CN116987203B

CN116987203B - P16 antigen, hybridoma cell strain, monoclonal antibody and application thereof

Info

Publication number: CN116987203B
Application number: CN202311273345.XA
Authority: CN
Inventors: 黄小军; 朱峻
Original assignee: Shenzhen Senying Bio Tech Co ltd
Current assignee: Shenzhen Senying Bio Tech Co ltd
Priority date: 2023-09-28
Filing date: 2023-09-28
Publication date: 2023-12-22
Anticipated expiration: 2043-09-28
Also published as: CN116987203A

Abstract

The invention provides a p16 antigen, a hybridoma cell strain, a monoclonal antibody and application thereof, wherein the p16 antigen comprises a first peptide segment, a second peptide segment, a third peptide segment, a first connecting peptide and a second connecting peptide; the first peptide fragment is a head peptide fragment of a human p16 antigen, the second peptide fragment is a middle peptide fragment of the human p16 antigen, and the third peptide fragment is a tail peptide fragment of the human p16 antigen; the first connecting peptide is respectively connected with the carboxyl end of the first peptide segment and the amino end of the second peptide segment; the second linking peptide is linked to the carboxy terminus of the second peptide fragment and the amino terminus of the third peptide fragment, respectively. The invention selects hypervariable region fragments of human p16 protein, forms fusion protein by connecting with connecting elements, is favorable for fully exposing p16 epitope, and prepares the p16 monoclonal antibody with good specificity and high sensitivity by constructing hybridoma cell strains.

Description

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

Technical Field

The invention belongs to the technical field of biomedicine, and relates to a p16 antigen, a hybridoma cell strain, a monoclonal antibody and application thereof.

Background

The tumor suppressor protein p16.sup.INK4a (p 16) localizes to the nucleus and cytoplasm and plays a key role in cell cycle regulation. The p16 protein specifically binds to CDK4 or the Cyclin-CDK4 complex, inhibiting CDK4 activity, thereby slowing the progression of the cell from G1 into S phase. When the p16 gene is deleted or mutated, the protein function is lost, CDK4 activity cannot be inhibited, and cells enter malignant proliferation to accelerate tumor generation.

Expression of the p16 protein in cells is generally regulated by two oncostatin proteins, p53 and retinoblastoma protein (RB), at low levels. In transformed cells of high-risk HPV (hrHPV), RB binds to HPV E7 and inactivates p16, and thus p16 overexpression can be used as an indicator of tumor transformation. The p16 protein is found to be abnormally expressed in various tumors, such as ovarian cancer, lung adenocarcinoma, rectal cancer, breast cancer, high-grade cervical intraepithelial tumors and the like.

The molecular weight of the p16 protein is about 16 kDa, and antibodies with different binding characteristics are prepared by selecting different antigens for immunization, so that the p16 cells can be accurately identified, and the method has an important role in tumor pathology research.

CN105001327a discloses an anti-p 16 monoclonal antibody, a preparation method and application thereof, the monoclonal antibody secreted by hybridoma (30859-S17) can identify recombinant protein p16 molecules and lymphocytes expressing p16, and can detect various tumor tissues such as skin squamous cell carcinoma, pancreatic cancer, melanoma, lymphoma, esophageal cancer, cervical cancer and the like which highly express p16 protein. However, the existing antigen preparation process often needs the steps of vector construction, transfection, expression, screening, purification and the like, the epitope is not easy to expose, and the obtained antibody has weak specific binding with the antigen.

Therefore, there is a need to optimize antigens to produce monoclonal antibodies against p16 antigens that are more specific and sensitive.

Disclosure of Invention

Aiming at the defects and actual demands of the prior art, the invention provides a p16 antigen, a hybridoma cell strain, a monoclonal antibody and application thereof, wherein the p16 antigen is a fusion protein formed by selecting a hypervariable region fragment of a human p16 protein and connecting by using a connecting element, which is favorable for fully exposing p16 antigenic determinants, and the monoclonal antibody with good specificity and high sensitivity is prepared by constructing the hybridoma cell strain.

To achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a p16 antigen, the p16 antigen comprising a first peptide fragment, a second peptide fragment, a third peptide fragment, a first linker peptide and a second linker peptide;

the first peptide fragment is a head peptide fragment of a humanized p16 antigen, and the amino acid sequence is shown as SEQ ID NO. 1;

the second peptide segment is a middle peptide segment of a humanized p16 antigen, and the amino acid sequence is shown as SEQ ID NO. 2;

the third peptide fragment is a tail peptide fragment of a human p16 antigen, and the amino acid sequence of the third peptide fragment is shown as SEQ ID NO. 3;

the first connecting peptide is respectively connected with the carboxyl end of the first peptide segment and the amino end of the second peptide segment;

the second connecting peptide is respectively connected with the carboxyl end of the second peptide segment and the amino end of the third peptide segment;

SEQ ID NO: 1：

MEPAAGSSMEPSADWLATAAARGR；

SEQ ID NO: 2：

PIQVMMMGSARVAELLLLHGAEPNCA；

SEQ ID NO: 3：

RPVHDAAREGFLDTLVVLHRAGARLDVRDA。

in the invention, a hypervariable region peptide segment of a human p16 protein is selected, a relatively conservative segment in the p16 protein is replaced by a connecting peptide, a p16 fusion protein with a structure of a first peptide segment-a first connecting peptide-a second peptide segment-a second connecting peptide-a third peptide segment is constructed, and the use of the connecting peptide is beneficial to fully exposing an epitope without being influenced by a biosynthesis process and improving the immunogenicity of an antigen, so that the anti-p 16 monoclonal antibody with strong specificity and sensitivity is prepared.

Preferably, the amino acid sequence of the first and second connecting peptides is (Gly Gly Gly Gly Ser) _n Or (Gly Gly Gly Gly Thr) _n Wherein n is an integer of 1 to 6.

Preferably, the amino acid sequence of the first connecting peptide is shown as SEQ ID NO. 4;

SEQ ID NO: 4：GGGGSGGGGSGGGGSGGGGS。

preferably, the amino acid sequence of the second connecting peptide is shown as SEQ ID NO. 5;

SEQ ID NO: 5：GGGGTGGGGTGGGGT。

in the invention, in order to ensure the conformation and the function of the p16 protein hypervariable region fragment and avoid mutual shielding caused by steric hindrance, the first peptide fragment and the second peptide fragment are connected by adopting the connecting peptide shown in SEQ ID NO. 4, and the second peptide fragment and the third peptide fragment are connected by adopting the connecting peptide shown in SEQ ID NO. 5, so that the p16 epitope is fully exposed.

Preferably, the amino acid sequence of the p16 antigen is shown as SEQ ID NO. 6;

SEQ ID NO: 6：

MEPAAGSSMEPSADWLATAAARGRGGGGSGGGGSGGGGSGGGGSPIQVMMMGSARVAELLLLHGAEPNCAGGGGTGGGGTGGGGTRPVHDAAREGFLDTLVVLHRAGARLDVRDA。

in a second aspect, the invention provides a nucleic acid molecule comprising a gene encoding the p16 antigen of the first aspect.

In a third aspect, the present invention provides a recombinant expression vector comprising a nucleic acid molecule according to the second aspect;

the empty carrier of the recombinant expression vector is a plasmid vector.

In the present invention, the empty vector of the recombinant expression vector is a vector for expressing a target gene commonly used in the art, for example, a pET plasmid vector, preferably pET-23a (+) or pET-22b (+).

In a fourth aspect, the present invention provides a method for constructing the recombinant expression vector of the third aspect, the method comprising:

inserting the coding gene of the p16 antigen of the first aspect and/or the nucleic acid molecule of the second aspect between restriction enzyme sites of an expression vector to obtain the recombinant expression vector.

In a fifth aspect, the present invention provides a recombinant engineering bacterium for preparing the p16 antigen according to the first aspect, the recombinant engineering bacterium comprising the recombinant expression vector according to the third aspect, and/or the recombinant engineering bacterium having the nucleic acid molecule according to the second aspect integrated in its genome.

