CN111410689B

CN111410689B - Monoclonal antibody 69E2 for resisting HPV16E7 protein, hybridoma cell strain, and preparation method and application thereof

Info

Publication number: CN111410689B
Application number: CN202010180043.8A
Authority: CN
Inventors: 胡仁建; 蔡家利
Original assignee: Chongqing University of Technology
Current assignee: Chongqing University of Technology
Priority date: 2020-03-16
Filing date: 2020-03-16
Publication date: 2021-11-02
Anticipated expiration: 2040-03-16
Also published as: CN111410689A

Abstract

The invention discloses an anti-HPV 16E7 protein monoclonal antibody 69E2, a hybridoma cell strain, a preparation method and application thereof, wherein the hybridoma cell is preserved in China center for type culture Collection with the preservation number as follows: CCTCC NO: c2019181, the hybridoma cell can secrete monoclonal antibodies with high titer, and the obtained antibodies can react with HPV16 positive cell strains CaSki and HPV16 positive cervical carcinoma squamous cell carcinoma paraffin tissue slices, so that the hybridoma cell has good specificity; can be used as detection reagents of ELISA kits, immunochemical kits, immunofluorescence kits, chemiluminescence immunoassay detection kits and the like.

Description

Monoclonal antibody 69E2 for resisting HPV16E7 protein, hybridoma cell strain, and preparation method and application thereof

Technical Field

The invention relates to the field of immunology, in particular to a hybridoma cell secreting monoclonal antibody 69E2 against HPV16E7 protein, and also relates to a monoclonal antibody secreted by the cell, and a preparation method and application thereof.

Background

Cervical cancer is the fourth most common cancer in women worldwide, with an annual increase of 530,000 cases and 275,000 deaths, of which about 85% originate in developing countries. In China, the incidence of cervical cancer is the first place of female genital tract malignant tumor. Persistent infection of High risk type human papillomavirus (HR-HPV) of the cervix is the direct cause of Cervical Intraepithelial Neoplasia (CIN) and cervical cancer, with HPV type 16 being the highest, 53%. The HPVE7 protein is a key molecule of HPV carcinogenesis, the E7 oncogenic protein is selectively expressed in tumor cells and is not expressed in normal cells, and as the cervical intraepithelial neoplasm CIN progresses from low grade to high grade (from CIN1 to CIN2 and CIN3 to erosive cervical cancer), the expression of the E7 oncogenic protein obviously advances to the outer layer and the expression amount is higher and higher, and finally the oncogenic protein E7 protein appears in cervical exfoliated cells. HR-HPVDNA detection combined with cytological examination (such as TCT) can increase the detection rate of cervical intraepithelial neoplasia CIN3 and reduce the occurrence of cervical cancer, and the detection rate of cervical in-situ adenocarcinoma and invasive cervical adenocarcinoma can be increased by the joint detection method. The method is used for carrying out cytological and HPVDNA joint detection on 30-65-year-old women, and a few of patients with negative cytological detection results and positive HR-HPVDNA detection results inevitably occur. The latest guidelines for cervical cancer screening in the united states provide 2 options: (1) the cytological and HR-HPVDNA co-tests were repeated at 12 months. Colposcopy is required if the result of HPV positive or cytological examination is LSIL or more severe when the joint detection is repeated; (2) HPV genotyping assays were performed immediately (HPvl6 or HPvl 8). The new screening guidelines lack a positive and consistent treatment regimen for positive HR-HPV DNA detection results, and colposcopy is a traumatic examination, which also increases the psychological and economic burden on patients, mainly because patients who are negative cytology and positive HR-HPV DNA detection cannot predict which patients will eventually develop cervical intratumoral CINs 1, CIN2, CIN3 and cervical invasive carcinoma, and if women who are negative cytological examination and positive HR-HPV DNA detection are supplemented with E7 oncoproteins such as HPV16E7, HPV18E7 oncoproteins for HR-HPV detection, it is likely that cancer cells will be found in the cervical intraepithelial neoplasia CIN2, CIN3 or even in the CIN1 stage, and the purposes of early warning, early diagnosis and early treatment of cervical cancer will be achieved.

Disclosure of Invention

In view of the above, the present invention aims to provide an anti-HPV 16E7 protein mab 69E 2; the second purpose of the invention is to provide hybridoma cells secreting monoclonal antibody 69E2 against HPV16E7 protein; the invention also aims to provide a preparation method of the anti-HPV 16E7 protein monoclonal antibody 69E 2; the fourth purpose of the invention is to provide the application of the monoclonal antibody 69E2 for resisting HPV16E7 protein in preparing a kit for detecting HPV16E7 protein; the fifth purpose of the invention is to provide a kit containing the anti-HPV 16E7 protein monoclonal antibody 69E 2.

In order to achieve the purpose, the invention provides the following technical scheme:

1. an anti-HPV 16E7 protein monoclonal antibody 69E2, wherein the monoclonal antibody 69E2 comprises a heavy chain and a light chain, and amino acid sequences of a variable region CDR1, CDR2 and CDR3 of the heavy chain are respectively shown as 50 th-54 th, 69 th-85 th and 118 th-127 th positions of SEQ ID NO. 6; the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the light chain are shown in positions 46-55, positions 71-77 and positions 110-118 of SEQ ID NO. 8.

Preferably, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6; the amino acid sequence of the light chain is shown as SEQ ID NO. 8.

Preferably, the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 5; the nucleotide sequence of the light chain is shown as SEQ ID NO. 7.

Preferably, the monoclonal antibody is secreted by a hybridoma cell strain HPV16E7-69E2, and the preservation number is CCTCC NO: C2019181.

2. the hybridoma cells secreting monoclonal antibody 69E2 resisting HPV16E7 protein are preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2019181.

3. the preparation method of the anti-HPV 16E7 protein monoclonal antibody 69E2 comprises the following steps: immunizing a mouse by taking HPV16E7 protein as an antigen, fusing SP2/0 with spleen cells or lymph node cells of an immunized BALB/c mouse by using a hybridoma technology, screening a hybridoma cell strain with high stability and high titer for resisting the HPV16E7 protein, preparing monoclonal antibody ascites, and purifying to obtain the monoclonal antibody 69E2 for resisting the HPV16E7 protein.

