P16 antigen, hybridoma cell strain, monoclonal antibody and preparation method and application thereof

Abstract

The invention discloses a p16 antigen, a hybridoma cell strain, a monoclonal antibody and a preparation method and application thereof, and belongs to the technical field of biology. A prokaryotic expression technology is used for expressing a human p16 protein amino terminal peptide fragment as a p16 antigen peptide immune mouse, a hybridoma cell strain of an anti-p16 monoclonal antibody is prepared through a subcloning technology, the anti-p16 monoclonal antibody capable of being combined with the p16 protein is obtained through serum-free culture, and the antibody can be used for preparing a kit for detecting the p16 protein in human tissues and body fluid samples.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a p16 antigen, a hybridoma cell line, a monoclonal antibody and a preparation method and application thereof. Background Art

[0002] The tumor suppressor protein p16INK4a (p16 for short) is a cyclin-dependent kinase inhibitor encoded by the Cdkn2a (Cyclin-dependent kinase 2a) gene. By binding to cyclin-dependent kinases 4 (CDK4) and 6 (CDK6), it effectively inhibits the formation of an active complex between CDK4 / 6 and cyclin D, thereby preventing the phosphorylation of the retinoblastoma protein (Rb). In its unphosphorylated state, Rb tightly binds to and inhibits the activity of E2F transcription factors, arresting the cell cycle in the G1 phase. This strictly restricts cell progression from G1 to S phase, achieving precise negative regulation of cell cycle progression and proliferation. This sophisticated regulatory mechanism is crucial for maintaining normal cell growth, differentiation, and tissue homeostasis.

[0003] High-risk human papillomavirus (HR-HPV) infection is a major pathogenic factor in numerous human malignancies, particularly cervical cancer. The HPV-encoded E7 protein binds to the Rb protein, disrupting the interaction between Rb and the E2F transcription factor. This binding leads to abnormal activation of the E2F transcription factor due to loss of Rb's inhibition. The activated E2F transcription factor activates a series of genes involved in cell cycle progression and DNA synthesis, accelerating the transition from G1 to S phase of the cell cycle and promoting hyperproliferation. Furthermore, in HR-HPV-infected cells, to cope with this abnormal cell cycle regulation imbalance, they overexpress the p16 protein. However, this compensatory overexpression of p16 in the nucleus and cytoplasm does not effectively inhibit cell proliferation, but it serves as a specific immunophenotypic characteristic of high-risk HPV-associated malignancies. Therefore, detecting p16 protein expression in human cells or body fluids can be used for the diagnosis and differential diagnosis of HPV-associated malignancies.

[0004] Although a variety of monoclonal antibodies targeting different epitopes of the p16 protein have been used to detect the p16 protein, the existing anti-p16 antibodies still cannot meet clinical needs due to the scattered distribution of mutation sites and truncation of the CDKN2a gene encoding the p16 protein.

[0005] Therefore, the preparation of highly specific and sensitive anti-p16 monoclonal antibodies targeting the amino-terminal antigen epitope of the p16 protein is of great practical significance. Such antibodies are expected to provide more effective tools for the accurate detection of p16 protein in human cells or body fluids, thereby providing a more reliable basis for the early diagnosis, disease monitoring and formulation of treatment plans for HPV-related malignant tumors. Summary of the Invention

[0006] Due to the sporadic genetic mutations and truncation deletions of the p16 protein, anti-p16 monoclonal antibodies targeting epitopes located in the middle and carboxyl regions of the p16 protein amino acid sequence lack specificity and sensitivity, making it difficult to meet the clinical needs of p16 protein detection. Therefore, the use of p16 protein amino-terminal antigenic peptides can effectively address this problem. To this end, the present invention provides a p16 antigenic peptide, hybridoma cell line, monoclonal antibody preparation, and their application.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a p16 antigen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The present invention provides a nucleic acid molecule, which is the coding gene of the p16 antigen.

[0009] The present invention provides a recombinant expression vector, which contains the nucleic acid molecule; the empty vector of the recombinant expression vector is a plasmid vector.

