Hpv56 subtype l1 protein, epitope peptide, monoclonal antibody, hybridoma cell and application thereof

Abstract

The application relates to an HPV56 subtype L1 protein, an epitope peptide, a monoclonal antibody, a hybridoma cell and application thereof, and aims to solve the technical problem that there is no commercial vaccine for the HPV56 subtype on the market at present. The prokaryotic expression vector of the HPV56 L1 is constructed, the HPV56 subtype L1 protein is induced and expressed, the recombinant HPV56 subtype L1 protein is used as an immunogen to immunize mice, the hybridoma cell strain is screened through cell fusion technology and ELISA identification, and the monoclonal antibody produced by the cell strain can specifically recognize and combine with the HPV56 subtype L1 protein 2 ATWRPSENKVYL 13 region. The monoclonal antibody, the antigen epitope peptide or the hybridoma cell provided by the application lay a foundation for the development of a rapid detection technology for the HPV56 and the L1 protein thereof, have wide research application value and commercial use value in the immune detection related to the HPV56 subtype L1 protein, and have important guiding significance for the development of a vaccine based on the HPV56 L1 protein.

Description

Technical Field

[0001] This invention application relates to the field of bioimmunotechnology, specifically to HPV56 subtype L1 protein, its epitope peptide, monoclonal antibody, hybridoma cells and their applications. Background Technology

[0002] Human papillomavirus (HPV) is a double-stranded, non-enveloped, highly conserved circular small DNA tumor virus belonging to the genus Papillomavirus in the family Papovaviridae. The HPV genome is approximately 8000 bp in length, with a molecular weight of approximately 5000 kDa and a diameter of approximately 55 nm. The HPV genome is divided into three regions, containing eight open reading frames (ORFs). The early expression region (E), late expression region (L), and long control region (LCR) together constitute the HPV genome. Currently, more than 200 HPV subtypes have been isolated and identified. Through gene sequencing and sequence alignment, the L1 gene sequence differences between different HPV subtypes exceed 10%, while the L1 sequence differences between HPV subtypes are less than 2%. Based on differences in carcinogenicity, HPV can be divided into three categories: low-risk human papillomavirus (LR-HPV), high-risk human papillomavirus (HR-HPV), and suspected high-risk types. HPV primarily infects humans through sexual transmission, damaging mucous membranes and epithelial tissues. It is the cause of most cervical cancers. Persistent infection with high-risk HPV can lead to cervical cancer, a highly prevalent malignant tumor among women worldwide, with high morbidity and mortality rates.

[0003] Currently, there are no commercially available vaccines targeting the HPV56 subtype. Therefore, developing a protein antigenic epitope and antibody against the HPV56 virus is of particular importance.

[0004] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] In view of at least one of the above technical problems, this disclosure provides an HPV56 subtype L1 protein, its epitope peptide, a monoclonal antibody, hybridoma cells and their applications, aiming to solve the technical problem of the lack of commercial vaccines targeting HPV56 subtypes on the market.

[0006] The first aspect disclosed in this application provides a monoclonal antibody against HPV56 subtype L1 protein, wherein the heavy chain variable region includes CDR H1, CDR H2, and CDR H3, the amino acid sequences of which are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively, and the light chain variable region includes CDR L1, CDR L2, and CDR L3, the amino acid sequences of which are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively.

[0007] The second aspect disclosed in this application is the application of the monoclonal antibody in the preparation of reagents for detecting HPV56 subtype virus.

[0008] A third aspect disclosed in this application involves using the monoclonal antibody in the preparation of reagents for immunological detection.

[0009] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages:

[0010] 1. This application describes the construction of a prokaryotic expression vector pE-SUMO-HPV 56-L1 for HPV56 L1, inducing the expression of HPV56 subtype L1 protein. Mice were immunized with the recombinant HPV56 subtype L1 protein as an immunogen. Hybridoma cell lines were obtained through cell fusion technology and ELISA identification. The monoclonal antibodies produced by these cell lines specifically recognize and bind to the L1 protein. 2 ATWRPSENKVYL 13 area.

[0011] 2. The hybridoma cells screened in this application have stable antibody secretion capabilities. The monoclonal antibodies secreted by these cells can rapidly and specifically recognize HPV56 and its L1 protein, laying the foundation for the development of rapid detection technology for HPV56 and its L1 protein. This technology has broad research and commercial application value in the immunoassay related to HPV56 subtype L1 protein and provides important guidance for the development of vaccines based on HPV56 L1 protein.