4. The anti-HPV 16E7 protein monoclonal antibody 69E2 is applied to the preparation of a kit for detecting HPV 16.

Preferably, monoclonal antibody 69E2 in the kit is used as a detection antibody.

5. A kit containing the anti-HPV 16E7 protein monoclonal antibody 69E 2.

Preferably, the kit is ELISA kit, immunochemical kit, immunofluorescence kit, chemiluminescence detection kit and Western Blot detection reagent.

The invention has the beneficial effects that: the invention discloses a hybridoma cell secreting monoclonal antibody 69E2 resisting HPV16E7 protein, which can secrete monoclonal antibody 69E2 resisting HPV16E7 protein, can be used for quantitative detection of HPV16E7 protein, has the advantages of good specificity and high sensitivity, and has important significance for early diagnosis of cervical cancer.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows the result of reaction of purified HPV16E7 protein with histidine-tagged antibody by Western Blot detection.

FIG. 2 is a 10% SDS-PAGE electrophoresis of purified mAb 69E2 (M: Marker;

lanes

1 and 2, two bands at molecular weights 50kDa and 25kDa, respectively).

FIG. 3 is the peak titer chart for mAb 69E 2.

FIG. 4 shows 4 pairs of mAbs that can be paired.

FIG. 5 is a graph showing affinity assay of mAb 69E 2.

FIG. 6 shows the results of immunocytochemistry of mAb 69E2 with CaSki cells and HeLa cells (10X 20).

FIG. 7 is a diagram of immunohistochemical results of monoclonal antibody 69E2 and HPV 16-positive cervical cancer squamous cell carcinoma and HPV 18-positive cervical cancer adenocarcinoma.

FIG. 8 is a graph showing the results of indirect immunofluorescence reactions of mAb 69E2 with CaSki cells and HeLa cells (10X 40).

FIG. 9 is a graph showing the results of Western Blot reaction of mAb 69E2 with total protein from CaSki cells and HeLa cells.

FIG. 10 shows the linear relationship between the ng-scale dilutions of the antigen HPV16E7 protein by the isobaric dilution method (A: determination coefficient R of straight line fitting of reference curves prepared when the antigen HPV16E7 protein is diluted by the isobaric dilution method (0, 25, 50, 100, 200, 400, 600, 800, 1000 ng/well) in ng-scale dilutions²＝0.9723，R²The judgment coefficient is also called a decision coefficient and is also called goodness of fit, and is established on the basis of regression analysis and used for researching the interpretation degree of one random variable to another random variable, the value is 0-1, and the closer to 1, the higher the interpretation degree of the independent variable to the dependent variable is; b: the coefficient of variation CV is the standard deviation/average of the original data, two points with low concentration content with antigen of 0 and coefficient of variation CV lower than 10% are used as the abscissa, the corresponding OD value is used as the ordinate, the equation for obtaining the calculation detection limit is Y0.01122X +0.6896 and the judgment coefficient R²The value is 0.9889; c: S/N is equal to the OD value of the Sample to be detected/the Negative control OD value, S is Sample of the Sample to be detected, N is Negative control Negative, and the OD of the lower limit of the quantification is accurately determined by the high standard of S/N equal to 3_450nm3 × 0.6896 ═ 2.0688, lower limit of quantitation (2.0688-0.6896)/0.01122 ═ 122.9234 ng.

FIG. 11 is a diagram showing a reference curve for detecting a cervical cancer specimen and HPV16E7 protein content in the specimen (A, B, C is a diagram showing straight line fitting, double logarithm fitting and signal-to-noise ratio of the reference curve for detecting a cervical cancer specimen, D is a reference curve and equation for calculating a detection limit, and E is a diagram showing HPV16E7 content of 3 groups of tissues including 4 cervical normal tissues, 12 HPV 16-positive cervical cancer tissues and 4 HPV 52-positive cervical cancer tissues).

FIG. 12 shows the epitope identification result of mAb 69E2 (A: the result of indirect ELISA using overlapping peptides; B: the result of indirect competition ELISA; C: the localization of the epitope specific to mAb 69E2 on the three-dimensional crystal structure map of HPV16E7 protein).

FIG. 13 is a graph showing the conservation analysis alignment of the specific epitope of mAb 69E 2.

Deposit description

Hybridoma cell strains secreting antibodies 79A11, 69A6 and 69E2 are sent to China center for type culture Collection, the collection addresses are Wuhan university in Wuhan, China, and the hybridoma cell strains are named as HPV16E7-79A11, HPV16E7-69A6 and HPV16E7-69E2 respectively; the preservation date of HPV16E7-79A11 is 2017, 4 and 10 months, and the preservation number is CCTCC NO: c201718, namely a monoclonal hybridoma cell strain HPV16E7-79A 11; the preservation date of HPV16E7-69A6 is 2017, 4 and 10 months, and the preservation number is CCTCC NO: c201720, namely a monoclonal hybridoma cell strain HPV16E7-69A 6; the preservation date of HPV16E7-69E2 is 8-21.2019, and the preservation number is CCTCC NO: c2019181, and is classified and named as a mouse hybridoma cell strain HPV16E7-69E 2.

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

The invention clones and expresses HPV16E7 gene by using gene engineering technology, takes purified HPV16E7 protein as antigen immune BALB/c mouse, fuses SP2/0 and spleen cell and lymph node cell of immune BALB/c mouse by using hybridoma technology, screens to obtain anti-HPV 16E7 protein monoclonal hybridoma cell strain with good stability and high titer, prepares monoclonal antibody ascites, and purifies by using salting out, affinity chromatography, gel chromatography and other comprehensive methods to obtain 3 monoclonal antibodies with higher purity and higher titer, which are named as 79A11, 69A6 and 69E 2.

