HPV16 type E6 oncoprotein monoclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to an HPV16 type E6 oncoprotein monoclonal antibody as well as a preparation method and application thereof. The monoclonal antibody specifically bound with the HPV16E6 recombinant antigen is obtained through screening, the monoclonal antibody is applied to a biotin-avidin amplification ELISA system, the E6 oncoprotein of HPV16 can be specifically detected, the sensitivity is 100 pg / mL, the monoclonal antibody does not intersect with oncoprotein of other high-risk HPV, and the E6 oncoprotein of HPV16 can be specifically detected. High specificity and sensitivity are shown for cervical exfoliated cell sample detection of normal people, CIN3 people and cervical cancer people, and the method can be applied to early screening of cervical cancer in the future; compared with traditional exfoliative cytology examination, the method is faster and more accurate, meanwhile, the risk of false positive and misdiagnosis caused by too high sensitivity of nucleic acid detection can be avoided, and the method has very high application value.

Description

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

[0002] Human papillomavirus (HPV) belongs to the category of papillomavirus DNA viruses. 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 correlated with anal and genital tumors in the general population. It is generally believed that persistent infection with high-risk human papillomavirus is the primary cause of cervical cancer and precancerous lesions. According to reports, about 600,000 new cervical cancer patients are diagnosed each year worldwide, and about 340,000 die from the disease. The economic burden caused by HPV-related diseases in my country is about 18 billion yuan each year, of which cervical cancer is about 7 billion yuan, which brings great health costs to individuals and the whole society.

[0003] The papillomavirus genome is divided into an early region (E), six open reading frames (ORFs) (E1, E2, E4, E5, E6 and E7) expressed in this region immediately after the initial infection of the host cell, and a late region (L) with major capsid protein L1 and minor capsid protein L2. 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, thereby changing metabolic pathways, such as targeting NFX1-91, which inhibits telomerase production. When NFX1-91 is degraded by E6, telomerase levels increase, inactivating the main mechanism for inhibiting cell growth. In addition, when E6 interacts with cellular transcription factors E2F1 / DP1, PDZ domains and MAGUK (membrane-associated guanylate kinase family) proteins. In summary, the E6 protein prevents normal protein activity in a variety of ways, allowing cells to grow and reproduce at an increased rate that is characteristic of cancer. The role of E6 expression in HPV-related cancers has also been considered an attractive target for therapeutic HPV vaccines against cervical cancer tumors. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a monoclonal antibody against HPV16 type E6 oncoprotein and its preparation method and application. The present invention screens and obtains a monoclonal antibody that specifically binds to HPV16 E6 recombinant antigen, and applies the monoclonal antibody to a biotin-avidin amplified ELISA system, which can specifically detect the E6 oncoprotein of HPV16 with a sensitivity of 100pg / mL, and has no cross-talk with other high-risk HPV oncoproteins. It shows high specificity and sensitivity in the detection of cervical exfoliated cell samples of normal people, CIN3 and cervical cancer people, and can be used for early screening of cervical cancer in the future; it is faster and more accurate than traditional exfoliated cytology, and can avoid the risk of false positives and misdiagnosis caused by the excessive sensitivity of nucleic acid detection, and has high application value.

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

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

[0007] 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;

[0008] 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.

[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 method for preparing a monoclonal antibody, comprising the following steps:

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

[0013] S2) taking all spleen cells of the immunized mice and fusing them with SP2 / 0 myeloma cells in the logarithmic growth phase, screening and culturing them to obtain hybridoma cells;

[0014] S3) The hybridoma cells obtained by screening are expanded and cultured, and injected intraperitoneally into female BALB / c mice, and the ascites is collected and purified after centrifugation to obtain monoclonal antibodies.

[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.

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

[0019] The ELISA detection system is used in detecting HPV16 type E6 oncoprotein.

