary to perform a reverse screen on the background components to screen out the specific cell lines targeting HPV16 E6 protein. Clone the positive cells to the monoclonal state by limiting dilution method, and then expand the culture and cryopreservation of the cell lines;

[0056] S3) Screening of positive clones by indirect ELISA method:

[0057] Coat the recombinant HPV16 E6 protein and pET32a-HPV18-E6 (histidine tag) of the pET32a vector in the microplate respectively. The coating buffer is carbonate buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water), and the coating concentration is 1 μg / mL. Incubate overnight at 4°C; Block with 1% gelatin, 150 μL per well, block at 37°C for 2 hours, wash the plate once with the washing solution, and pat dry; Add 50 μL of cell culture supernatant and react at 37°C for 30 min. Discard the liquid in the well, wash the plate 4 times with PBST washing solution, pat dry, then add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS), react at 37°C for 30 min, wash the plate 4 times again, pat dry, add 50 μL / well of TMB chromogenic solution and develop color at room temperature for 10 min, and finally add 0.5 M sulfuric acid to terminate the reaction. Measure the OD450nm value with an enzyme-linked immunosorbent assay (ELISA) reader. Select the positive cell lines that react only with the recombinant HPV16E6 protein and not with the control antigen for subsequent experiments.

[0058] S4) Preparation of monoclonal antibody ascites

[0059] After expanding the culture of the screened monoclonal cell lines, inject 0.2 mL (containing 2.5×10 6Female BALB / c mice pretreated with Freund's incomplete adjuvant (about 10 days later, when the abdomen of the mice was significantly swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000 r / min for 10 minutes, and the middle layer was collected.)

[0060] S5) Affinity chromatography purification of monoclonal antibodies

[0061] The ascites was centrifuged at 12000 r / min for 5 minutes, and the supernatant was diluted 10 times with binding buffer (20 mM PBS, 150 mM NaCl, pH 7.4), filtered through a 0.22 μm filter, and the filtered sample was slowly pumped into a Protein G (Cytiva) purification column equilibrated with binding buffer using a peristaltic pump. The AKTA purifier was connected, and the column was washed with binding buffer for 5 - 10 column volumes until the UV absorption peak was washed flat, and then eluted with elution buffer (0.1 M glycine, pH 2.7). The elution peak was collected, and the collected sample was adjusted to neutral with 1 M Tris-HCl, pH 9, loaded into a dialysis bag (MW: 8000 - 14000), and dialyzed in 0.01 M PBS pH 7.4 solution at 2 - 8 °C for 14 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 r / min for 5 minutes. The supernatant was the purified monoclonal antibody. The concentration of the purified monoclonal antibody was measured using a micro-spectrophotometer and stored in aliquots.)

[0062] Identification of purified monoclonal antibodies:

[0063] Referring to the operation method of indirect ELISA in S3, 13 purified monoclonal antibodies screened were identified. The results are as Figure 5 shown. It can be seen that the purified monoclonal antibodies all had good binding activity with HPV16 E6 protein at a concentration of 1 μg / mL.)

[0064] Example 4 Application of HPV16 E6 monoclonal antibody

[0065] (I) Pairing of double-antibody sandwich ELISA

[0066] HRP labeling of antibodies: The screened monoclonal antibodies were labeled according to the instructions of G-Biosciences' HOOK TM HRP PLUS LabelingKit (Cat#: 786 - 313). Finally, the labeled antibodies were dialyzed overnight in 0.01 M PBS, pH 7.4 buffer, and glycerol was added in a volume ratio of 1:1, and stored in aliquots at -20 °C.)

[0067] Specifically: Dilute the antibody to be labeled with coupling buffer to a final concentration of 2 mg / mL. Subsequently, add the diluted antibody solution to the tube containing HRP (supplied with the kit), and pipette to mix well. Incubate at room temperature for 1 h, and mix well regularly during the incubation. Add 50 μL of termination solution, mix well for 15 min to terminate the labeling reaction, dialyze overnight in PBS buffer, and add an equal volume of glycerol for storage.

[0068] Screening of paired antibodies: Coat the purified monoclonal antibodies at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL with 50 μL / well of coating buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water, pH 9.6) overnight at 4°C. The next day, discard the coating solution, and block with 1 - 2% BSA or gelatin, 150 μL / well, incubate at 37°C for 2 h, discard the blocking solution, dilute the antigen to be detected, HPV16 E6 protein, and the negative control antigen, HPV18 E6 protein, with PBS to 100 ng / mL and add them to the ELISA plate, 50 μL / well, incubate at 37°C for 35 min, wash the plate 4 times with PBST washing solution, add the HRP-labeled monoclonal antibody diluted 500, 1000, and 2000 times with PBS, 50 μL / well, incubate at 37°C for 35 min, wash the plate 4 times again, pat dry, add 50 μL / well of TMB chromogenic solution, develop color at room temperature for 10 min, finally add 50 μL of 0.5 M sulfuric acid to terminate the reaction, and measure the OD450nm value with an ELISA reader. Select the antibody combination with a higher detection value for HPV16 E6 and no reaction with HPV18 E6 protein as the optimal pair for the double antibody sandwich.