Example 1 preparation of HPV16E7 protein

Specific primers with 6 histidine tags were designed based on the full length gene of HPV16E7, and the sequences of primers F and R (underlined endonucleases) were as follows:

F:5'-gcccatggaccatcaccatcaccatatgcatggagatacacctac-3’(SEQ ID NO.1)；

R:5'-gcaagcttttatggtttctgygaacagatggggcacac-3’(SEQ ID NO.2)。

using CaSki cell genome total DNA as a template, carrying out PCR amplification on the HPV16E7 full-length gene under the following PCR reaction conditions: pre-denaturation at 94 ℃ for 5min, pre-denaturation at 94 ℃ for 30s, pre-denaturation at 55-70 ℃ for 30s, pre-denaturation at 72 ℃ for 60s, pre-denaturation at 72 ℃ for 35 cycles, and extension at 72 ℃ for 7 min. The PCR product HV16E7 gene fragment and the plasmid pET-28a (+) are subjected to restriction enzyme digestion by Nco I and Hind III, a correct recombinant plasmid pET-28a (+) -HPV16E7 is identified to transform competent cells Rosetta (DE3) pLysS, the engineering bacteria pET-28a (+) -HPV16E7-Rosetta (DE3) pLysS is induced in LB culture medium by 1mM IPTG for 4h at 37 ℃, the lysate is centrifuged, 12% SDS-PAGE electrophoresis is used for determining the expression amount and the expression form of HPV16E7 protein, and the 16E7 protein is purified by nickel column affinity chromatography and anion exchange chromatography to obtain the HPV16E7 protein with the purity of more than 95%. Western Blot analysis As shown in FIG. 1, histidine-tagged antibodies were reactive with the expressed HPV16E7 protein, indicating that the expressed HPV16E7 protein carries a histidine tag. The 12% SDS-PAGE electrophoresis result shows that the expressed HPV16E7 protein has high expression amount and is in soluble expression, the molecular weight is about 18kDa, the predicted theoretical molecular weight of the HPV16E7 protein is about 11988.32Da and is close to 12kDa, and PI is 4.59 (figure 2). However, the molecular weight of the HPV16E7 protein expressed at present is 18kDa, which is larger than the theoretical molecular weight of HPV16E7 protein of 12kDa, and the HPV16E7 protein has electrophoresis abnormal behavior phenomenon, as the wild-type HPV16E7 protein reported in the literature (Armstrong DJ and Roman A. biochem Biophys Res Commun, 1993) has a large amount of negative charges, the HPV16E7 protein can migrate at 14-21 kDa during electrophoresis, and the HPV16E7 protein expressed in the research has a molecular weight of about 18kDa, which is exactly consistent with the migration of HPV16E7 protein at 14-21 kDa reported in the literature.

Wherein the sequence of the PCR amplification product is shown as (SEQ ID NO. 3):

gcccatggaccatcaccatcaccatatgcatggagatacacctacattgcatgaatatatgttagatttgcaaccagagacaactgatctctactgttatgagcaattcaatgacagctcagaggaggaggatgaaatagatggtccagctggacaagcagaaccggacagagcccattacaatattgtaaccttttgttgcaagtgtgactctacgcttcggttgtgcgtacaaagcacacacgtagacattcgtactttggaagacctgttaatgggcacactaggaattgtgtgccccatctgttcacagaaaccataa

the coded amino acid sequence is shown as SEQ ID NO. 4:

MDHHHHHMHGDTPTLHEYMLDLQPETTDLYCYEQFNDSSE

EEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP

example 2 preparation of monoclonal antibody against HPV16E7 protein

The hybridoma technology is used for immunizing a BALB/c mouse by taking HPV16E7 protein as an antigen, rear leg inguinal lymph node B lymphocytes and spleen cells of the immunized mouse are respectively fused with myeloma cells SP2/0-Ag14, and hybridoma cell strains of the anti-HPV 16E7 protein monoclonal antibody are obtained through culture screening and multiple cloning, and the results are shown in tables 1 and 2.

Table 1 shows the names of spleen-derived positive polyclonal hybridoma cell lines with high titer, monoclonal antibody hybridoma cell lines selected after multiple cloning, and OD of cell supernatant antibody_450nmValues and antibody subtypes, among which monoclonal antibodies with high titer and well-defined subtypes are 79A11(IgM), 38E11(IgM), 74F3 (mainly IgG2a) and 72E6 (subtype undefined). It can be seen that the subtype of spleen-derived monoclonal antibody is mainly IgM.

TABLE 1 Positive polyclonal and monoclonal hybridoma cell lines from splenocytes

TABLE 2Shows the names of the positive polyclonal hybridoma cell strains with high titer derived from inguinal lymph node, the hybridoma cell strains of the monoclonal antibodies screened after multiple cloning, and the OD of the antibody of the cell supernatant_450nmValues and antibody subtypes, of which monoclonal antibodies 69E2, 69D10, 69B11, 54D5, 54F4 and 54G5 are IgG2a, but the subtype of lymph node-derived monoclonal antibody 69a6 is IgM.

TABLE 2 Positive polyclonal and monoclonal hybridoma cell lines from lymph node cells

And (4) identifying the monoclonal antibody subtype, preparing monoclonal antibody ascites, and purifying the monoclonal antibody according to IgM and IgG purification schemes respectively.

Subtype identification of monoclonal antibody: the subtype of the positive monoclonal antibody cell supernatant identified by the monoclonal antibody subtype identification kit has two types: IgG2a and IgM. The IgG2a subtype positive monoclonal hybridoma cell strain is mainly derived from lymph nodes, including 69B11, 69D10, 69E2, 54D5, 54F4, 54G5 and the like; while the subtype of lymph node derived mab 69A6 is IgM; the positive monoclonal hybridoma cell lines of the IgM subtype mainly originated from the spleen, and mainly contained 79a11, 56E4, 38E11, etc., whereas the subtype of the spleen-derived mab 74F3 was unknown, mainly IgG2a, and the subtype of the spleen-derived mab 72E6 was also unknown (table 3).

TABLE 3 OD of monoclonal antibody subtype identification_450nmValue and subtype results thereof

Preparing, collecting and storing monoclonal antibody ascites:

the monoclonal antibody ascites is prepared by an animal in vivo induction method, and the result shows that the number of hybridoma cells of a mouse actually injected with BALB/c is as follows: 1.5*10⁶-2.7*10⁶The volume of ascites generated by most mice is between 3 and 5 mL/mouse, the volume of ascites generated by a small part can reach 8 to 10 mL/mouse, and the injection is carried outTo 2.0 x 10⁶Production of ascites more than 1.5 x 10⁶A/only. Disinfecting abdomen with 9 # needle, introducing into abdominal cavity, extracting ascites, collecting ascites, centrifuging at 4 deg.C, adding cell freezing medium to cells, freezing, recovering cells, culturing, inactivating supernatant, adding glycerol, and storing at-80 deg.C.