In a sixth aspect, the present invention provides a method of preparing the p16 antigen of the first aspect, the method comprising:

culturing and inducing the recombinant engineering bacteria of the fifth aspect;

collecting bacterial liquid, centrifuging and re-suspending to obtain bacterial suspension;

and carrying out ultrasonic disruption on the bacterial suspension, and then carrying out protein purification to obtain the free p16 antigen.

In a seventh aspect, the present invention provides a monoclonal antibody against the p16 antigen, the monoclonal antibody being prepared from the p16 antigen of the first aspect as an immunogen, and the antigen binding fragment of the monoclonal antibody specifically binding to the human p16 antigen.

Preferably, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises a CDR1 shown in SEQ ID NO. 7, a CDR2 shown in SEQ ID NO. 8 and a CDR3 shown in SEQ ID NO. 9;

the light chain variable region comprises a CDR1 shown in SEQ ID NO. 10, a CDR2 with an amino acid sequence of KAS and a CDR3 shown in SEQ ID NO. 11;

SEQ ID NO: 7：GYTFTRYY；

SEQ ID NO: 8：IYNPDSYT；

SEQ ID NO: 9：TQYDY；

SEQ ID NO: 10：QHIVHNNGIIY；

SEQ ID NO: 11：FQGPHVPWT。

in the present invention, monoclonal antibodies against p16 antigen comprising heavy chain variable region CDRs and light chain variable region CDRs have specifically good p16 antigen binding activity.

Preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 12;

the light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 13;

SEQ ID NO: 12：

VQLLFSGAELVRPGASVKPSYKASGYTFTRYYIPWVKQRPGQGLKWNGNIYNPDSYTNYNQPFKDKVTLTVDKSSSTVYMQISSPTSEDSAVTYCTQYDYWGPGTTLTHSS；

SEQ ID NO: 13：

LMWQTPLSAPVSLGDQASISCRSSQHIVHNNGIIYLEWTLQKPGQSPKPLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDEGVYFCFQGPHVPWTFGAGTKLEIK。

preferably, the monoclonal antibody further comprises a constant region;

the constant regions include heavy chain constant regions and light chain constant regions.

Preferably, the heavy chain constant region subtype of the monoclonal antibody is IgG.

Preferably, the light chain constant region subtype of the monoclonal antibody is Kappa.

Preferably, the antigen binding fragment comprises any one or a combination of at least two of ScFv, (Fab') 2, fab, fv, scFv, diabodies, linear antibodies and multispecific antibodies.

In an eighth aspect, the present invention provides a hybridoma cell line, which secretes the monoclonal antibody of the seventh aspect, named 10F4, and is deposited with the China center for type culture collection, with a deposit number of cctccc NO: c202335, the preservation date is 2023, 3 and 8, and the preservation address is university of Wuhan in Wuhan, china (post code: 430072).

According to the invention, the p16 antigen is adopted to immunize a female BalB/c mouse with the age of 6-8 weeks, spleen cells of the mouse are fused with SP2/0 cells, monoclonal cells are obtained by a limiting dilution method, positive hybridoma cells are screened by an indirect ELISA method, and the positive hybridoma cells are used as hybridoma cell strains secreting monoclonal antibodies against the p16 antigen and used for biological preservation.

In a ninth aspect, the invention provides a kit for detecting p16 antigen, the kit comprising a monoclonal antibody according to the seventh aspect.

Preferably, the kit further comprises any one or a combination of at least two of a solid phase carrier, a coating buffer, a blocking solution, a sample processing solution or a color developing solution.

In a tenth aspect, the invention provides the use of the p16 antigen of the first aspect, the nucleic acid molecule of the second aspect, the recombinant expression vector of the third aspect, the recombinant engineering bacterium of the fifth aspect, the monoclonal antibody of the seventh aspect, the hybridoma cell line of the eighth aspect or the kit of the ninth aspect in the preparation of a disease diagnostic reagent.