[0069] (2) Application of paired monoclonal antibodies in the biotin-avidin amplification ELISA system

[0070] Biotin can specifically bind to avidin or biotin monoclonal antibody and has a multi-level amplification effect. The binding between the two has high stability and strong specificity. In practical applications, it can not only greatly improve the sensitivity of the detection method but also minimize the non-specific binding of reaction reagents. Therefore, using the biotin-avidin amplification system can effectively improve the detection performance of antibody pairs.

[0071] 1. Coupling of biotin and antibody

[0072] The molar ratio of biotin (Thermo, EZ-Link NHS Biotin, 20217) conjugated to the antibody was 20:1. First, 2.0 mg of activated biotin was added to 360 μL of ultrapure water and dissolved to prepare a 10 mM biotin solution. 2 mg of antibody and 26.6 μL of 10 mM biotin were reacted by shaking at room temperature for 3 h (the reaction system was controlled to be about 2 mL). Then, the biotin-antibody mixture was dialyzed with 0.01 M PBS to remove the excess free biotin. After dialysis, the antibody concentration was measured, and an equal volume of glycerol was added, and it was stored at -20 °C.

[0073] 2. Establishment of biotin-avidin amplified ELISA system

[0074] The monoclonal antibody combinations screened above were used for coating and biotin conjugation respectively. Then, by exploring the coating antibody, biotin-antibody concentration and dilution degree of HRP-labeled avidin, the most suitable reaction conditions for the amplification system were determined. Monoclonal antibody was coated in the microplate (coating buffer carbonate buffer: 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate were fixed to 1 L of pure water), and the coating concentration gradients were 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 50 μL / well, overnight at 4 °C; the microplate was washed once with the washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, blocked with 3% sucrose and 1% BSA, 150 μL per well, at 37 °C for 2 h, patted dry and stored for later use after drying; the recombinant protein HPV16 E6 was diluted to a concentration of 10 ng / mL with PBS, and 50 μL was added to the microplate coated with monoclonal antibody. At the same time, the recombinant protein HPV18 E6 was diluted to 10 ng / mL as a negative control, and reacted at 37 °C for 30 min. The liquid in the wells was flicked out, and the microplate was washed 4 times with PBST washing solution and patted dry; the biotin-conjugated monoclonal antibody was diluted to concentrations of 1 μg / mL, 2 μg / mL, 4 μg / mL with PBS, and 50 μL / well was added to the microplate and reacted at 37 °C for 30 min. The liquid in the wells was flicked out, the microplate was washed 4 times with PBST washing solution and patted dry, then 50 μL / well of HRP-polymeric streptavidin (BIOSYNTH, 65R-S105PHRP, diluted 10000, 20000, 40000 times with PBS) was added and reacted at 37 °C for 30 min, then washed 4 times again, patted dry, 50 μL / well of TMB chromogenic solution was added and developed color at room temperature for 10 min, and finally the termination solution was added to terminate the reaction, and the OD450nm value was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The coating concentration, biotin-monoclonal antibody concentration and dilution degree of HRP-labeled avidin with the most obvious difference between negative and positive were selected as the optimal reaction conditions.

[0075] 3. Detection specificity and sensitivity of the biotin-avidin amplified ELISA system After determining the optimal conditions such as the antibody coating concentration, the concentration of biotin monoclonal antibody, and the dilution factor of HRP-labeled avidin in the biotin-avidin amplified ELISA detection system, according to the detection method established above, the recombinant protein of HPV16 E6 was serially diluted to identify the detection sensitivity of the method for the recombinant antigen. At the same time, the recombinant antigens of HPV16 E7, HPV18 E6, and HPV18 E7 were added to detect whether there was cross-reactivity with other high-risk E6 / E7 oncoproteins. Finally, the optimal conditions were determined as follows: the antibody coating concentration was 1 μg / mL, the concentration of biotin monoclonal antibody was 2 μg / mL, and the dilution factor of HRP-labeled avidin was 20,000-fold. As Figure 2 shown, this combination can specifically detect the recombinant protein of HPV16 E6 at 100 pg / ml and does not cross-react with other irrelevant proteins, showing good detection specificity and high sensitivity.

[0076] The full-length nucleotide sequence of the variable region of the heavy chain (H chain) of the monoclonal antibody 14D10 is shown in SEQ ID No.6, and the amino acid sequence is shown in SEQ ID No.7.