2 mug/mL of envelope antigen HPV16E7 protein is used for detecting 25 monoclonal antibody ascites altogether by an indirect ELISA method, the dilution degree of the antibody is diluted from 1:5000 to 1:5120000, the signal-to-noise ratio S/N value is more than or equal to 2.1, the signal-to-noise ratio S/N is the OD value of a sample to be detected/the OD value of negative control, 11 monoclonal antibody ascites with the highest titer of more than 1:128000 in 25 monoclonal antibody ascites are detected: 79A11, 69E2, 69A6, 69A11, 69B11, 38E11, 54D5, 54F4, 54G5, 56E4, and 56E5 (see Table 4).

TABLE 4 maximum potency of 11 monoclonal antibody ascites with potency not less than 1:128000

And (3) measuring the titer of the purified monoclonal antibody by indirect ELISA:

the 8 purified monoclonal antibodies were determined to be positive by indirect ELISA method with S/N ratio of 2.1 or more, and the highest titers of the monoclonal antibodies 79A11, 69A6, 69E2, 54G5, 54D5, 54F4, 74F3 and 72E6 were 6.25ng, 0.78ng, 0.19ng, 1.56ng, 0.39ng, 1.56ng, 6.25ng and 6.25ng in sequence (Table 5).

TABLE 5 potency of the purified 8 monoclonal antibodies

The purity of the IgG2a subtype monoclonal antibody obtained by the two-step method of caprylic acid-ammonium sulfate precipitation and Protein G purification is as high as more than 95%, and the titer is high, wherein the titer of one monoclonal antibody 69E2 is the highest (0.19 ng); the purity of the IgM subtype monoclonal antibody purified by the four-step method of ammonium sulfate precipitation, gel filtration chromatography, IgM column and gel filtration chromatography is up to more than 95%, and the titer of the monoclonal antibody 79A11 is higher, about 6.25 ng. The high purity and high activity of the monoclonal antibody are important for the establishment of the subsequent characterization analysis and detection method of the monoclonal antibody.

3 hybridoma cell strains 79A11, 69A6 and 69E2 which stably secrete monoclonal antibodies are sent to China center for type culture collection, the collection addresses are Wuhan university in Wuhan, China, and the collection addresses are respectively named as HPV16E7-79A11, HPV16E7-69A6 and HPV16E7-69E 2; the preservation date of HPV16E7-79A11 is 2017, 4 and 10 months, and the preservation number is CCTCC NO: c201718, namely a monoclonal hybridoma cell strain HPV16E7-79A 11; the preservation date of HPV16E7-69A6 is 2017, 4 and 10 months, and the preservation number is CCTCC NO: c201720, namely a monoclonal hybridoma cell strain HPV16E7-69A 6; the preservation date of HPV16E7-69E2 is 8-21.2019, and the preservation number is CCTCC NO: c2019181, and is classified and named as a mouse hybridoma cell strain HPV16E7-69E 2.

Example 3 identification of monoclonal antibody 69E2

Monoclonal antibody 69E2 of IgG2a subtype purified by two-step method-caprylic acid-ammonium sulfate precipitation and

Protein G. Lanes

1 and 2 of 10% SDS-PAGE show the elution peaks of mAb 69E2 after purification with Protein G, and two bands at molecular weights of 50kDa and 25kDa were found in

lanes

1 and 2, respectively, which correspond to the molecular weights of the heavy and light chains of the IgG subtype antibodies, and no other bands were found (see FIG. 2). The purity of the monoclonal antibody 69E2 purified by the two-step method, namely caprylic acid-ammonium sulfate precipitation and Protein G is higher, more than 95 percent, and the monoclonal antibody can be used for identifying and detecting the monoclonal antibody 69E 2.

Nanjing Kinshire Biotechnology Limited uses hybridoma cell line 69E2 to determine the nucleotide sequence of monoclonal antibody 69E2, uses the nucleotide sequence of monoclonal antibody 69E2 to predict the amino acid sequence of monoclonal antibody 69E2, and the results are as follows:

the heavy chain nucleic acid sequence of the monoclonal antibody 69E2 is shown as SEQ ID NO.5(1407bp), the structure is Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon, and the bold type represents the CDR sequence:

the coded amino acid sequence is shown as SEQ ID NO.6(468aa), the structure is Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon, and the bold is the CDR sequence:

the light chain nucleotide sequence is shown in SEQ ID NO.7(708bp), the Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon, and the CDR sequences are bold:

the light chain amino acid sequence is shown in SEQ ID NO.8(235aa), the structure is Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region-Stop codon, and the bold is CDR sequence:

example 4 detection of monoclonal antibody 69E2

1. Indirect ELISA method for detecting monoclonal antibody 69E2 titer

(1) Coating antigen: coating concentration of antigen HPV16E7 protein is 1 mug/mL, 100 mug/hole, MH-1 micropore plate oscillator of the forest shellfish shakes the ELISA plate for 10min at room temperature, then covers the plastic plate, is placed at 37 ℃ for standing reaction for 1h, is transferred to 4 ℃ for standing reaction overnight for 12-14 h, and is washed by PBST for 3 times the next day, and each time is 3-5 min;

(2) and (3) sealing: dissolving 0.25% BSA in PBST as a blocking solution, shaking the ELISA plate for 10min at 300. mu.L/well and MH-1 microplate oscillator room temperature, standing at 37 ℃ for reaction for 2h, and washing the plate 3 times with PBST, 3-5min each time;

(3) adding a primary antibody: 79A11 and 69E2 were diluted with blocking solution at a concentration of 1. mu.g/mL in the first well and 7.8ng/mL in the second well at 100. mu.L/well, while 4 controls were set: positive serum control, negative serum control, control with addition of only secondary antibody, goat anti-mouse IgG-HRP, and blank control. Vibrating the ELISA plate for 10min at room temperature with MH-1 microplate oscillator, standing at 37 deg.C for reaction for 1h, washing the plate for 3 times with PBST, each time for 3-5 min;

(4) adding a secondary antibody: diluting goat anti-mouse IgG-HRP (immunoglobulin G-horse radish) with blocking solution 0.25% BSA at a ratio of 1:10000 at a concentration of 100 muL/well, shaking the microplate with MH-1 microplate oscillator at room temperature for 10min, standing at 37 ℃ for reaction for 1h, and washing the plate with PBST for 3 times, 3-5min each time;

(5) adding a substrate TMB for color development: mixing the solution A and the solution B in the TMB two-component developing solution kit according to a ratio of 1:1, shaking an enzyme label plate at the room temperature of an MH-1 micropore plate oscillator for 10min at a rate of 100 mu L/hole, and standing and reacting for 20min at 37 ℃;

(6) adding a stop solution: 2MH₂SO₄Terminating the reaction, shaking the ELISA plate at room temperature for 2min at 50 mu L/hole;

(7) determination of OD with full-wavelength microplate reader_450nmAnd (4) processing values and data.