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

[0021] The present invention screens and obtains a monoclonal antibody that specifically binds to the HPV16 E6 recombinant antigen. The monoclonal antibody is applied to a biotin-avidin amplified ELISA system, and can specifically detect the E6 oncoprotein of HPV16 with a sensitivity of 100 pg / mL, without crosstalk with oncoproteins of other high-risk HPVs. The detection of cervical exfoliated cell samples of normal people, CIN3 and cervical cancer people shows high specificity and sensitivity, and can be applied to the early screening of cervical cancer in the future. Compared with traditional exfoliated cytology examination, it is faster and more accurate, and can avoid the risks of false positives and misdiagnosis caused by the excessive sensitivity of nucleic acid detection, and has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[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 antigens;

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

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

[0027] Figure 5 The figure shows the identification results of 13 purified HPV16 E6 monoclonal antibodies (1 μg / mL) screened by the indirect ELISA method. DETAILED DESCRIPTION

[0028] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0029] The above technical solution is described in detail below in conjunction with specific embodiments.

[0030] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0031] Example 1 Preparation of recombinant antigens

[0032] This embodiment provides a recombinant antigen of 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 type E6 oncoprotein, comprising the following steps:

[0038] (1) Plasmid construction:

[0039] A partial fragment of the HPV16 E6 gene (1-147 nt) was selected, as shown in SEQ ID No. 2, and synthesized by Anhui General Biotechnology after optimization, and cloned into the pET32a expression vector to obtain the recombinant plasmid Pet32a-HPV16 / E6 expressing HPV16 E6;

[0040] (2) Induced expression:

[0041] The recombinant plasmid Pet32a-HPV16 / E6 was transformed into BL21 (DE3) competent cells, and the transformed bacteria were spread on LB agar plates containing 100 μg / mL ampicillin and cultured at 37°C overnight. A single colony was picked and inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin, and cultured at 37°C and 220 rpm overnight. 1% of the total volume of the culture medium was inoculated into LB medium containing 100 μg / mL ampicillin, and cultured at 37°C and 220 rpm for about 3 hours, until the OD600 was 0.6-0.9, and a final concentration of 0.1 mM IPTG was added, and the bacteria were collected after induction at 30°C and 200 rpm for 4 hours.

[0042] (3) Purification of recombinant protein:

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

[0044] Specifically: A buffer is 50mM PB, 300mM NaCl, pH 8.0, B buffer is 50mM PB, 300mM NaCl, 0.5M imidazole, pH 8.0, the chromatography column is balanced with A buffer, then the fermented bacterial solution is centrifuged at 8000rpm for 10min, the precipitate is resuspended with A solution, ultrasonically broken in ice water for 30min, ultrasonicated for 5 seconds at intervals of 5 seconds, centrifuged at 12000rpm for 30min, the supernatant is filtered with a 0.22 micron filter, loaded, the chromatography column is washed with A buffer, and finally gradient eluted with B buffer, and the purification is observed by SDS-PAGE protein gel electrophoresis. The target protein is selected to collect the peak, dialyzed to buffer A, the protein concentration is determined by Nanodrop, and the tube is divided into 1ml / tube and stored at -20℃.

[0045] (4) Identification of recombinant protein:

[0046] The purified recombinant HPV16 E6 protein was coated (1 μg / mL), and the reaction with the positive monoclonal antibody was identified by indirect ELISA. The positive monoclonal antibody was purchased commercially (Santa Cruz, C1P5). First, the recombinant protein was coated in a microplate: the coating buffer was carbonate buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate were fixed to 1 L pure water), the coating concentration was 1 μg / mL, 50 μl / well, 4°C overnight; 1% BSA was blocked, 100 μl per well, 37°C for 2 hours, the plate was washed once with washing buffer PBST (PBS containing 0.05% Tween-20), and patted dry; the monoclonal antibody was diluted with PBS according to a gradient of 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL, and 50 μL was added to the microplate coated with the antigen. At the same time, BK polyomavirus VP1 mouse monoclonal antibody was used as a negative control, and the reaction was carried out at 37°C for 30 minutes. Shake out the liquid in the wells, wash the plate 4 times with PBST washing solution, pat dry, add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 in PBS), react at 37°C for 30 min, wash the plate 4 times again, pat dry, add 50 μL / well of TMB color developing solution, develop 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 reader.

[0047] like Figure 1 As shown in the figure, "C1P5" represents the commercialized HPV16 E6 mouse monoclonal antibody, and "Ctrl" represents the BK polyomavirus VP1 mouse monoclonal antibody. It can be seen from the figure that the recombinantly expressed HPV16 E6 protein can bind specifically to the commercialized monoclonal antibody very well, but does not react with the control monoclonal antibody (BK polyomavirus VP1 mouse monoclonal antibody), indicating that the recombinant antigen is successfully expressed.