[0010] The present invention provides a method for constructing the above-mentioned recombinant expression vector, the construction method comprising: The p16 antigen coding gene and / or the nucleic acid molecule are inserted between the restriction enzyme cutting sites of the expression vector to obtain the recombinant expression vector.

[0011] The present invention provides a recombinant engineered bacterium for preparing the p16 antigen. The recombinant engineered bacterium comprises the recombinant expression vector, and / or the nucleic acid molecule is integrated into the genome of the recombinant engineered bacterium.

[0012] The present invention provides a method for preparing the p16 antigen, the method comprising: Cultivating and inducing the recombinant engineered bacteria; The bacterial solution was collected, centrifuged and resuspended to obtain a bacterial suspension; The bacterial suspension is ultrasonically disrupted and then protein purified to obtain free p16 antigen.

[0013] The present invention provides a monoclonal antibody against p16 antigen, wherein the monoclonal antibody is prepared by using the p16 antigen as an immunogen; the antigen-binding fragment of the monoclonal antibody specifically binds to the p16 antigen, and the monoclonal antibody comprises a heavy chain variable region (V H ) and light chain variable region (V L ),in, The heavy chain variable region comprises: CDR1 amino acid sequence: TSGVGVG (as shown in SEQ ID NO. 2); CDR2 amino acid sequence: HIWWDDDKHYNPALKS (as shown in SEQ ID NO. 3); CDR3 amino acid sequence: LGDYLFPY (as shown in SEQ ID NO. 4); The light chain variable region comprises: CDR1 amino acid sequence: RSSRNIVHNNGITYLE (as shown in SEQ ID NO. 5); CDR2 amino acid sequence: KVSNRFS (as shown in SEQ ID NO. 6); CDR3 amino acid sequence: FQGSHVPPT (as shown in SEQ ID NO.7).

[0014] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.9.

[0015] The monoclonal antibody further comprises a constant region, which includes a heavy chain constant region and a light chain constant region.

[0016] Furthermore, the heavy chain constant region subtype of the monoclonal antibody is IgG1; the light chain constant region subtype of the monoclonal antibody is Kappa.

[0017] The present invention provides a hybridoma cell line, which secretes the monoclonal antibody, is named hybridoma cell line 3G3B5, and is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with accession number CGMCC No. 46037 and a deposit date of August 14, 2024.

[0018] The p16 antigen, the nucleic acid molecule, the recombinant expression vector, the recombinant engineered bacteria, the monoclonal antibody against the p16 antigen or the hybridoma cell line are used in the preparation of a detection reagent for detecting the expression of p16 protein in tumor and normal tissue cells, and the detection reagent is used in at least one detection method selected from immunohistochemistry, immunoblotting and enzyme-linked adsorption assay.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The p16 antigen provided by the present invention selects the 90 amino acid residues at the amino terminus of the human p16 protein as an immunogen, effectively reducing the possibility of antigen epitope loss caused by gene truncation or gene mutation at the carboxyl terminus of the p16 protein, thereby improving the specificity and sensitivity of the anti-p16 monoclonal antibody.

[0020] The recombinant expression vector provided herein can be a prokaryotic expression vector commonly used in the art for expressing a target gene, preferably the low-temperature-inducible promoter vector pCzn1-His. This vector carries the promoter of the cold shock protein cspA gene, which can be induced at temperatures as low as 11°C, promoting protein solubility and increasing the probability of soluble protein expression. Furthermore, the pCzn1-His expression vector itself carries a 6xHis-tagged protein, facilitating affinity purification using a Ni purification system to obtain high-purity recombinant protein.

[0021] The recombinant engineered bacteria provided by the present invention contain the recombinant pCzn1-His expression vector described in the second paragraph, and / or the nucleic acid molecule described in the p16 protein antigen peptide is integrated into the genome of the recombinant engineered bacteria.