And (3) judging the result of the indirect ELISA by using the ratio of the Sample to the Negative control, judging the indirect ELISA to be positive when the signal-to-noise ratio S/N is more than 2.1, wherein S is the Sample, and N is the Negative control Negative. 2 mu g/ml of antigen-coated HPV16E7 holoprotein, diluting monoclonal antibody 69E2 from 4 mu g/ml to 0.1953 mu g/ml, 100 mu l/well, reacting 12 concentrations of monoclonal antibody 69E2 with antigen-coated HPV16E7 holoprotein respectively, and obtaining a positive result that the S/N value of monoclonal antibody 69E2 at 0.19ng is 8.9257 and is far greater than 2.1, so that the maximum titer of monoclonal antibody 69E2 is at least 0.19ng, see Table 6 and FIG. 3.

TABLE 6 results of the highest titer of monoclonal antibody 69E2 determined by indirect ELISA method

2. Direct ELISA for detecting antibody titer of conjugate HRP-69E2

Separating the undegraded antibody IgG2a and the degraded small molecular fragment from the detection antibody 69E2(IgG2a) after standing by using a gel chromatography column, and Labeling HRP with about 1mg of the undegraded detection antibody 69E2 by using a Proxidase Labeling Kit-NH with the product number of LK51 Kit₂HRP-labeled detection antibody in (for 1mg)Body 69E2 was about 1 mg. The signal to noise ratio S/N value is more than or equal to 2.1, the positive judgment is made to determine that the antibody titer of the conjugate HRP-69E2 is at least 0.78ng (Table 7), which indicates that the effect of the detection antibody 69E2 after being marked with HRP is good, and the method can be used for establishing a quantitative method.

TABLE 7 Signal-to-noise ratio S/N and antibody titer for direct ELISA detection of conjugate HRP-69E2

3. Method and result of pairing monoclonal antibody 69E2 with other monoclonal antibodies

The monoclonal antibody 69E2 is not marked with Horse Radish Peroxidase (HRP) and is used as a capture antibody, and is matched with other 7 monoclonal antibodies (79A11-HRP, 69A6-HRP, 54D5-HRP, 54G5-HRP, 54F4-HRP, 74F3-HRP and 72E6-HRP) which are marked with HRP and are used as detection antibodies, so that a double-antibody sandwich ELISA experiment is carried out; the monoclonal antibody 69E2 labeled horseradish peroxidase HRP was used as a detection antibody, and paired with other 7 monoclonal antibodies (79a11, 69a6, 54D5, 54G5, 54F4, 74F3, and 72E6) against HPV16E7 protein which is not labeled with HRP as capture antibodies, to perform a double antibody sandwich ELISA experiment.

As a result: of the 56 pairs of paired monoclonal antibodies, 4 pairs of monoclonal antibodies can be paired: 79A11 and 69E2-HRP, 79A11 and 74F3-HRP, 79A11 and 69A6-HRP, 79A11 and 54D5-HRP (see FIG. 4). For the monoclonal antibody 69E2, only when the monoclonal antibody 79A11 is a capture antibody, and the monoclonal antibody 69E2-HRP is a detection antibody pair for detecting the antigen HPV16E7 protein, a stronger positive signal is obtained; when the monoclonal antibody 69E2 is a capture antibody, no positive signal is generated when the monoclonal antibody is matched with other 7 monoclonal antibodies. Therefore, the monoclonal antibody 69E2 is not suitable for being used as a capture antibody and is suitable for being used as a detection antibody in the double-antibody sandwich ELISA.

4. Affinity assay for monoclonal antibody 69E2

The monoclonal antibody 69E2 with a purity of more than 95% and its antigen HPV16E7 complete protein were sent to southern jinslei biotechnology limited to determine the affinity of monoclonal antibody 69E2 and antigen HPV16E7 complete protein, and the results are shown in table 8 and fig. 5. The result shows that the affinity of the antigen HPV16E7 and the monoclonal antibody 69E2 is 5.60E-09M, which indicates that the affinity of the monoclonal antibody 69E2 is high.

TABLE 8 affinity assay results for mAb 69E2

5. Preliminary application of monoclonal antibody 69E2 in immunocytochemistry and immunohistochemistry

A. Primary application of monoclonal antibody 69E2 in immunocytochemistry

The method comprises the following steps: preparing cell slide of cervical cancer cell strains CaSki and HeLa, preparing monoclonal antibody 69E2 into different concentrations to react with CaSki and HeLa cells respectively, adding DAB to develop color, the result is shown in FIG. 6. Immunocytochemistry results show that when the content of the monoclonal antibody 69E2 is 1 mug/hole, 0.5 mug/hole, 0.25 mug/hole, 0.125 mug/hole, 0.0625 mug/hole, 0.03125 mug/hole, 0.015625 mug/hole and 0.0078125 mug/hole in sequence, the monoclonal antibody reacts with CaSki cells (HPV16 positive) respectively, and when the content of the monoclonal antibody 69E2 is from 7.8125 ng/hole to 1 mug/hole, the CaSki cells have gradually enhanced brown yellow particles, namely the reaction sensitivity is at least 7.8125 ng/hole, and the effectiveness of the monoclonal antibody 69E2 is verified; when the monoclonal antibody is reacted with HeLa cells (positive HPV 18), no brown yellow particles appear in the HeLa cells when the content of the monoclonal antibody 69E2 is from 7.8125 ng/hole to 1 mu g/hole, the specificity of the monoclonal antibody 69E2 is verified, and the monoclonal antibody 69E2 has potential application value in detecting natural HPV16E7 protein in immunocytochemistry.