[0048] Example 2

[0049] This example provides another method for preparing a recombinant antigen of HPV16 type E6 oncoprotein, which is different from Example 1 only in that the expression vector is different, and the expression vector of this example is pET40b.

[0050] Example 3 Screening of monoclonal antibodies

[0051] This embodiment provides a monoclonal antibody obtained by screening based on the recombinant antigen, and the specific operation is as follows:

[0052] S1) Mouse immunization

[0053] 200 μL of the mixture obtained by mixing the purified recombinant protein with an equal volume of Freund's complete adjuvant was injected subcutaneously at multiple points to immunize 6-week-old female BALB / c mice at a dose of 30 μg / mouse. Then, at the 2nd and 4th weeks, the above dose was mixed with an equal volume of MF59 adjuvant and then injected intramuscularly for another immunization. At the 5th week, the mouse serum was collected to test the antibody titer. The mouse with the highest titer was selected and boosted with 20 μg of recombinant HPV16 E6 protein by intraperitoneal impact. Three days later, the mouse spleen was taken for the preparation of hybridoma cells;

[0054] S2) Screening of hybridoma cell lines

[0055] All spleen cells of immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and placed in HAT medium for screening and culture. When the fused cells grew to 1 / 2 of the bottom of the well, clones positive for HPV16 E6 protein were screened by indirect ELISA. Since the immunogen was derived from prokaryotic expression and contained a His tag, background components needed to be screened to screen out specific cell lines targeting HPV16 E6 protein. Positive cells were cloned to a monoclonal state by limiting dilution, and the cell lines were expanded and frozen;

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

[0057] HPV16 E6 recombinant protein and pET32a-HPV18-E6 (histidine tag) of pET32a vector were coated in microplates respectively. The coating buffer was carbonate buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water), the coating concentration was 1 μg / mL, and the plate was incubated at 4°C overnight; 1% gelatin was used for blocking, 150 μL per well, and the plate was blocked at 37°C for 2 hours. The plate was washed once with washing solution and patted dry; 50 μL cell culture supernatant was added and reacted at 37°C for 30 minutes. The liquid in the wells was shaken off, and the plate was washed 4 times with PBST solution. After patting dry, 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 in PBS) was added. The plate was reacted at 37°C for 30 min, and then washed 4 times. After patting dry, 50 μL / well of TMB colorimetric solution was added for color development at room temperature for 10 min. Finally, 0.5 M sulfuric acid was added to terminate the reaction, and the OD450 nm value was measured with an enzyme-labeled instrument. Positive cell lines that reacted only with HPV16E6 recombinant protein but not with control antigen were selected for subsequent experiments.

[0058] S4) Preparation of monoclonal antibody ascites

[0059] After the selected monoclonal cell lines were expanded and cultured, 0.2 mL (containing 2.5×10 6Cells) were pretreated with Freund's incomplete adjuvant in female BALB / c mice. After about 10 days, when the abdomen of the mice was obviously swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000r / min for 10 minutes to collect the middle layer.

[0060] S5) Affinity chromatography purification of monoclonal antibodies

[0061] The ascites was centrifuged at 12000r / min for 5 minutes, the supernatant was diluted 10 times with binding buffer (20mM PBS, 150mM NaCl, pH7.4), filtered through a 0.22μm filter, and the filtered sample was pumped into the Protein G (Cytiva) purification column equilibrated with binding buffer at a low speed through a peristaltic pump, connected to an AKTA purifier, washed with binding buffer for 5-10 column volumes until the UV absorption peak was washed flat, and then eluted with elution buffer (0.1M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1M Tris-HCl, pH 9, and placed in a dialysis bag (MW: 8000-14000), and dialyzed in 0.01M PBS pH7.4 solution at 2-8°C for 14 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000r / 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 indirect ELISA method in S3, the 13 purified monoclonal antibodies screened were identified. Figure 5 It can be seen that the purified monoclonal antibody has 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) Double antibody sandwich ELISA pairing

[0066] Antibody HRP labeling: The screened monoclonal antibodies were labeled according to the G-Biosciences HOOK TM The labeled antibody was dialyzed overnight in 0.01 M PBS, pH 7.4 buffer, and glycerol was added at a volume ratio of 1:1. The antibody was stored at -20°C in aliquots.