[0077] SEQ ID No.6 is as follows:

[0078] CAGGTGCAGCTGCAGCAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGCAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATACCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGACTGGGTCGCAACCATTAGTAGTGGTGGTAGTTTCACCTACTATTCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGGCAAGAACACCCTGTACCTGCAAATGACCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTACAAGAGATGATGGTTACTCCCTTGACTGGGGCCAAGGCACCACTCTCACCGTCTCCTCA.

[0079] SEQ ID No.7 is as follows:

[0080] QVQLQQSGGGLVKPGGSLQLSCAASGFTFSDYTMSWVRQTPEKRLDWVATISSGGSFTYYSDSVKGRFTISRDNGKNTLYLQMTSLKSEDTAMYYCTRDDGYSLDWGQGTTLTVSS。

[0081] The three CDR sequences of the heavy chain (H chain) are as follows:

[0082] Amino acid sequence of CDR-H1: GFTFSDYT (SEQ ID No.1);

[0083] Amino acid sequence of CDR-H2: ISSGGSFT (SEQ ID No.2);

[0084] Amino acid sequence of CDR-H3: TRDDGYSLD (SEQ ID No.3).

[0085] The full-length nucleotide sequence of the variable region of the light chain (L chain) of the monoclonal antibody 14D10 is as shown in SEQ ID No.8, and the amino acid sequence is as shown in SEQ ID No.9.

[0086] SEQ ID No.8 is as follows:

[0087] GACATCCAGATGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAGCATACACTGGTTTCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGACATTGCAGATTATTACTGTCAACAAAGTAATAGCTGGCCAATGTACACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAACGTACGGTG。

[0088] SEQ ID No.9 is as follows:

[0089] DIQMTQSPAILSVSPGERVSFSCRASQSIGTSIHWFQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPMYTFGGGTKLELKRTV。

[0090] The sequences of the three CDRs of the light chain (L chain) are as follows:

[0091] Amino acid sequence of CDR-L1: QSIGTS (SEQ ID No.4);

[0092] Amino acid sequence of CDR-L2: YAS;

[0093] Amino acid sequence of CDR-L3: QQSNSWPMYT (SEQ ID No.5).

[0094] (III) Identification of the binding activity of paired monoclonal antibodies

[0095] Referring to the aforementioned indirect ELISA method, monoclonal antibodies were serially diluted at concentrations of 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 0.1 ng / mL. The murine monoclonal antibody against BK polyomavirus VP1 was used as a negative control to determine the binding activity of the anti-HPV16 E6 monoclonal antibody (14D10).

[0096] As Figure 3 shown, the monoclonal antibody (14D10) still reacted with the recombinant HPV16 E6 antigen at a concentration of 1 ng / ml and did not react with the control monoclonal antibody (Ctrl, BK VP1 monoclonal antibody), indicating that this strain of monoclonal antibody has high binding activity.

[0097] (IV) Reactivity with cell lines expressing HPV16 E6 / E7 oncoproteins

[0098] TC-1 cells are mouse lung epithelial cells containing the E6 / E7 genes of HPV type 16 and are commonly used to construct disease animal models of cervical cancer. The TC-1 cell line can continuously express E6 and E7 proteins and has biological behaviors similar to those of cervical cancer cells. The reaction of the monoclonal antibody with the TC-1 cell line was identified by Western Blot. The cultured TC-1 cells in the logarithmic growth phase were digested and collected with 0.25% EDTA-trypsin, resuspended after washing twice with PBS, and sonicated on ice for 10 minutes under an ultrasonic disruptor with a power of 200 W. The disrupted supernatant was separated by electrophoresis according to the aforementioned SDS-PAGE method, transferred to a nitrocellulose membrane at the same time, and blocked overnight at 4°C with 5% skim milk (diluted with TBST). After washing three times with TBST, purified anti-HPV16 E6 monoclonal antibody (14D10 monoclonal antibody, 1 μg / ml, diluted with the blocking solution) was added, and at the same time, monoclonal antibody against BK VP1 with the same concentration was added as a control. After incubating at room temperature for 1 h, HRP-labeled goat anti-mouse IgG was added after washing three times with TBST, and the reaction was carried out in the dark at room temperature for 1 h. After thorough washing, DAB was added for color development.