Preferably, the disease comprises any one or a combination of at least two of cutaneous squamous cell carcinoma, pancreatic carcinoma, melanoma, lymphoma, breast carcinoma, esophageal carcinoma or cervical carcinoma.

Compared with the prior art, the invention has the following beneficial effects:

(1) The p16 antigen is a fusion protein obtained by adding the connecting peptide between hypervariable region fragments of the humanized p16 protein, so that the immunogenicity of the biosynthesis p16 antigen is improved, and the conservative fragments in the p16 antigen are replaced by the connecting peptide, thereby being beneficial to fully exposing p16 antigen epitopes;

(2) The hybridoma cell strain is prepared by immunizing a BalB/C mouse with p16 fusion protein as an antigen, and can secrete an anti-p 16 monoclonal antibody with strong specificity and high sensitivity;

(3) The anti-p 16 monoclonal antibody has the characteristics of strong specificity and high sensitivity in the in-vitro detection of the p16 protein expression condition of a biological sample.

Drawings

FIG. 1 is a schematic structural diagram of a p16 antigen fusion protein;

FIG. 2 shows the results of the potency detection of murine anti-human p16 monoclonal antibodies against p16 antigen;

FIG. 3 shows the results of specific detection of p16 antigen by murine anti-human p16 monoclonal antibody;

FIG. 4 shows the results of immunocytochemical staining of cervical cell samples using murine anti-human p16 monoclonal antibodies.

Detailed Description

The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

Example 1 according to the p16 sequence and structure published by Uniprot database, the head (SEQ ID NO: 1), middle (SEQ ID NO: 2) and tail (SEQ ID NO: 3) of p16 protein were selected as the first, second and third peptide fragments, respectively, and were linked using the first linker peptide (SEQ ID NO: 4) and the second linker peptide (SEQ ID NO: 5), respectively, to form a p16 fusion protein as shown in FIG. 1 (amino acid sequence shown in SEQ ID NO: 6).

The nucleic acid encoding the p16 fusion protein (SEQ ID NO: 6) was added at both ends with an EcoRI upstream and HindIII downstream cleavage site and a 6 Xhistidine tag at the tail. The coding sequence is subjected to double digestion by using restriction endonucleases EcoRI and HindIII, the digested product containing a sticky end is recovered by agarose gel electrophoresis, and the digested product is connected into a linearized pET-23a (+) expression vector which is also subjected to double digestion by EcoRI and HindIII, so that a recombinant plasmid vector containing a p16 antigen is obtained.

And (3) transforming BL21 (DE 3) escherichia coli competence with the recombinant plasmid vector to construct recombinant engineering bacteria for expressing the p16 antigen.

In addition, fragments of p16.sup.INK4 were selected and the following control groups were set:

control group (1): the amino acid sequence of the fusion protein is shown as SEQ ID NO. 14;

control group (2): the amino acid sequence of the fusion protein is shown as SEQ ID NO. 15;

control group (3): the amino acid sequence of the fusion protein is shown as SEQ ID NO. 16;

control group (4): the amino acid sequence of the fusion protein is shown as SEQ ID NO. 17;

control group (5): the amino acid sequence of the fusion protein is shown as SEQ ID NO. 18;

control group (6): conventional human p16 antigen;

SEQ ID NO: 14：

MEPAAGSSMEPSADWLATAAARGRVGGGGSGGGGSGGGGSGGGGSTAAARGRVEEVRALLEAGALPNAPNSYGGGGTGGGGTGGGGTLPNAPNSYGRRPIQVMMMGSARVAELL；

SEQ ID NO: 15：

MEPAAGSSMEPSADWLATAAARGRVGGGGSGGGGSGGGGSGGGGSLPNAPNSYGRRPIQVMMMGSARVAELLGGGGTGGGGTGGGGTVMMMGSARVAELLLLHGAEPNCADPATLTR；

SEQ ID NO: 16：

MEPAAGSSMEPSADWLATAAARGRVGGGGSGGGGSGGGGSGGGGSVMMMGSARVAELLLLHGAEPNCADPATLTRGGGGTGGGGTGGGGTADPATLTRPVHDAAREGFLDTLVVLHRAGARL；