[0022] The hybridoma cell line 3G3B5 of the present invention is obtained by immunizing mice with the amino-terminal truncated antigen peptide of human p16 protein as an immunogen and by an optimized monoclonal screening method, and can secrete anti-p16 monoclonal antibodies that can specifically bind to p16 protein.

[0023] The anti-p16 antigen monoclonal antibodies provided by the present invention can specifically recognize and bind to the p16 protein, avoiding cross-reactions with other non-target proteins, thereby improving the accuracy of detection. Due to the high affinity of the monoclonal antibodies, they can detect the presence of the p16 protein at extremely low concentrations, which is particularly important for the diagnosis of early tumors.

[0024] The anti-p16 monoclonal antibody of the present invention can specifically bind to the p16 protein in vitro and has good sensitivity. It can be used as a primary antibody to prepare various detection methods and kits for detecting the p16 protein in human tissue and body fluid samples, such as immunohistochemical staining, immunoblotting and enzyme-linked immunosorbent assay, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1This is the SDS-PAGE analysis of the present invention to identify the induced expression of the recombinant pCzn1-p16 expression vector in the engineering bacteria; wherein, lane M represents the protein molecular weight standard, lane 1 represents pCZN1 induction (empty load), lane 2 represents pCzn1-p16 without induction, lane 3 represents pCzn1-p16 after induction, lane 4 represents the supernatant after pCzn1-p16 induction and fragmentation, lane 5 represents the precipitate after pCzn1-p16 induction and fragmentation, and the position indicated by the red arrow is the band position of the target protein (p16 protein); Figure 2 Western Blot analysis of the p16 protein purified by Ni column in the present invention, wherein lane M represents the protein molecular weight standard, and lane 1 represents the purified sample (p16 protein); Figure 3 The purified anti-p16 monoclonal antibody (clone 3G3B5) of the present invention was analyzed by SDS-PAGE electrophoresis, wherein lane M is a protein molecular weight standard, lane 1 represents the purification analysis results of the anti-p16 monoclonal antibody (clone 3G3B5), 55KD is the antibody heavy chain, and 25KD is the antibody light chain; Figure 4 The detection of the variable region VH VL of the anti-p16 monoclonal antibody (clone number 3G3B5) of the present invention, wherein lane M is DL2000 DNA Marker; lane 1 represents the VH VL of the p16 monoclonal antibody (clone number 3G3B5) H gene, lane 2 represents p16 monoclonal antibody (clone number 3G3B5) V L Gene; Figure 5 These are the immunohistochemical staining results of p16 protein in human samples detected by the anti-p16 monoclonal antibody of the present invention, wherein A represents the expression of p16 protein in HeLa cells, B represents the expression of p16 protein in cancer cells with glandular involvement in cervical carcinoma in situ, C represents the expression of p16 protein in cervical squamous cell carcinoma, D represents the expression of p16 protein in high-grade endometrial carcinoma, E represents the expression of p16 protein in colon adenocarcinoma, F represents the expression of p16 protein in gastric adenocarcinoma, G represents the expression of p16 protein in metastatic adenocarcinoma, and H represents the expression of p16 protein in small cell lung carcinoma. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0027] The hybridoma cell line used in the present invention (clone number 3G3B5) was deposited on August 14, 2024, at the General Microbiology Center of the China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with the deposit number CGMCC No. 46037. The proposed classification name is: Balb / c mouse monoclonal antibody hybridoma cell line.

[0028] The present invention is described in further detail below with reference to the accompanying drawings: Example 1 According to the nucleotide and amino acid sequence and structure of the p16 gene DNA on human chromosome 9 (GenBank: AH005371.3) published in the PubMed database, the polypeptide represented by the amino-terminal 90 amino acids of the p16 protein (SEQ ID NO.1) was selected.

[0029] SEQ ID NO.1: MEPAAGSSMEPSADWLATAAARGRVEEVRALLEAGALPNAPNSYGRRPIQVMMMGSARVAELLLLHGAEPNCADPATLTRPVHDAAREGF.