B. Application of monoclonal antibody 69E2 in immunohistochemistry

The monoclonal antibody 69E2 is respectively subjected to immunohistochemical reaction with the HPV16 positive cervical cancer squamous cell carcinoma paraffin section and the HPV18 positive cervical cancer adenocarcinoma paraffin section to preliminarily verify the effectiveness and specificity of the 3 monoclonal antibodies against HPV16E7 protein in detecting HPV16E7 protein in cervical cancer tissues, and is used for preliminarily analyzing the application value of the monoclonal antibody 69E2 in immunohistochemical reaction. The method comprises the following specific steps:

(1) baking slices: taking cervical cancer squamous carcinoma (pathology No. 7532) and cervical cancer adenocarcinoma paraffin tissue (pathology No. 2255) provided by nine-hospital pathology department in Chongqing, baking at 60 deg.C for 2 h;

(2) dewaxing and hydrating: sequentially dewaxing and hydrating with xylene I15 min, xylene II 15min, 1/2 xylene (xylene: anhydrous ethanol 1:1)10min, anhydrous ethanol 10min, 95% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, and water 5min, washing with PBS for 5min for 3 times;

(3) cell permeation: treating with 0.01MPBS containing 0.5% Triton X-100 at pH7.2 at room temperature for 20min, washing with PBS for 5min for 3 times;

(4) antigen retrieval: repairing with sodium citrate solution with working concentration in 85 deg.C water bath for 45min, naturally cooling, washing with PBS for 3 times, each for 5 min;

(5) inactivation of endogenous peroxidase and biotin: adding 3% of H to each tablet₂O₂Reacting at room temperature for 20min to eliminate endogenous peroxidase and biotin, washing cells with PBS for 5min for 3 times;

(6) and (3) sealing: treating with 0.01MPBS containing 5% BSA or 5% goat serum at pH7.2 at room temperature for 60min, washing cells without PBS, and directly adding primary antibody;

(7) adding a primary antibody: using wax to draw a circle around each section of tissue, wherein the concentration of 3 monoclonal antibodies 79A11, 69E2 and 69A6 is 8.95 mu g/ml, 50 mu g/ml and the like in sequence, the concentration is 200 mu l/slice, setting a negative control which does not add primary antibody and only adds secondary antibody, adding PBS (a blank control), keeping overnight at 4 ℃, washing 3 times with PBS, and 5min each time;

(8) addition reagent 1 (polymer adjuvant or reaction enhancer): adding reagent 1 (polymer adjuvant) into the mouse hypersensitivity two-step detection kit of Zhongxie Jinqiao with product number PV-9002 into each slice, allowing reaction at room temperature for 20min at 200 μ l/slice, and washing with PBS for 3 times, 5min each time;

(9) reagent 2 (horseradish-enzyme-labeled anti-mouse IgG multimer) was added: adding 200 μ l/piece of reagent 2, washing with PBS for 3 times, each time for 5 min;

(10) DAB color development: performing color development with the working concentration of DAB color development kit (20X) with the product number of ZLI-9018 of Chinese shirt golden bridge under a microscope;

(11) counterdyeing: counterstaining with hematoxylin staining solution with ZLI-9615 of Zhongshirt Jinqiao for 2-6 min;

(12) water rinsing for 10-15min, repeated dehydration (in reverse order of dewaxing), clear, neutral gum mounting and photographing under microscope.

The results are shown in FIG. 7. Immunohistochemical results show that typical brown yellow particles can appear when the monoclonal antibody 69E2 reacts with the cervical cancer squamous carcinoma positive by HPV16, and brown yellow particles can not appear when the monoclonal antibody reacts with the cervical cancer adenocarcinoma positive by HPV 18. The monoclonal antibody 69E2 can detect HPV16E7 protein in the HPV 16-positive cervical cancer squamous cell carcinoma, but cannot detect HPV18E7 protein in the HPV 18-positive cervical cancer squamous cell carcinoma, and the monoclonal antibody 69E2 has potential application value in immunohistochemical detection of HPV 16-positive cervical cancer.

C. Primary application of monoclonal antibody 69E2 in indirect immunofluorescence method

A primary antibody, namely monoclonal antibody 69E2 is combined with corresponding antigens in cells on CaSki cells and HeLa cell crawlers in a self-made wet box to form an antigen-antibody complex, then a secondary antibody marked by fluorescein is combined with the primary antibody, and the reaction result is observed under a fluorescence microscope after the reaction, so that the HPV16E7-69E 2-based immunofluorescence kit is initially established. Locating the expression of HPV16E7 protein in the CaSki cells which are positive for HPV16 by utilizing an immunofluorescence technique, wherein primary antibodies with the dilution degree of 1 ug/well, 0.5 ug/well, 0.25 ug/well, 0.125 ug/well, 0.0625 ug/well, 0.03125 ug/well, 0.015625 ug/well and 0.0078125 ug/well react with the CaSki cells, when the dilution degree of the primary antibodies with 69E2 reaches the maximum, namely 7.8125 ng/well, the nucleus green fluorescence of the CaSki cells is still obvious, namely the reaction sensitivity is at least 7.8125 ng/well; whereas no green fluorescence signal was observed in response to HPV 18-positive HeLa cells at the same concentrations of primary 69E2 and secondary antibody (see fig. 8).

D. Western Blot reaction of monoclonal antibody 69E2 with CaSki and HeLa cell Total protein

CaSki and HeLa cells are cultured and collected, and total protein in CaSki and HeLa cells is extracted by RIPA lysate, wherein the total protein in CaSki cells contains HPV16E7 protein, and the total protein in HeLa cells contains HPV18E7 protein, so that the total protein in CaSki and HeLa cells and other protein c-myc (his-tag-c-myc, molecular weight 62kDa) with a histidine tag are separated by 12% SDS-PAGE electrophoresis, glue is transferred onto an NC membrane, then the NC membrane is sealed by 5% skim milk, primary antibody 69E2 is reacted with the protein on the NC membrane, secondary antibody goat anti mouse IgG-HRP is added, and pictures are taken after exposure, and the results are shown in figure 9.