[0012] 3. Based on the heavy chain variable region sequence and light chain variable region sequence of the monoclonal antibody disclosed in this application, one or more amino acids can be added, deleted, or replaced through conventional genetic engineering and protein engineering to obtain its active fragment or conserved variant. However, it can still specifically bind to HPV56 subtype L1 protein, laying the foundation for further modifying its antibody variable region sequence to prepare genetically engineered antibodies in different combinations, so as to further improve the specificity of the antibody. Attached Figure Description

[0013] Figure 1 This is a prediction of the transmembrane region of the L1 protein in one embodiment of this application.

[0014] Figure 2 This is a PCR of the L1 target gene in one embodiment of this application.

[0015] Figure 3 Identification of the recombinant vector pE-SUMO-HPV 56-L1 in one embodiment of this application.

[0016] Figure 4 The purification and identification of L1 protein in one embodiment of this application.

[0017] Figure 5 The results of mouse polyclonal antibody serum titers are shown in one embodiment of this application.

[0018] Figure 6 This is the result of ascites purification of monoclonal antibody in one embodiment of this application.

[0019] Figure 7 The results of titer determination of different monoclonal antibodies after purification are shown in one embodiment of this application.

[0020] Figure 8 This is the result of IPMA identification of monoclonal antibodies in one embodiment of this application.

[0021] Figure 9 In one embodiment of this application, indirect ELISA and Dot-ELISA are used for preliminary screening of L1 B cell epitopes.

[0022] Figure 10 This is the ELISA detection result of the reactivity of the conjugated polypeptide and L1 monoclonal antibody in one embodiment of this application.

[0023] Figure 11 This is the ELISA detection result of the coupled polypeptide and positive serum reactivity in one embodiment of this application.

[0024] Figure 12 This is the spatial structure of the HPV56 subtype L1 protein antigenic epitope peptide in one embodiment of this application. Detailed Implementation

[0025] The following examples illustrate specific implementations of the present invention. However, these examples are merely for illustrative purposes and do not limit the scope of the invention in any way.

[0026] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and materials involved are all commercially available conventional products; unless otherwise specified, the test and detection methods involved are all conventional methods.

[0027] Example 1: Amino acid sequence analysis, optimization, and gene synthesis of HPV56 subtype L1 protein

[0028] Based on the HPV56 L1 gene sequence information (GeneBank accession number: KU298919.1), bioinformatics analysis was performed on the gene sequence encoding the HPV56 subtype L1 protein. Using TMHMM Server v.2.0, the transmembrane region of the L1 protein was analyzed based on the amino acid sequence (SEQ ID NO.1), such as... Figure 1 As shown, all 1 to 499 aa of the L1 protein are located outside the cell membrane, therefore, the full-length L1 protein sequence needs to be expressed. The L1 protein gene sequence and the coding region corresponding to the 6×His tag were optimized and synthesized according to codon preference. L1 gene primers were designed using SnapGene software, and BamH Ⅰ and Xho I were selected as restriction enzyme sites to synthesize the HPV56 L1 gene (SEQ ID NO.2).

[0029] After the L1 gene amplification was completed, the products were identified by electrophoresis using a 1% nucleic acid gel, and the results were as follows: Figure 2 As shown, the PCR products with bands close to the target band were finally purified and recovered using a DNA purification and recovery kit. The concentration of the recovered DNA solution was determined using a NanoDrop 2000c and stored at -20℃ for later use.

[0030] Example 2: Construction of the pE-SUMO-HPV 56-L1 protein recombinant vector

[0031] The L1 gene and pE-SUMO vector were double-digested with enzymes. The digestion products were recovered using a gel extraction kit. 2 μL of the recovered DNA was measured in a centrifuge tube and ligated overnight at 16°C. 5 μL of the ligation product was added to 100 μL of DH5α competent cells, followed by 900 μL of antibiotic-free LB liquid medium. The cells were incubated at 37°C with shaking at 220 rpm for 50 min. After incubation, the solution was centrifuged at 4000 rpm at room temperature for 4 min, the supernatant was discarded, and the cell pellet was resuspended in 100 μL of liquid LB medium. The resuspended pellet was evenly spread on LB solid medium containing ampicillin, incubated horizontally at room temperature for 20 min, and then inverted in a 37°C incubator overnight. Pick a single colony and inoculate it into LB liquid medium containing ampicillin resistance. Then, incubate at 37°C and 220 rpm for 6 h. Centrifuge the bacterial suspension at 12000 rpm at room temperature for 2 min, discard the supernatant, and resuspend the precipitate in an appropriate amount of 1×PBS. Centrifuge the resuspended suspension at the maximum speed (about 12000 rpm) for 2 min, and use the supernatant as a template for bacterial PCR.