[0030] (1) Recombinant pCzn1-p16 expression vector After codon optimization according to the prokaryotic protein expression system, a 6×histidine tag and TAA (stop codon) were added to the nucleotide sequence corresponding to the amino-terminal 90-amino acid peptide of the p16 protein. NdeI and XbaI restriction sites were then added upstream and downstream of the sequence. The coding sequence was double-digested with the restriction endonucleases NdeI and XbaI. The digested product, containing sticky ends, was recovered by agarose gel electrophoresis and cloned into the linearized pCzn1 expression vector, which had also been double-digested with NdeI and XbaI. This resulted in the recombinant pCzn1-p16 expression vector containing the p16 antigen, with a molecular weight of 10.89 kd (including the tag).

[0031] (2) Construction of engineering bacteria with the recombinant pCzn1-p16 expression vector Transform 1 μL of the recombinant pCzn1 expression vector into 100 μL of competent Arctic-Express (DE3) E. coli and place on ice for 20 minutes. Heat shock the cells at 42°C for 90 seconds, quickly place the cells on ice for 5 minutes, and add 600 μL of LB culture medium. Incubate the cells at 37°C with shaking at 220 rpm for 1 hour. After centrifugation, spread the entire cell onto an LB plate containing 50 μg / mL Amp / Kan and incubate the cells in an inverted manner at 37°C overnight to obtain a single clone of the recombinant pCzn1-p16 expression vector engineered bacteria.

[0032] (3) Induced expression of recombinant pCzn1-p16 expression vector in engineered bacteria The monoclonal engineered bacteria on the transformation plate were picked and inoculated into a test tube containing 3 mL LB culture medium containing 50 μg / mL Amp / Kan, and shaken at 37°C 220 r / min overnight. The next day, the cells were inoculated into 30 mL LB culture medium containing 50 μg / mL Amp / Kan at a ratio of 1:100, and shaken at 37°C 220 r / min until the bacterial OD600 reached 0.6-0.8. 1 mL of culture was removed and centrifuged at 10,000 r / min at room temperature for 2 min. The supernatant was discarded and the bacterial pellet was resuspended in 100 μL 1× loading buffer. The remaining culture was used to induce fusion protein expression, centrifuged at 4,000 r / min for 10 min, the supernatant was discarded, the cells were resuspended in PBS and ultrasonically disrupted, the supernatant and pellet were taken and added to the loading buffer, mixed, and analyzed by 12% SDS-PAGE. The bands were visualized by Coomassie Brilliant Blue staining ( Figure 1 ).

[0033] By the attached Figure 1 It can be seen that by comparing lanes 2 and 3, a band consistent with the expected molecular weight appeared in lane 3 after pCzn1-p16 was induced, and there was no obvious band at this position in lane 2 when pCzn1-p16 was not induced, indicating that pCzn1-p16 successfully expressed the target protein (p16 protein) under induced conditions; by comparing lanes 4 and 5, a target protein (p16 protein) band was detected in the supernatant after pCzn1-p16 was induced and broken in lane 4, indicating that the target protein (p16 protein) was expressed in a soluble manner; a target protein (p16 protein) band was also detected in the precipitate after pCzn1-p16 was induced and broken in lane 5, indicating that the target protein (p16 protein) can exist in the form of inclusion bodies.

[0034] (4) Ni column affinity purification of p16 fusion protein and analysis of results 1) Ni column affinity purification of p16 fusion protein Using a low-pressure chromatography system, the supernatant solution was loaded onto a Ni-IDA-Sepharose Cl-6B affinity chromatography column (purchased from Novagen) pre-equilibrated with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min. The column was flushed with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min until the OD value of the effluent reached 0. 280 Wash with Ni-IDA Washing-Buffer (20 mM Tris-HCl, 30 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 mL / min until the OD value of the effluent reaches 0.1%. 280 The target protein was eluted with Ni-IDA Elution Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 mL / min, and the flow-through was collected. The collected protein solution was placed in a dialysis bag, dialyzed against PBS overnight, and analyzed by 12% SDS-PAGE.