SEQ ID NO: 17：

MEPAAGSSMEPSADWLATAAARGRVGGGGSGGGGSGGGGSGGGGSLPNAPNSYGRRPIQVMMMGSARVAELLGGGGTGGGGTGGGGTHRAGARLDVRDAWGRLPVDLAEELGHRDVAR；

SEQ ID NO: 18：

MEPAAGSSMEPSADWLATAAARGRVGGGGSGGGGSGGGGSGGGGSVMMMGSARVAELLLLHGAEPNCADPATLTRGGGGTGGGGTGGGGTHRAGARLDVRDAWGRLPVDLAEELGHRDVAR。

example 2 single colony is selected from recombinant engineering bacteria, inoculated in LB liquid medium containing 4 mL ampicillin (50 mug/mL) for culture, added with IPTG with final concentration of 0.5 mM, and strains expressing target proteins are screened;

the strain expressing the target protein is inoculated in LB liquid medium containing 350 mL ampicillin (50 mu g/mL) and cultured at 37 ℃ until OD ₆₀₀ 0.8, IPTG was added at a final concentration of 0.5. 0.5 mM and induced to express 4 h at 37 ℃;

the thalli are collected by centrifugation, resuspended in precooled 10 mM PBS, sonicated for 30 min (work 3s, rest 8 s) at 900W, centrifuged for 10 min at 12000g at 4 ℃, and the supernatant is collected;

filtering the supernatant with 0.45 μm filter, loading the filtrate to Ni-IDA column, and controlling flow rate to 0.5 mL/min; washing with washing solution (20 mmol/L Tris-HCl,1 mol/L NaCl, 8M urea) to remove unadsorbed protein, gradient eluting with washing solution (20 mmol/L Tris-HCl,1 mol/L NaCl, 8M urea) and PBS containing imidazole (0.05 mM, 0.1 mM and 0.25 mM) at different concentrations, collecting different elution peaks for SDS-PAGE to identify protein, adding renaturation buffer into sample with purity higher than 90% according to volume ratio of 1:3 for renaturation, and changing the solution to 10 mM PBS,PEG 20000 by using a dialysis bag for concentration to obtain p16 fusion protein antigen.

Example 3, 6-8 week old BalB/c mice with the appropriate weights were selected and kept in a standard animal house for 3 days, if no abnormal conditions were found, immunization was initiated:

adding 30 mu g p antigen into 0.5 mL autoclaved normal saline, fully and uniformly mixing the mixture by a micro vortex oscillator, adding 0.5 mL Freund's complete adjuvant, pushing and pulling the mixture by a syringe to fully mix and emulsify the mixture, and subcutaneously injecting the mixture to immunize 6-8 week old BalB/c mice, wherein the immunization dose is 100 mu g/mouse; performing secondary immunization at intervals of two weeks, emulsifying the antigen by adopting Freund's incomplete adjuvant, wherein the immunization dose is the same as that of the first immunization dose; taking tail blood after the second immunization, performing gradient dilution measurement on serum titer by using an indirect ELISA method, and selecting a mouse with the highest antibody titer for boosting;

preparing single cell suspension from spleen of mice after immunization by aseptic extraction, washing with cell culture solution for 1 time, grinding, sieving with stainless steel screen, centrifuging the obtained cells, and washing with cell culture solution for 2 times; mixing spleen cells of mice with BalB/c-derived SP2/0 myeloma cells in logarithmic phase at a ratio of 1:10, dropwise adding 1 mL 50% PEG-1500 (pH=8), incubating at room temperature for 90 seconds, adding incomplete culture medium to terminate reaction, centrifuging, removing supernatant, adding HAT culture medium, suspending, mixing uniformly, fixing volume to 150 mL, dropwise adding into 96-well cell culture plate, standing at 37deg.C and 5% CO ₂ Culturing in a constant temperature incubator for 7 days;

and screening the obtained hybridoma cells within 7-10 days after fusion by adopting an ELISA method, observing the growth condition of the cells on the 5 th day after fusion, detecting the titer of a cell culture supernatant by adopting an indirect ELISA method on the 10 th-14 th day, performing expanded culture on positive hybridoma cells with the strongest titer until the cell positive rate reaches 100%, and obtaining a hybridoma cell strain 10F4 by fixed strain, and freezing in liquid nitrogen for later use.