[0032] After bacterial PCR, the products were identified using a 1% nucleic acid gel, and the results were as follows. Figure 3 As shown, monoclonal strains with band sizes matching the target gene of HPV56 L1 were selected and sent to General Biotech for sequencing. Successfully sequenced positive strains were cultured overnight at 37°C and 220 rpm in ampicillin-containing liquid LB medium. Recombinant plasmids were extracted using the Plasmid Miniprep Plus Purification Kit according to the kit's instructions. 1 μL of the recombinant plasmid was added to 100 μL of BL21 competent cells, followed by 900 μL of ampicillin-free LB liquid medium. The cells were incubated at 37°C with shaking for 50 min. The bacterial pellet was resuspended in 100 μL of liquid LB medium. The resuspended pellet was then evenly spread onto ampicillin-containing LB solid medium and incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing ampicillin resistance. The cultures were then incubated at 37°C and 220 rpm for 6 h. The culture was centrifuged at 12000 rpm for 2 min at room temperature, the supernatant was discarded, and the precipitate was resuspended in 1×PBS. The resuspended culture was centrifuged at maximum speed (approximately 12000 rpm) for 2 min, and the supernatant was used for colony PCR. The positive strain was named pE-SUMO-HPV 56-L1-BL21 and stored at -20°C.

[0033] Example 3: Expression, purification and identification of HPV56 subtype L1 protein

[0034] The recombinant expression strain pE-SUMO-HPV 56-L1-BL21, frozen in Example 2, was inoculated into LB liquid medium (ampicillin-resistant) at a ratio of 1:1000 and cultured overnight at 37°C with shaking for 14 h. 2 mL of the bacterial culture was inoculated into 200 mL of LB medium (ampicillin-resistant) at a ratio of 1:100 and cultured at 37°C with shaking for 2.5 h. A small amount of the bacterial culture was taken to measure the OD value; when it reached approximately 0.6, IPTG was added to achieve a final concentration of 0.2 mmol / L. The culture was then incubated overnight at 16°C with shaking for 14 h, and a sample was collected before induction. The bacterial culture was aliquoted into centrifuge tubes, centrifuged at 8000 rpm for 5 min, and the supernatant was discarded to obtain the bacterial cells. An appropriate amount of 1×PBS solution (w / v ratio of 1:20) was added to the bacterial cells, and the cells were resuspended and thoroughly mixed. 100 μL of the bacterial culture was collected as a sample. The mixed bacterial culture was placed in an ice-water bath and then subjected to ultrasonic disruption. Ultrasonication program: Total time 30 min, 2 s working time, 4 s interval. After completion, the bacterial suspension changed from turbid to clear. The centrifuge was pre-cooled to 4℃. The sample was centrifuged at 12000 rpm for 15 min. The supernatant and precipitate were collected. The ultrasonic precipitate was resuspended in an equal volume of 1×PBS solution. The ultrasonic supernatant and precipitate were collected again. Finally, SDS-PAGE and Western Blot identification techniques were used to identify HPV56 subtype L1 protein. The results of SDS-PAGE and Western Blot identification are as follows: Figure 4 As shown.

[0035] SDS-PAGE identification process:

[0036] (1) Sample preparation: Take 100 μL of each of the above-mentioned samples, including empty vector expression bacteria, add 25 μL of 5×SDS loading buffer, boil in water for 10 min, centrifuge for 1 min after water bath, and store at 4℃ for later use.

[0037] (2) Electrophoresis: First, add protein markers to the wells, then add samples in sequence, 20 µL of sample to each well, and start the instrument to begin electrophoresis. Electrophoresis conditions: 160 V, 1 h.

[0038] (3) Staining and decolorization: The protein gel was stained with Coomassie brilliant blue staining solution and then decolorized.

[0039] (4) Photographing: Scan the protein gel with a gel imaging device, take a picture and save it.

[0040] Western Blot identification process:

[0041] (1) Preparation: Perform SDS-PAGE on the sample. After the process, remove the lower layer of the protein gel and cut the PVDF membrane to the size of the protein gel. Activate the membrane in methanol. Then, place the PVDF membrane, the lower layer of the protein gel, and the upper and lower filter membranes into the transfer buffer for 5 min.

[0042] (2) Transfer: Turn on the transfer apparatus, wipe it clean with an alcohol swab and let it dry. Stack the membranes in the following order: lower filter, PVDF membrane, lower protein gel, and upper filter. Press and squeeze out air bubbles after each placement. Connect the power supply and perform the transfer at 15 V for 55 min.