[0035] 2) Detection of purified p16 protein by Western Blot 5 μL of sample purified by Ni column was loaded and subjected to polyacrylamide gel electrophoresis at a constant voltage of 100 V and a constant current of 250 mA for about 1.5 h. The membrane was then washed with PBST and blocked with 5% skim milk powder blocking solution at 37°C for 1 h. Anti-His antibody was used as the primary antibody and the membrane was washed 4 times with PBST after overnight reaction at 4°C. Goat anti-mouse anti-HIS tag antibody was used as the secondary antibody and the membrane was reacted for 1 h at 37°C. The membrane was then washed 4 times and developed by ECL. Figure 2 ).

[0036] By the attached Figure 2 The data show that a band consistent with the expected molecular weight appeared in lane 1, indicating that the sample purified by the Ni column contains the target protein (p16 protein).

[0037] Example 2 Eight 6-8 week old BALB / c mice (Zhongding Bio, batch number: 20221124) were immunized with the p16 protein antigen peptide. The first immunization was performed with an immune antigen dose of 100 μg / mouse / time, and the booster immunization was performed four times, with an immune antigen dose of 50 μg / mouse / time every 2-3 weeks. 0.5 mL of Freund's complete adjuvant was used for the first immunization, and 0.5 mL of Freund's incomplete adjuvant was used for the booster immunization. The immunogen and immune adjuvant were fully mixed and emulsified in a 1:1 ratio to prepare a stable "oil-in-water" liquid, which was injected subcutaneously at multiple points. After 5 immunizations, blood was collected from the retrobulbar region, and the serum titer was determined by serial dilution using an indirect ELISA method. The steps for indirect ELISA to detect the titer of antiserum are as follows: (1) Dilute the p16 protein antigen peptide to 1 μg / mL with coating buffer, coat with 100 μL / well, and incubate at 4°C overnight. (2) Wash the plate three times with PBST, add 200 μL of blocking solution to each well, incubate at 37°C for 1 hour, discard the blocking solution, and wash the plate once. (3) Dilute the antiserum 3-fold to 1:500, add 100 μL to each well, and incubate at 37°C for 1 hour. Discard the solution in the wells, wash the plate three times, add 100 μL of diluted enzyme-labeled secondary antibody (enzyme-labeled secondary antibody preparation: goat anti-mouse-HRP, 1:20,000) to each well, and incubate at 37°C for 1 hour. Discard the solution in the wells, wash the plate four times, add 100 μL of TMB colorimetric solution to each well, and incubate at 37°C for 15 minutes. Add 100 μL of 1 M HCL solution to each well to terminate the reaction. Immediately read the plate on a microplate reader at a wavelength of 450 nm, and the maximum dilution factor of the antiserum corresponding to the well with an OD value 2.1 times greater than the set negative control OD value was determined as the titer of the sample.

[0038] Through ELISA titer detection of mouse antiserum and p16 protein after the fifth immunization, the antiserum titer of 4# and 8# mice was about 40.5K, the antiserum titer of 3#, 5# and 6# mice was about 121.5K, and the antiserum titer of 1#, 2# and 7# mice was about 364.5K.

[0039] Example 3 Cell fusion was performed to obtain a specific fusion cell line targeting p16 protein.

[0040] (1) Electrofusion of myeloma cells and spleen cells 1) Myeloma cell preparation One week before fusion, SP2 / 0 myeloma cells (Zhongding Bio, batch number: 20180305) were expanded in DMEM medium containing 10% fetal bovine serum (FBS). On the day of fusion, SP2 / 0 cells were collected, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded before adding 20 mL of DMEM basal medium for counting.

[0041] The DMEM culture medium contains: 4.5 g / L glucose, 0.584 g / L L-glutamine, phenol red, sodium pyruvate, sodium bicarbonate, etc.

[0042] 2) Splenocyte Preparation On the day of fusion, mice were killed by cervical dislocation, and spleens were aseptically removed to prepare single-cell suspensions. Splenocytes were collected after centrifugation at 1500 rpm for 5 min and then added to cell culture medium for counting.