The results of the Western Blot reaction show: showing that no exposed band appeared after the monoclonal antibody 69E2 reacted with a histidine-tagged other protein, c-myc (his-tag-c-myc), suggesting that monoclonal antibody 69E2 did not react with histidine-tag; monoclonal antibody 69E2 reacted with CaSki cell total protein with a strong exposure band, whereas reacted with total protein in HeLa cells with no strong exposure band. Because the total protein of CaSki cells contains HPV16E7 protein and the total protein of HeLa cells contains HPV18E7 protein, the HPV16E7 protein in the total protein of CaSki cells can be detected by the monoclonal antibody 69E2, and the HPV18E7 protein in the total protein of HeLa cells cannot be detected by the monoclonal antibody 69E 2. The 69E2 is suggested to have potential application value in detecting Western Blot of HPV16E7 protein.

E. Conventional double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) method for quantitatively detecting HPV16E7 protein based on monoclonal antibody 79A11 and 69E2-HRP + TMB detection systems

The optimization conditions of the detection system are as follows: the optimal coating concentration of the capture antibody 79A11 was 2. mu.g/ml, the optimal working concentration of the detection antibody HRP-69E2 was 1. mu.g/ml, the background was low and the signal was strong for a 2h blocking with 5% skim milk. The double antibody sandwich ELISA showed that the decision coefficient R of the reference curve fitted with straight lines prepared when the antigen HPV16E7 protein was diluted in ng scale by the equal ratio equinormal method (0, 25, 50, 100, 200, 400, 600, 800, 1000 ng/well)²0.9723 (fig. 10, a), R²The judgment coefficient, also called a decision coefficient, also called goodness-of-fit, is established on the basis of regression analysis and used for researching the interpretation degree of one random variable to another random variable, and the value is 0-1, and the closer to 1, the higher the interpretation degree of the independent variable to the dependent variable is. The coefficient of variation CV is the standard deviation of the original data/the average of the original data, two points with the antigen of 0 and the low concentration of 25ng with the coefficient of variation CV lower than 10% are used as the abscissa, the corresponding OD value is used as the ordinate, and the equation for obtaining the calculation detection limit is Y0.01122X +0.6896 and R²The value was 0.9889 (fig. 10, B). S is Sample to be tested, N is Negative control Negative, and signal to noise ratio S/NOD value of sample to be tested/negative control OD value, and OD value with S/N of signal-to-noise ratio being 3 as lower limit of quantification_450nmWhen the assay is carried out at 3 × 0.6896 ═ 2.0688, and the lower limit of quantitation is (2.0688-0.6896)/0.01122 ═ 122.9234ng (fig. 10 and C), the sensitivity is not satisfactory, and a more sensitive method needs to be searched.

F. Preliminary analysis of chemiluminescence immunoassay method for quantitatively detecting HPV16E7 protein established based on monoclonal antibody 79A11 and 69E2-Biotin + HRP-Streptavidin detection system

The optimization conditions of the chemiluminescence immunoassay method are as follows: the coating concentration of the capture antibody 79A11 is 2ug/mL, the blocking is performed for 2h by 0.25% BSA blocking solution, the antigen HPV16E7 protein is diluted at ng level by an equal ratio equal difference method and reacts with the capture antibody at 37 ℃ for 2h, the working concentrations of the detection antibodies Biotin-69E2 and HRP-Streptavidin are both 1ug/mL, and the luminol reaction is performed for 10 min. The contents of 9 concentrations of antigen HPV16E7 protein, 0, 25, 50, 100, 200, 400, 600, 800 and 1000 ng/hole, are used as abscissa, and the corresponding OD_425nmThe values are plotted on the ordinate, and a reference curve is obtained. The results are shown in FIG. 11, where the regression equation for the straight line fit is Y53.35X +10294, R for the straight line fit and the log-log fit²0.9666 and 0.956 in that order; the contents of 0 and 25ng in low concentration of antigen HPV16E7 protein are used as abscissa, and the corresponding OD_425nmThe value is ordinate, a regression equation Y of 363.1X +3441 for calculating the lower limit of detection is obtained, and a coefficient R is determined²0.9823, OD 3S/N_425nm3 x 3441 to 10324, the detection limit (10324 and 3441)/363.1 to 18.9562ng, the coefficient of variation CV is 1.5184% to 11.9649%, which is less than 20%, which shows that the lower limit of detection is also the lower limit of quantification, and compared with the lower limit of quantification 122.9234ng of HPV16E7 oncoprotein by the conventional double antibody sandwich ELISA method, the sensitivity is improved by 6.48 times. OD of the specimen_425nmThe value was put into the equation Y-53.35X +10294 to obtain HPV16E7 protein less than the lower quantification limit of 18.9562ng for both 4 normal cervical tissues and for cervical cancer tissues in 4 cases positive to HPV 52; the content of 12 HPV16 positive cervical carcinoma HPV16E7 protein fluctuates between 102.1743 and 438.2473ng, the signal-to-noise ratio S/N values are all larger than 3.0, the positive cervical carcinoma HPV16E7 protein is positive, and the difference is obvious (P is less than 0.01) compared with a normal tissue and an HPV52 positive cervical carcinoma tissue, which shows that the chemical development established by the subjectThe photoimmunoassay method was preliminarily effective in detecting HPV16E7 protein in HPV 16-positive cervical cancer tissues.

Example 6 epitope identification of monoclonal antibody 69E2

1. Synthesis of HPV16E7 stepped overlap 18 peptide

(1) Finding the HPV16E7 amino acid sequence: the HPV16E7 protein sequence in the cell line of CaSki was found at the NCBI website (Seguence ID: NC-001526.2). The full-length 98 amino acids of the HPV16E7 protein have the following specific amino acid sequences:

MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP(SEQ ID NO.9)。

(2) short peptide synthesis: the method comprises the steps of moving 6 amino acids from amino acid No. 1 at one time, designing overlapping peptide containing 18 amino acids, entrusting Shanghai QiaoYao company to synthesize 15 overlapping peptides with purity higher than 95%, dissolving the synthesized overlapping peptide with DMSO to a storage concentration of 1mg/ml, subpackaging, and storing at-80 ℃.