[0067] Specifically: dilute the antibody to be labeled with coupling buffer to a final concentration of 2 mg / mL, then add the diluted antibody solution to the tube containing HRP (provided with the kit), mix it by pipetting, and incubate it at room temperature for 1 hour. Mix it regularly during the incubation period. Add 50uL of stop solution, mix it for 15 minutes to terminate the labeling reaction, dialyze it in PBS buffer overnight, and add an equal volume of glycerol for freezing.

[0068] Screening of paired antibodies: The purified monoclonal antibodies were coated with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water, pH 9.6) at a concentration of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, respectively, at 4°C overnight. The coating solution was discarded the next day, and the plate was blocked with 1-2% BSA or gelatin at 150 μL / well, incubated at 37°C for 2 h, and the blocking solution was discarded. The antigen HPV16 E6 protein to be tested and the negative control antigen HPV18 E6 protein were diluted with PBS at 100 ng / mL and added to the ELISA plate at 50 μL / well, incubated at 37°C for 35 min, and the plate was washed 4 times with PBST washing solution, and PBS was added. 500, 1000, 2000 times diluted HRP labeled monoclonal antibody, 50 μL / well, incubate at 37°C for 35 min, wash the plate 4 times, pat dry and add 50 μL / well of TMB colorimetric solution, color at room temperature for 10 min, finally add 50 μL of 0.5M sulfuric acid to terminate the reaction, and measure the OD450nm value with an enzyme marker. The antibody combination with a high HPV16 E6 detection value and no reaction with HPV18 E6 protein was selected as the optimal pairing of the double antibody sandwich.

[0069] (II) Application of paired monoclonal antibodies in biotin-avidin amplified ELISA system

[0070] Biotin and avidin or biotin monoclonal antibody can specifically bind and have a multi-stage amplification effect. The combination of the two is highly stable and specific. In practical applications, it can not only greatly improve the sensitivity of the detection method, but also minimize the non-specific binding of the reaction reagents. Therefore, the use of the biotin-avidin amplification system will effectively improve the detection performance of the antibody pair.

[0071] 1. Biotin-antibody conjugation

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

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

[0074] The above-screened monoclonal antibody combinations were used for coating and biotin coupling, and then the most suitable reaction conditions for the amplification system were determined by exploring the coating antibody, biotin antibody concentration and the dilution of HRP-labeled avidin. The monoclonal antibody was coated in a microplate (coating buffer carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate fixed to 1 L pure water), the coating concentration gradient was 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 50 μL / well, 4°C overnight; the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, blocked with 3% sucrose, 1% BSA, 150 μL per well, 37°C for 2 hours, patted dry and stored for later use; 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 the 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 shaken off, the plate was washed 4 times with PBST washing solution, and patted dry; the biotin-conjugated monoclonal antibody was diluted with PBS to a concentration of 1μg / mL, 2μg / mL, and 4μg / mL, and 50μL / well was added to the microplate, and reacted at 37℃ for 30min. The liquid in the wells was shaken off, the plate was washed 4 times with PBST washing solution, and 50μL / well of HRP-polymer streptavidin (BIOSYNTH, 65R-S105PHRP, diluted with PBS 10000, 20000, and 40000 times) was added after patting dry, and reacted at 37℃ for 30min, and the plate was washed 4 times again, and 50μL / well of TMB colorimetric solution was added after patting dry to develop color at room temperature for 10min, and finally the stop solution was added to terminate the reaction, and the OD450nm value was measured with an enzyme reader. The coating concentration, biotin monoclonal antibody concentration, and HRP-labeled avidin dilution with the most obvious positive and negative differences were selected as the optimal reaction conditions.