[0043] 3) Cell fusion Mouse spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 1:10, electroporated, and fused. The fused cells were plated into a 96-well plate at 100 μL per well. The cell culture plate was then placed in a CO2 incubator for incubation.

[0044] (2) Cell fusion screening 1) Screening of positive cells Observe cell growth on day 5 after fusion. On days 10-14, assay the titer of cell culture supernatants using an indirect ELISA assay. The method is as follows: Coat an ELISA plate with 1 μg / mL antigen in PBS overnight the day before testing. The next day, aspirate 100 μL of post-fusion cell supernatant per well for ELISA testing. Based on the ELISA results, identify positive wells (OD value of the sample well divided by the OD value of the negative well ≥ 2.1). Use a single-channel pipette to select positive wells from the entire plate for a second confirmation assay. After further confirmation, dilute and count the cells in the positive wells for subcloning.

[0045] 2) Cell subcloning to obtain antigen-specific monoclonal cell lines After confirming the positive cells using indirect ELISA, subclone the cells. Expand the culture of the hybridoma cells with the highest titer until the positive rate reaches 100%. The resulting hybridoma cell line, designated anti-p16 monoclonal antibody hybridoma cell line 3G3B5, was frozen in liquid nitrogen until further use.

[0046] At the same time, the screened hybridoma cell line (3G3B5) was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number: CGMCC No. 46037 and the deposit date of August 14, 2024.

[0047] Example 4 Expression and purification of p16 monoclonal antibody (clone 3G3B5) Anti-p16 monoclonal antibody monoclonal hybridoma cell line (clone number 3G3B5) was revived and expanded for antibody expression in serum-free cell culture. Antibody purification was performed using a Protein A / G agarose gel affinity purification column to obtain the desired purified antibody, which was then dialyzed against PBS at 4°C overnight. The protein concentration of the monoclonal antibody secreted by the hybridoma cell line was determined to be 1.15 mg / mL using the BCA assay. The purified antibody was then analyzed by SDS-PAGE electrophoresis ( Figure 3 ).

[0048] By the attached Figure 3The data show that bands of 55 KD and 25 KD, which are consistent with the expected molecular weight, appeared in lane 1, indicating that the anti-p16 monoclonal antibody monoclonal hybridoma cell line (clone number 3G3B5) can express the antibody.

[0049] The affinity of the mouse anti-p16 monoclonal antibody (clone 3G3B5) and a control monoclonal antibody for the p16 antigen was tested by indirect ELISA using the same method as above. The test results are shown in Table 1. The results showed that the prepared mouse anti-human p16 monoclonal antibody had strong binding ability to the p16 antigen, indicating that the monoclonal antibody can specifically recognize and bind to the p16 antigen, indicating that the antibody preparation process was relatively successful and that the antibody has good affinity and specificity. The titer for the p16 antigen reached 1:3280.5KD (OD value > 0.5) (Table 1), indicating that the antibody has high activity.

[0050] Table 1: ELISA detection of p16 monoclonal antibody (clone number 3G3B5) titer

[0051] Example 5 Gene sequencing of 3G3B5 monoclonal cell line (1) Hybridoma cell RNA extraction and reverse transcription Hybridoma cells were centrifuged at 1000 rpm for 5 minutes, and RNA was extracted by adding an adequate amount of TRIpure Reagent. RNA was precipitated with isopropanol, centrifuged, and then removed from impurities with 80% ethanol. The RNA precipitate was then dissolved in an appropriate amount of RNase-free ddH2O. The RNA concentration was determined to be 180.20 ng / μL. Resuspended RNA was then reverse transcribed using the following reaction conditions: 25°C for 5 minutes, 37°C for 45 minutes, and 85°C for 5 seconds. cDNA was obtained after cooling to 4°C and stored at -80°C.