[0099] After electrophoretic separation of the lysate of TC-1 cells, WB reactions were carried out with 14D10 and the control monoclonal antibody BK VP1 monoclonal antibody, and the results are as Figure 4 shown: "M" in the figure represents the marker, 1 and 2 represent lane 1 and lane 2 respectively. The samples loaded in lanes 1 and 2 are both the lysate of TC-1 cells. Among them, the primary antibody incubated in lane 1 is the anti-HPV16 E6 monoclonal antibody 14D10, and lane 2 is the control BK VP1 mouse monoclonal antibody. The monoclonal antibody 14D10 can react with TC-1 cells and has a band at about 22 kDa, which is consistent with the size of E6, while it does not react with the control monoclonal antibody, indicating that this monoclonal antibody can react with the natural oncoprotein expressed by the cells and is more authentic. Western blot showed that the monoclonal antibody 14D10 could recognize TC-1 cells (expressing HPV16 E6 / E7) and had an obvious band at about 22 kDa, while the control did not react.

[0100] (V) Clinical application in detecting cervical exfoliated cell swab samples

[0101] Using the established ELISA amplification detection system, the cervical brush samples collected clinically were detected. The sample collection was implemented by the collaborating hospitals. Cervical swab samples were collected from the physical examination population and patients with suspected / confirmed cervical cancer. Specifically: Using a commercial cervical sampling brush, exfoliated cells in the cervical transformation zone and endocervical canal were collected according to the operation instructions, and the brush head was removed and placed in a physiological saline buffer. The specific DNA of 15 high-risk viruses such as HPV16, 18, 31, 35, etc. in the collected samples was qualitatively detected using a high-risk HPV virus typing nucleic acid detection kit (Shanghai ZJ Bio-Tech Co., Ltd.). 30 normal samples with negative HPV16 nucleic acid in the physical examination population, 18 samples with positive HPV16 nucleic acid in confirmed cervical cancer patients, and 12 samples with positive HPV16 nucleic acid in confirmed CIN3 (precancerous lesion of cervical cancer) patients were randomly selected from them. The above samples were detected using the established amplified ELISA system. Specifically: The antibody with a coating concentration of 1 μg / mL was coated overnight at 4°C, and the plate was washed and then blocked; the sampling tubes to be tested were vortexed and mixed evenly, 50 μL was taken and added to the microwell plate coated with monoclonal antibody. At the same time, 2 wells of recombinant protein HPV16 E6 (10 ng / mL) were set as positive controls for the reaction, 2 wells of physiological saline preservation solution were set as negative controls, and the reaction was carried out at 37°C for 30 min. After washing the plate, 2 μg / mL of biotin-conjugated monoclonal antibody was added, 50 μL / well, and the reaction was carried out at 37°C for 30 min. After washing and patting dry, 20,000-fold diluted HRP-polymerized streptavidin was added, and after reacting at 37°C for 30 min, color development was carried out and the reaction was terminated, and the OD450nm value was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The Cut-off value for detection was 2.1 times the mean value of the negative control, that is, greater than this value was positive, indicating the presence of HPV16 E6 oncoprotein in the sample to be tested, and less than or equal to this value was negative, indicating the absence of HPV16 E6 oncoprotein in the sample to be tested or the content was lower than the minimum detection limit. Based on this, it can be inferred whether the patient is infected with HPV16, which is applicable to the early screening of cervical cancer and precancerous lesions in health examinations. As shown in Table 1, the sensitivity of the detection method of the present invention for detecting CIN3 was 91.7%, the sensitivity for detecting cervical cancer was 94.4%, and the detection specificity was 100%, indicating that this method has high accuracy in the detection of cervical cancer and precancerous lesions and can play a major role in the early screening of cervical cancer.

[0102] Table 1 - Evaluation Results of Clinical Sample Detection

[0103] HPV16 E6 Normal CIN3 Cervical cancer Total Negative 30 1 1 32 Positive 0 11 17 28 Total 30 12 18 60

[0104] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. A monoclonal antibody against HPV16 type E6 oncoprotein, characterized in that: The antibody comprises a VH domain and a VL domain; The VH domain includes CDR-H1 of the amino acid sequence shown in SEQ ID No.1, CDR-H2 of the amino acid sequence shown in SEQ ID No.2, and CDR-H3 of the amino acid sequence shown in SEQ ID No.3; The VL domain includes CDR-L1 with the amino acid sequence shown in SEQ ID No.4, CDR-L2 with the amino acid sequence of YAS and CDR-L3 with the amino acid sequence shown in SEQ ID No.

5.

2. The HPV16 type E6 oncoprotein monoclonal antibody according to claim 1, characterized in that: The VH domain has the nucleotide sequence shown in SEQ ID No.6 and the amino acid sequence shown in SEQ ID No.

7.

3. The HPV16 type E6 oncoprotein monoclonal antibody according to claim 1, characterized in that: The VL domain has the nucleotide sequence shown in SEQ ID No.8 and the amino acid sequence shown in SEQ ID No.

9.

4. An ELISA detection system constructed based on the monoclonal antibody described in any one of claims 1 to 3.

5. The ELISA detection system according to claim 4, characterized in that The ELISA detection system is a biotin-avidin amplified ELISA system.

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