The screened 10F4 hybridoma cell strain is preserved in China Center for Type Culture Collection (CCTCC) NO: c202335, the preservation date is 2023, 3 and 8, and the preservation address is university of Wuhan in Wuhan, china (post code: 430072).

The protein concentration of the monoclonal antibody secreted by the hybridoma cell line was measured by the BCA method and was 2.839 mg/mL.

Example 4, after centrifugation of hybridoma cells at 1000 rpm for 5 min, adding cell lysate to extract RNA, precipitating RNA from aqueous phase with isopropanol, washing the precipitated RNA after centrifugation, removing impurities, and carrying out reverse transcription after resuspension to obtain cDNA;

PCR was performed using universal primers and using hybridoma cell cDNA as a template to amplify the heavy and light chain variable region genes of the antibody, and a 50. Mu.L system containing 2. Mu.L cDNA, 25. Mu.L Taq enzyme, 2. Mu.L upstream primer, 2. Mu.L downstream primer, 16. Mu.L DEPC water was subjected to PCR amplification under the following conditions: pre-denaturation at 95℃for 5 min;95 ℃ for 15 s,60 ℃ for 15 s,72 ℃ for 5 s,35 cycles; 72 ℃ for 5 min; and (3) identifying the obtained PCR product by using 1% agarose gel electrophoresis, recovering the target fragment, and carrying out sample feeding and sequencing to obtain the monoclonal antibody variable region gene fragment.

Homologous recombination primers are used for respectively adding homologous recombination arms at two ends of an antibody heavy chain variable region gene and two ends of a light variable region gene, and expression plasmids containing a mouse antibody heavy chain IgG1 constant region and a light chain Kappa constant region are linearized by using double enzymes to generate homologous recombination arms; the variable region gene fragment added with the homologous recombination arm and the linearized plasmid are connected in a homologous recombination mode to form a complete expression vector, the recombination product is transformed into escherichia coli competence, and the plasmid is amplified.

Collecting bacterial liquid, purifying the expressed antibody supernatant by adopting an affinity purification (Protein A) method to obtain the mouse anti-human p16 monoclonal antibody, measuring the purified monoclonal antibody by an ultraviolet spectrophotometer, adjusting the concentration to 1.0 mg/mL, sub-packaging and freezing at-20 ℃.

The heavy chain variable region of the mouse anti-human p16 monoclonal antibody comprises CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8 and CDR3 shown in SEQ ID NO. 9 through sequencing, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 12; the light chain variable region comprises CDR1 shown in SEQ ID NO. 10, CDR2 with the amino acid sequence KAS and CDR3 shown in SEQ ID NO. 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 13.

Based on the same experimental operation, the monoclonal antibody is obtained by immunizing a mouse with the control group protein, expressed and purified.

Example 5 the affinity activity (potency) of murine anti-human p16 monoclonal antibodies and control monoclonal antibodies to p16 antigen was measured by ELISA as follows:

the p16 antigen was diluted to 1 ng/. Mu.L with PBS and added to a 96-well ELISA plate at 100. Mu.L per well, coated at 37℃for 2h; the supernatant was discarded, the plate was washed 3 times with 0.01. 0.01M PBST to prepare a blocking solution containing 1% BSA, 100. Mu.L of the blocking solution was added to each well, and the wells were blocked at 37℃for 2 hours;

the supernatant was discarded, washed 5 times with PBST, the purified and concentrated antibody was subjected to gradient dilution at a concentration of 1:2560000 from 1:1000-fold dilution, wells were added with 100. Mu.L each, and incubated at 37℃for 1 h;

discarding the antibody diluent, washing with PBST for 6 times, diluting goat anti-mouse IgG-HRP with 1:5000 blocking solution, adding 100 μl of the mixture into each well, and incubating at 37deg.C for 1 h; discarding secondary anti-dilution, cleaning with PBST for 6 times, adding TMB,100 μl/hole, and standing at 37deg.C for 15 min in dark; the reaction was stopped by adding 50. Mu.L of 1M dilute sulfuric acid to each well, and the absorbance was measured at 450 nm.