[0052] (2) Gene amplification of the heavy and light chain variable regions of hybridoma cell antibodies PCR was performed using universal primers and 3G3B5 hybridoma cell cDNA as a template to amplify the heavy and light chain variable region genes of the antibody. A 25 μL system contained 0.5 μL of cDNA template, 12.5 μL of Green Taq Mix, 0.5 μL of VH-F mix (VL-F), and 0.5 μL of VH-R mix (VL-R). 11 μL of RNase-free ddH2O was added to bring the total reaction volume to 25 μL.

[0053] The PCR primer sequences are as follows: VH-F: 5'-CAGGTGCAGCTGGTGCAGTCTGG-3' (SEQ ID NO.10) VH-R: 5'-GGAAGTGGTGGTGGTGGTGGTGG-3' (SEQ ID NO.11) VL-F: 5'-GAGGTGCAGCTGGTGCAGTCTGG-3' (SEQ ID NO.12) VL-R: 5'-CAGGAGACAGAGTTCAGACAGG-3' (SEQ ID NO.13) PCR amplification was performed under the following conditions: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 40 s, 35 cycles; and extension at 72°C for 7 min. The obtained PCR products were identified by 1% agarose gel electrophoresis ( Figure 4 ), and recovered the target fragments and sent them for sequencing.

[0054] By the attached Figure 4 The data show that bands of 55KD and 25KD appeared in lane 1, which are consistent with the expected molecular weight, indicating that the anti-p16 monoclonal antibody monoclonal hybridoma cell line (clone number 3G3B5) can express the antibody.

[0055] (3) Cloning and sequencing of heavy and light chain variable region genes of hybridoma cell antibodies The recovered antibody fragments were subjected to TA cloning and sequencing. The PCR tube reaction system was: pMD19-T Vector 1 μL, V H / V L PCR fragments (0.1 pmol to 0.3 pmol) were added to a total volume of 5 µL with RNase-free ddH2O. 5 µL of Solution I was added and the reaction was allowed to proceed overnight at 16°C. The ligation product was added to 100 µL of DH5α competent cells and placed on ice for 30 minutes. The cells were heated at 42°C for 45 seconds and then placed on ice for 1 minute. 890 µL of SOC medium was added and the cells were revived and incubated at 37°C at 200 rpm for 60 minutes. The cells were plated onto LB-Amp medium and incubated overnight at 37°C. A single colony was picked and sequenced to confirm the amino acid sequence of the heavy chain variable region of the anti-p16 monoclonal antibody (clone 3G3B5) as shown in SEQ ID NO. 8, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO. 9.

[0056] SEQ ID NO.8 QVQLEESGPGILQPSQTLSLTCSFSGFSLSTSGVGVGWIRQPSGKGLEWLAHIWWDDDKHYNPALKSRLTISKDTSSNQVFLKIASVDTADTATYYCTRLGDYLFPYWGQGTLVTVSE SEQ ID NO.9 DIVMTQTPLSLPVSLGDPASISCRSSRNIVHNNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIK.

[0057] The SOC medium composition is as follows: 20 g / L tryptone, 5 g / L yeast extract, 0.5 g / L sodium chloride, 0.186 g / L potassium chloride, 10 mL / L magnesium chloride (1M solution), 10 mL / L magnesium sulfate (1M solution), and 20 mL / L glucose (20% solution).

[0058] LB-Amp medium is composed of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15-20 g / L agar, 100 μg / mL ampicillin, and the pH is adjusted to 7.0-7.2.