[0043] (3) Sealing: After the transfer is completed, the PVDF membrane is sealed with 5% skim milk at 37°C for 2 hours.

[0044] (4) Primary antibody: Discard the skim milk, wash three times with PBST solution, add anti-His monoclonal antibody (1:4000), incubate at 37°C for 1 h.

[0045] (5) Secondary antibody: The primary antibody was recovered and placed at -20℃. The membrane was washed three times with PBST solution and then placed in Goat Anti-Mouse IgG / HRP (1:4000) and incubated at 37℃ for 1 h.

[0046] (6) Color development: After incubation, wash the membrane with PBST solution several times, use AEC kit for color development, and record the data after the reaction is terminated after about 10 minutes.

[0047] Example 4: Preparation of Monoclonal Antibodies

[0048] 1. Animal immunization process

[0049] (1) HPV56 subtype L1 protein was injected into two BALB / c mice as an immunogen. The HPV56 L1 recombinant protein was mixed with Freund's adjuvant, emulsified, and then used to immunize the mice. The mice were immunized once every 14 days for a total of three immunizations.

[0050] (2) One week after the third immunization, blood was collected from the veins of mice. 10 μL of tail blood was mixed with 990 μL of PBS solution, centrifuged, and the supernatant was collected. The serum titer was determined. Serum from unimmunized mice was used as a negative control.

[0051] (3) Three days before cell fusion, select mice with the best immune response and inject them with 200 μL of protein into their peritoneum to enhance immunity.

[0052] Immunization procedure steps:

[0053] (1) Addition of antigen protein: The concentration of purified antigen protein is 0.25 mg / mL. Take 100 μL of antigen protein and mix it with an equal volume of Freund's complete adjuvant, and emulsify it using an emulsifier.

[0054] (2) Emulsification: Before using the emulsifier, clean the instrument step by step with 75% alcohol, ultrapure water and PBS solution. Emulsify the antigen protein for about half an hour. When oil droplets are formed and do not immediately dissipate in water, it indicates that emulsification is complete.

[0055] (3) Immunize mice: Use a 1 mL sterile syringe to draw up the emulsion, making sure to remove air, and then inject it into the back of the mouse, injecting about 50 μL at each location.

[0056] The results of polyclonal antibody serum titers in mice after immunization are as follows: Figure 5 As shown.

[0057] 2. Cell fusion

[0058] (1) Mix the counted myeloma cells and mouse spleen cells at a ratio of 1:8, balance the centrifuge tubes, and centrifuge at 1100 rpm for 10 minutes.

[0059] (2) After centrifugation, discard the supernatant, place the centrifuge tube in a 37°C water bath, use a pipette to draw 1 mL of PEG1500 (preheated to 37°C in advance), and slowly and evenly add it to the centrifuge tube within 90 seconds. After the centrifugation is complete, continue the water bath for 90 seconds.

[0060] (3) Take 15 mL of GNK buffer, add 1 mL evenly over 30 seconds, then add 3 mL over 30 seconds, and finally add 11 mL over 30 seconds. After the addition is complete, add 25 mL of GNK buffer, continue to bathe in a 37°C water bath for 5 minutes, and then centrifuge at 1100 r / min for 10 minutes;

[0061] (4) After centrifugation, discard the supernatant, gently disperse the cells, and resuspend the cells in HAT medium. Add 100 μL / well to the culture plate and incubate at 37°C.

[0062] (5) Starting from day 5, observe the number of cell clusters in each well and add fresh culture medium in a timely manner.

[0063] 3. Screening of hybridoma cells

[0064] Hybridoma cell lines with positive results were screened by indirect ELISA using HPV56 L1 recombinant protein and pE-SUMO empty vector protein (negative control) as coating agents and cell supernatant as primary antibody. Hybridoma cell lines with high OD values ​​and good specificity were selected. After a second round of retesting, positive hybridoma cells were screened for subcloning and expanded culture.

[0065] Subcloning steps:

[0066] (1) In a clean bench, replace the culture medium in the wells of hybridoma cells that require subcloning, and resuspend and disperse the cells. Then, take a small amount of cell sap, dilute and stain with trypan blue, and count the cells under a microscope.

[0067] (2) After counting, dilute the original cell solution with HT medium at a ratio of 1-2 cells per well. Add 100 μL to each well of a new 96-well plate and incubate at 37°C for one week.

[0068] (3) One week later, observe and screen the number of cell clusters in each well.