As shown in FIG. 2 and Table 1, the prepared murine anti-human p16 monoclonal antibody has stronger binding capacity to p16 antigen, and the potency of the prepared murine anti-human p16 monoclonal antibody to p16 antigen reaches 1:2560000 (OD value is more than 0.5), which is obviously superior to that of the anti-p 16 monoclonal antibody of a control group.

TABLE 1

Dilution factor	10000	20000	40000	80000	160000	320000	640000	1280000	2560000
										OD450	4.687	4.392	4.198	3.930	3.329	2.839	1.592	0.921	0.638

Example 6, ELISA plates were coated with p16 protein and CK7 protein, respectively, at a coating per well of 50 ng; the mouse anti-human p16 monoclonal antibody is diluted to 10 ng/mL and added to each ELISA plate, 100 mu L is added to each hole, and the mixture is incubated at 37 ℃ for 1 h; after washing, adding HRP-labeled goat anti-mouse secondary antibody, adding 100 mu L of the secondary antibody into each hole, and incubating at 37 ℃ for 0.5 h; after washing TMB was added and incubated at 37℃for 15 min, the reading was terminated. The results are shown in FIG. 3, which shows that the mouse anti-human p16 monoclonal antibody does not cross react with other proteins, and has strong specificity.

Example 7, immunocytochemical staining of cervical cell samples was performed using a murine anti-human p16 monoclonal antibody coated slide, as follows:

(1) Placing a cervical cell sample taken from a donor in a cell preservation solution for preservation 1 h;

(2) Placing the cell sample preserved by the cell preservation solution on a mouse anti-human p16 monoclonal antibody coated glass slide, incubating for 20 min at 37 ℃, preparing a sheet from the cell sample by using a cell adhesive, and cleaning by using a cleaning solution;

(3) Dripping the sealing liquid, incubating for 20 min at room temperature, and adding the washing liquid for washing;

(4) Dripping DAB staining solution, incubating for 5 min at room temperature, and adding washing solution for cleaning;

(4) Dropwise adding hematoxylin color development liquid, and incubating for 1-3 min at room temperature;

(5) Staining results of the cell samples were observed under a microscope.

Wherein, the final concentration of each component of the cell preservation solution is as follows: nitrilotriacetic acid 3.5 mM, gluconic acid 3.6 mM, sorbitol 22 mM, aminobutanol 3 mM, paraformaldehyde 0.18 mM, PEG 400.8 mM, naCl 75 mM, KCl 12 mM and PBS buffer, the pH of the cell preservation solution being 7.2;

the final concentrations of the components of the blocking solution were as follows: skim milk powder 95 g/L, tritonX-100.8 g/L, proclin300 265 g/L and PBS buffer, wherein the pH of the blocking solution is 7.2;

the washing liquid is PBS buffer solution, and the pH value of the washing liquid is 7.2;

the staining solution comprises DAB staining solution, which consists of DAB substrate, DAB substrate buffer solution and DAB color developing agent, wherein the DAB substrate comprises peroxidase glucan polymer, tris/HCl, sodium chloride, bovine serum albumin, 5-bromo-5-nitro-1, 3-dioxane, 4-aminoantipyrine, polyethylene glycol (PEG 3000), casein and tween-20, and the DAB substrate has a pH value of 7.6; the DAB substrate buffer solution comprises imidazole, N' -ethyl bis (2- [ 2-hydroxyphenyl ] glycine) (EDHPA), ethylphenyl polyethylene glycol (Nonidet P-40), hydrogen peroxide and benzodimethammonium chloride, and has a pH of 7.5; the DAB color developing agent is prepared by dissolving 3, 3-diaminobenzidine carbon tetrachloride in 80% propylene glycol;