[0075] 3. Detection specificity and sensitivity of biotin-avidin amplified ELISA system After determining the optimal conditions of the biotin-avidin amplified ELISA detection system, including the antibody coating concentration, biotin monoclonal antibody concentration, and HRP-labeled avidin dilution, the HPV16 E6 recombinant protein was graded diluted according to the detection method established above to identify the detection sensitivity of the method for recombinant antigens. At the same time, HPV16 E7, HPV18 E6, and HPV18 E7 recombinant antigens were added to detect whether the method had cross-reactions with other high-risk E6 / E7 oncoproteins. The optimal conditions were finally determined to be: antibody coating concentration of 1 μg / mL, biotin monoclonal antibody concentration of 2 μg / mL, and HRP-labeled avidin dilution of 20,000 times. Figure 2 As shown, the combination can specifically detect 100pg / ml HPV16 E6 recombinant protein and does not cross with other unrelated proteins, with good detection specificity and high sensitivity.

[0076] The full-length nucleotide sequence of the heavy chain (H chain) variable region 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] CAGGGTGCAGCTGCAGCAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGCAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATACCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGACTGGGTCGCAACCATTAGTAGTGGTGGTAGTTTCACC TACTATTCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGGCAAGAACACCCTGTACCTGCAAATGACCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTACAAGAGATGATGGTTACTCCCTTGACTGGGGCCAAGGCACCACTCTCACCGTCTCCTCA.

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

[0080] QVQLQQSGGGLVKPGGSLQLSCAASGFTFSDYTMSWVRQTPEKRLDWVATISSGGSFTYYSDSVKGRFTISRDNGKNTLYLQMTSLKSEDTAMYYCTRDDGYSLDWGQGTTLTVSS.

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

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

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

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

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

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

[0087] GACATCCAGATGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAGCATACACTGGTTTCAGCAAAGAACAAATGGTTCTCCAAGGCTTTCCATAAAGTATGCTTCTGAGTCTATCTC TGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGACATTGCAGATTATTACTGTCAACAAAGTAATAGCTGGCCAATGTACACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAACGTACGGTG.

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

[0089] DIQMTQSPAILSVSPGERVSFSCRASQSIGTSIHWFQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPMYTFGGGTKLELKRTV.

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

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

[0092] CDR-L2 amino acid sequence: YAS;

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

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

[0095] Referring to the above-mentioned indirect ELISA method, the monoclonal antibody was serially diluted at 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 0.1 ng / mL, and the binding activity of the HPV16 E6 monoclonal antibody (14D10) was determined using the BK polyomavirus VP1 mouse monoclonal antibody as a negative control.

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

[0097] (IV) Reaction with cell lines expressing HPV16 E6 / E7 oncoprotein

[0098] TC-1 cells are mouse lung epithelial cells that contain HPV16 E6 / E7 genes and are often used to construct animal models of cervical cancer. The TC-1 cell line can continuously express E6 and E7 proteins and has similar biological behaviors to 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 with 0.25% EDTA-trypsin and collected. The cells were washed twice with PBS and resuspended. The cells were ultrasonically disrupted in an ice bath at a power of 200 W for 10 minutes. The supernatant was electrophoretically separated according to the aforementioned SDS-PAGE method, and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk powder (diluted with TBST) at 4°C overnight. After washing with TBST three times, purified HPV16E6 monoclonal antibody (14D10 monoclonal antibody, 1 μg / ml, diluted with blocking solution) was added. At the same time, the same concentration of BK VP1 monoclonal antibody was added as a control. The membrane was incubated at room temperature for 1 hour. After washing with TBST three times, HRP-labeled goat anti-mouse IgG was added. The membrane was reacted at room temperature in the dark for 1 hour. After sufficient washing, DAB was added for color development.

[0099] After electrophoresis separation of TC-1 cell lysate, WB reaction was performed with 14D10 and control monoclonal antibody BK VP1. The results are as follows: Figure 4 As shown: "M" in the figure represents marker, 1 and 2 represent lanes 1 and 2 respectively, and the samples in lanes 1 and 2 are all TC-1 cell disruption solutions, of which the primary antibody incubated in lane 1 is HPV16E6 monoclonal antibody 14D10, and lane 2 is the control BK VP1 mouse monoclonal antibody. Monoclonal antibody 14D10 can react with TC-1 cells, with a band at around 22kDa, which is consistent with the size of E6, but does not react with the control monoclonal antibody, indicating that the monoclonal antibody can react with the natural cancer protein expressed by the cells, which is more authentic. Western immunoblotting shows that 14D10 monoclonal antibody can recognize TC-1 cells (expressing HPV16 E6 / E7), with a clear band at around 22kDa, while the control does not react.