[0069] (4) Screen out the wells containing single-cell clusters for indirect ELISA identification, using the same method as above.

[0070] (5) Continue screening until a single cell cluster is found that can specifically react with HPV56 subtype L1 protein. Finally, freeze the selected cell line in liquid nitrogen.

[0071] 4. Large-scale preparation of monoclonal antibodies

[0072] The preparation steps for monoclonal antibodies are as follows:

[0073] (1) Use a syringe to draw 500 μL of sterile liquid paraffin and inject it into the abdominal cavity of multiparous female mice.

[0074] (2) Remove the hybridoma cell line in advance and culture and passage it. One week after injection of paraffin, blow away the cells with serum-free 1640 medium or PBS solution, and collect the cells by centrifugation. Gently flick the cells by hand, add medium to dilute the cells, stain the cells with trypan blue and count them, and inject 1×106 cells into each mouse intraperitoneally.

[0075] (3) After cell injection, closely monitor the health status and abdominal changes of the mice. Collect ascites fluid when the mouse's abdomen becomes distended and it becomes difficult to move. Centrifuge at 6000 rpm for 20 min, and gently aspirate the intermediate supernatant from the centrifuge tube using a pipette. Then aliquot and store in a low-temperature freezer for purification.

[0076] The following steps were taken to purify mouse ascites using a Protein A affinity chromatography column:

[0077] (1) Balancing: Balancing Protein A columns with a binding buffer of 10 times the column volume.

[0078] (2) Sample loading: Dilute the ascites fluid 4 times with Binding buffer, mix and add to the chromatography column, load the column at a flow rate of 1 mL / min, and repeat the sample loading three times.

[0079] (3) Washing: Use binding buffer to wash away proteins that are not bound to the protein indicator until the protein indicator no longer turns blue.

[0080] (4) Elution: Add an appropriate amount of Neutralization buffer (to adjust pH) to the Ep tube for collecting monoclonal antibodies, and then use Elution buffer to elute the target protein.

[0081] (5) Dialysis: Pre-treat the dialysis bag, boil it in a water bath for 10 minutes, use 1×PBS as the dialysis buffer, change the dialysis solution multiple times, and then measure the concentration and aliquot and store at -20℃.

[0082] The results of monoclonal antibody ascites purification, monoclonal antibody titer, and IPMA identification are as follows: Figure 6 , Figure 7 , Figure 8 As shown.

[0083] RNA was extracted from the HPV56 subtype L1 protein monoclonal antibody, and its heavy chain variable region (VH) and light chain variable region (VL) were amplified using conventional PCR methods.

[0084] Example 5: Identification of Antigenic Epitopes Recognized by Monoclonal Antibodies

[0085] 1. Design of overlapping peptides

[0086] Peptides were designed and synthesized using the overlapping peptide method. In the first round, five peptides were synthesized, covering the entire L1 sequence, with adjacent peptides overlapping by 50 amino acids. In the second round, two peptides were synthesized, with adjacent peptides overlapping by 8 amino acids. In the third round, three peptides were synthesized, with adjacent peptides overlapping by 12 amino acids. Preliminary identification of the antigenic epitopes recognized by the monoclonal antibody was performed, and the results are as follows: Figure 9 As shown, its amino acid sequence is shown in SEQ ID NO.9.

[0087] 2. Reactivity detection of conjugated peptides, L1 monoclonal antibodies, and positive sera.

[0088] The peptide conjugated with BSA was coated onto an ELISA plate. The reactivity of the peptide with monoclonal antibodies and mouse positive serum was detected by indirect ELISA and Dot-ELISA. Results are as follows: Figure 10 , Figure 11 As shown, the results indicate that peptide T1 reacts strongly with monoclonal antibody against HPV56 subtype L1 protein, and also reacts with HPV-positive serum.

[0089] Example 6: Spatial Structure Analysis of HPV56 L1 Antigen Epitope Peptide

[0090] A 3D model of the HPV56 subtype L1 protein was built using the software SWISS-MODEL. The results are as follows: Figure 12 As shown, the selected HPV56 subtype L1 protein B-cell epitopes were displayed on the L1 protein model using PyMOL software, indicating that the epitope region recognized by the monoclonal antibody is located on the protein surface.

[0091] Although some preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0092] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of its inventive concept. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. An antigenic epitope peptide, characterized in that, It is the L1 protein 2-13 peptide segment of HPV56 subtype, and its amino acid sequence is shown in SEQ ID NO.

9.

2. The use of the antigenic epitope peptide of claim 1 in the preparation of an immunological detection reagent for detecting HPV56 virus.

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