the hematoxylin color developing solution is prepared by the following steps:

dissolving 1 part of hematoxylin in 100 parts of absolute ethyl alcohol, and heating to obtain a solution A;

dissolving 60 g alum in 100 parts of distilled water, adding 100 parts of glycerin, and heating to obtain a solution B;

mixing the solution A and the solution B, and adding 10 parts of glacial acetic acid to obtain a solution C;

and exposing the solution C to air and sunlight for 3-4 weeks to obtain the hematoxylin color development liquid.

The staining results are shown in fig. 4, the staining effect of the cervical cell sample is good, the cervical cell is complete in shape and clear in staining, the cell nucleus is not enlarged, the shape is regular, and the nucleolus and the nucleus are not divided.

The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims

1. A p16 antigen, wherein the p16 antigen consists of a first peptide fragment, a second peptide fragment, a third peptide fragment, a first connecting peptide and a second connecting peptide;

the amino acid sequence of the first connecting peptide is shown as SEQ ID NO. 4;

the amino acid sequence of the second connecting peptide is shown as SEQ ID NO. 5.

2. The p16 antigen according to claim 1, wherein the amino acid sequence of the p16 antigen is shown in SEQ ID No. 6.

3. A nucleic acid molecule, wherein said nucleic acid molecule is a gene encoding the p16 antigen of claim 1 or 2.

4. A recombinant expression vector comprising the nucleic acid molecule of claim 3;

the empty carrier of the recombinant expression vector is a plasmid vector.

5. A method of constructing a recombinant expression vector according to claim 4, comprising:

inserting the coding gene of the p16 antigen according to claim 1 or 2 and/or the nucleic acid molecule according to claim 3 between restriction sites of an expression vector to obtain the recombinant expression vector.

6. A recombinant engineering bacterium for preparing the p16 antigen according to claim 1 or 2, wherein the recombinant engineering bacterium comprises the recombinant expression vector according to claim 4 and/or the nucleic acid molecule according to claim 3 is integrated into the genome of the recombinant engineering bacterium.

7. A method of preparing the p16 antigen of claim 1 or 2, comprising:

culturing and inducing the recombinant engineering bacteria of claim 6;

8. A monoclonal antibody against the p16 antigen, wherein the monoclonal antibody is prepared from the p16 antigen of claim 1 or 2 as an immunogen, and an antigen binding fragment of the monoclonal antibody specifically binds to a human p16 antigen;

the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

the light chain variable region comprises a CDR1 shown in SEQ ID NO. 10, a CDR2 with an amino acid sequence of KAS and a CDR3 shown in SEQ ID NO. 11.

9. The monoclonal antibody of claim 8, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID No. 12;

the light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 13.

10. The monoclonal antibody of claim 8, further comprising a constant region;

11. The monoclonal antibody of claim 10, wherein the heavy chain constant region subtype of the monoclonal antibody is IgG; the light chain constant region subtype of the monoclonal antibody is Kappa.

12. A hybridoma cell line, wherein the hybridoma cell line secretes the monoclonal antibody of any one of claims 8-11, designated as hybridoma cell line 10F4, and deposited with the chinese collection center under the accession number cctccc NO: c202335, with a date of storage of 2023, 3 and 15.

13. A kit for detecting p16 antigen, comprising the monoclonal antibody of any one of claims 8-11.

14. The kit of claim 13, further comprising any one or a combination of at least two of a solid support, a coating buffer, a blocking solution, a sample processing solution, or a color developing solution.

15. Use of the p16 antigen of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant expression vector of claim 4, the recombinant engineering bacterium of claim 6, the monoclonal antibody of any one of claims 8-11, the hybridoma cell line of claim 12, or the kit of claim 13 or 14 in the preparation of a disease diagnostic reagent;

the disease is cervical cancer.