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

[0101] The cervical brush samples collected clinically were tested using the established ELISA amplification detection system. Sample collection was carried out by the cooperative hospital, and cervical swab samples were collected from the physical examination population and patients with suspected / confirmed cervical cancer. Specifically, the commercial cervical sampling brush was used to collect exfoliated cells in the cervical transition zone and endocervical canal according to the operating instructions, and the brush head was removed and placed in a physiological saline buffer. The high-risk HPV virus typing nucleic acid detection kit (Shanghai Zhijiang Biotechnology Co., Ltd.) was used to qualitatively detect the specific DNA of 15 high-risk viruses such as HPV16, 18, 31, and 35 in the collected samples. Thirty normal samples with negative HPV16 nucleic acid, 18 samples with positive HPV16 nucleic acid and confirmed cervical cancer, and 12 samples with positive HPV16 nucleic acid were randomly selected from the physical examination population. The samples were tested using the established amplified ELISA system, specifically: coating with an antibody concentration of 1 μg / mL, overnight at 4°C, and blocking after washing; vortexing the sampling tube to be tested, taking 50 μL and adding it to the microplate coated with monoclonal antibody, and setting 2 wells of recombinant protein HPV16 E6 (10 ng / mL) as a positive control for the reaction, setting 2 wells of physiological saline storage solution as a negative control, and reacting at 37°C for 30 minutes. After washing the plate, add biotin-conjugated monoclonal antibody 2 μg / mL, 50 μL / well, and react at 37°C for 30 minutes. After washing, pat dry and add 20000 times diluted HRP-polymer streptavidin, react at 37°C for 30min, then color develop and terminate the reaction, and measure the OD450nm value with an enzyme marker. The cut-off value detected is 2.1 times the mean of the negative control, that is, greater than this value is positive, indicating that HPV16E6 oncoprotein exists in the sample to be tested, and less than or equal to this value is negative, indicating that HPV16 E6 oncoprotein does not exist in the sample to be tested or the content is below the minimum detection limit. Based on this, it can be inferred whether the patient is infected with HPV16, which is suitable for early screening of cervical cancer and precancerous lesions in health examinations. As shown in Table 1, the detection method of the present invention has a sensitivity of 91.7% for detecting CIN3, a sensitivity of 94.4% for detecting cervical cancer, and a detection specificity of 100%, indicating that the method has a high accuracy in detecting cervical cancer and precancerous lesions, and can play a significant role in the early screening of cervical cancer.

[0102] Table 1 - Clinical sample testing and evaluation results

[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 a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A monoclonal antibody against HPV16 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. A method for preparing the monoclonal antibody according to any one of claims 1 to 3, characterized in that: The specific operations are: S1) immunizing female BALB / c mice with the purified recombinant antigen; S2) taking all spleen cells of the immunized mice and fusing them with SP2 / 0 myeloma cells in the logarithmic growth phase, screening and culturing them to obtain hybridoma cells; S3) The hybridoma cells obtained by screening are expanded and cultured, and injected intraperitoneally into female BALB / c mice, and the ascites is collected and purified after centrifugation to obtain monoclonal antibodies.

5. The method for preparing a monoclonal antibody according to claim 4, characterized in that: In step (S1), an adjuvant is also added to the purified recombinant antigen.

6. The method for preparing a monoclonal antibody according to claim 5, characterized in that: The volume ratio of the purified recombinant antigen to the adjuvant is 1:1 。 7. The method for preparing a monoclonal antibody according to claim 5, characterized in that: The adjuvant is any one of Freund's complete adjuvant, MF59 aqueous adjuvant, and aluminum hydroxide adjuvant.

8. An ELISA detection system is constructed based on the monoclonal antibody according to any one of claims 1 to 3 or the monoclonal antibody prepared by the method according to any one of claims 4 to 7.

9. The ELISA detection system according to claim 8, characterized in that The ELISA detection system is a biotin-avidin amplified ELISA system.

10. Use of the ELISA detection system according to claim 8 or 9 in detecting HPV16 type E6 oncoprotein.

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