[0059] Example 6 Mouse anti-human p16 monoclonal antibody was used as the primary antibody in the immunohistochemical staining kit for immunohistochemical staining of human tissue samples to detect the expression of p16 protein in the above tissue samples. The specific steps are as follows: Human tissue wax blocks fixed with 10% formalin and embedded in paraffin were selected, and 4 μm paraffin sections were prepared using a paraffin slicer. After conventional dewaxing and hydration, they were placed in sodium citrate antigen retrieval solution for high-temperature antigen retrieval, and washed with PBS three times for 5 minutes each time. 100 μL of 3% H2O2 was added dropwise to remove endogenous peroxidase, and the cells were washed with PBS three times, 5 min each time; Add 100 μL of blocking solution containing goat serum, incubate at room temperature for 30 min, and wash three times with PBS, each time for 5 min; Add the mouse anti-human p16 monoclonal antibody dropwise, incubate at room temperature for 30 min (the amount used should be sufficient to submerge the sample area), and wash with PBS three times, 5 min each time; Add anti-horseradish peroxidase (HRP) labeled secondary antibody, incubate at room temperature for 30 min, and wash with PBS three times, 5 min each time; Add 100 μL of fresh 3,3'-diaminobenzidine (DAB) staining solution, incubate at room temperature, monitor the optimal color development time under a microscope, and terminate the color development by rinsing with deionized water. Counterstain with hematoxylin for about 3 minutes, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for 1 second, and rinse with tap water; Dehydrate with 80%, 95%, and 100% gradient alcohol, make the slides transparent, and seal with neutral gum. Observe the expression of p16 protein under a microscope, see Appendix Figure 5 As shown, the results showed that anti-p16 monoclonal antibody (clone number 3G3B5) can be used as a primary antibody for the detection of p16 protein in tissue samples ( Figure 5 ) and showed high specificity and sensitivity.

[0060] The above content is only for explaining the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A p16 antigen, characterized in that The amino acid sequence of the p16 antigen is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule is the gene encoding the p16 antigen according to claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid molecule according to claim 2; the empty vector of the recombinant expression vector is a plasmid vector.

4. A method for constructing the recombinant expression vector according to claim 3, characterized in that: The construction method comprises: The p16 antigen encoding gene according to claim 1 and / or the nucleic acid molecule according to claim 2 are inserted between the restriction enzyme cutting sites of the expression vector to obtain the recombinant expression vector.

5. A recombinant engineered bacterium for preparing the p16 antigen according to claim 1, characterized in that: The recombinant engineered bacteria comprises the recombinant expression vector according to claim 3, and / or the nucleic acid molecule according to claim 2 is integrated into the genome of the recombinant engineered bacteria.

6. A monoclonal antibody against p16 antigen, characterized in that: The monoclonal antibody is prepared using the p16 antigen according to claim 1 as an immunogen. The antigen-binding fragment of the monoclonal antibody specifically binds to the human p16 antigen. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises: The amino acid sequence of CDR1 is shown in SEQ ID NO. 2; The amino acid sequence of CDR2 is shown in SEQ ID NO.3; The amino acid sequence of CDR3 is shown in SEQ ID NO.4; The light chain variable region comprises: The amino acid sequence of CDR1 is shown in SEQ ID NO.5; The amino acid sequence of CDR2 is shown in SEQ ID NO.6; The amino acid sequence of CDR3 is shown in SEQ ID NO.

7.

7. The monoclonal antibody against p16 antigen according to claim 6, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

9.

8. The monoclonal antibody against p16 antigen according to claim 6, characterized in that: The monoclonal antibody further comprises a constant region; the constant region comprises a heavy chain constant region and a light chain constant region.

9. A hybridoma cell line, characterized in that The hybridoma cell line secretes the monoclonal antibody according to any one of claims 6 to 8, is named hybridoma cell line 3G3B5, and is deposited in the General Microbiology Center of the China Culture Collection Administration under the accession number CGMCC No. 46037, with a deposit date of August 14, 2024.

10. Use of the p16 antigen according to claim 1, the nucleic acid molecule according to claim 2, the recombinant expression vector according to claim 3, the recombinant engineered bacteria according to claim 5, the anti-p16 antigen monoclonal antibody according to any one of claims 6 to 8, or the hybridoma cell line according to claim 9 in the preparation of a detection reagent for detecting the expression of p16 protein in tumor and normal tissue cells, wherein the detection reagent is used in at least one detection method selected from immunohistochemistry, immunoblotting, and enzyme-linked absorbance assay.

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