2. Indirect ELISA method for overlapping peptide screening dominant linear B cell epitope of anti-HPV 16E7 monoclonal antibody

(1) Coating antigen: BSA was set as a negative control protein and pool of 15 peptides mixed together at a ratio of 1:1 was set as a positive control protein. Coating contents of 15 overlapping peptides, negative control protein and positive control protein are all 5 mu g/hole and 100 mu l/hole, the temperature is 4 ℃ overnight, and the plate is washed by PBST for 4 times in the second day, and each time lasts for 3 min;

(2) and (3) sealing: preparing 1.5% BSA with PBST, 250. mu.l/well, washing the plate 3 times with PBST at 37 ℃ for 2h, 3 minutes each time;

(3) adding a primary antibody: the concentration of the monoclonal antibody 69E2 is 1.84mg/ml, and the dilution is 1: 8000; washing the plate with PBST for 3min 4 times at 37 deg.C in 100 μ l/well for 1 h;

(4) adding a secondary antibody: anti-mouse IgG, HRP-linked Antibody, available from CST, 7076S, was diluted at 1:3000, 100. mu.l/well, 45 minutes at 37 ℃ and washed 4 times for 3min each with PBST;

(5) adding a TMB color development liquid: diluting solution A and solution B in a TMB two-component color development kit of Beijing Solaibao PR1210 at a ratio of 1:1, 100 mul/hole, and 5-8min at 37 ℃;

(6) final (a Chinese character of 'gan')Stopping reaction: plus 2MH₂SO₄50 μ l/well;

(7) determination of OD_450nmAnd analyzing the data.

The results are shown in FIG. 12A. The results show that indirect ELISA method of overlapping peptide shows that monoclonal antibody 69E2 and overlapping peptide HPV16E7_49-66The reaction signal of (A) is strongest (P)<0.01) with the overlapping peptide HPV16E7_49-66The anteroposterior overlapping peptide HPV16E7_43-60And HPV16E7_55-72、HPV16E7_61-78All react according to the rule of overlapping peptide reaction.

3. Indirect competitive ELISA method for overlapping peptide to screen dominant linear B cell epitope of anti-HPV 16E7 monoclonal antibody 69E2

Because the linear epitope screened by the indirect ELISA method for overlapping peptides has the advantage that overlapping peptides with strong hydrophobic amino acids can be screened, but has the disadvantage that overlapping peptides with strong hydrophilic amino acids are omitted. Since indirect ELISA relies on the immobilization efficiency of overlapping peptides, steric hindrance, and affinity of monoclonal antibodies, whereas indirect competition ELISA relies on affinity and steric hindrance only, the sensitivity of indirect competition ELISA will decrease, but the reliability will increase, so the dominant linear B cell epitope is co-screened in conjunction with indirect competition ELISA. Prior to indirect competition ELISA experiments, a checkerboard experiment was performed to determine the optimal envelope concentration of the antigen HPV16E7 holoprotein and the optimal reactive concentration of the monoclonal antibody, as OD_450nmValues were determined between 0.7 and 0.8.

A. Chessboard experiment of indirect ELISA

(1) Coating antigen: HPV16E7 holoprotein: the HPV16E7 holoprotein was formulated at 4. mu.g/ml, 2. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 12 wells per concentration, 100. mu.l/well, overnight at 4 ℃ and the plate was washed 4 times with PBST the second day, 3 minutes each;

(3) adding a primary antibody: respectively preparing monoclonal antibody 69E2 into a first gradient of 4 mu g/ml, sequentially diluting 8 gradients in a multiple ratio manner, respectively adding antigen holes with 4 concentrations, 100 mu l/hole, sequentially adding positive serum, negative serum, secondary antibody control and blank control of HPV16E7 in the 9 th, 10 th, 11 th and 12 th columns, 100 mu l/hole, 1h at 37 ℃, washing the plate for 4 times by PBST, and 3min each time;

(4) adding a secondary antibody: anti-mouse IgG-HRP, cat # 7076S from CST corporation, was diluted at 1:3000, 100. mu.l/well, 45min at 37 ℃ and washed 4 times for 3min each with PBST;

(6) and (3) terminating the reaction: plus 2MH₂SO₄50 μ l/well;

(7) determination of OD_450nmAnd analyzing the data.

B. Indirect competitive ELISA method of overlapping peptide at 1 mu mol/L for preliminary screening dominant linear B cell epitope of monoclonal antibody 69E2

(1) Envelope antigen HPV16E7 holoprotein: OD in the selection chessboard experiment_450nmHPV16E7 holoprotein was coated at an antigen to antibody ratio of 0.7-0.8. The coating concentration of the antigen HPV16E7 protein for the monoclonal antibody 69E2 was 0.25. mu.g/ml, the concentration of the corresponding monoclonal antibody 69E2 reacted with the overlapping peptide was 0.125. mu.g/ml, 100. mu.l/well, and the plate was washed 4 times with PBST on the second day overnight at 4 ℃ for 3 minutes each time;

(2) and (3) sealing: preparing 1.0% BSA with PBST, 250. mu.l/well, washing the plate 3 times with PBST at 37 ℃ for 2h, 3 minutes each time;

(3) mixture of plus primary antibody and overlapping peptide reaction: respectively preparing 2 mu g/ml of monoclonal antibody 69E2, preparing 2 mu mol/L of 15 overlapping peptides, preparing 2mg/ml of BSA, respectively mixing the monoclonal antibody with the overlapping peptides, the BSA and the PBST in a ratio of 1:1, carrying out shake reaction at 25 ℃ for 30 minutes, adding the mixture into a plate, washing the plate for 4 times with the PBST at 100 mu L/hole for 1 hour at 37 ℃, and washing the plate for 3 minutes each time;

(4) adding a secondary antibody: anti-mouse IgG-HRP, cat # 7076S from CST corporation, was diluted at 1:3000, 100. mu.l/well, 45 minutes at 37 ℃ and washed 4 times for 3 minutes each with PBST;

(5) adding a TMB color development liquid: diluting solution A and solution B in a TMB two-component color development kit of Beijing Solaibao PR1210 at a ratio of 1:1, 100 mul/hole, and 5-8 minutes at 37 ℃;

(6) and (3) terminating the reaction: plus 2MH₂SO₄50 μ l/well;

(7) determination of OD_450nmAnd analyzing the data.

C. Indirect competitive ELISA method of overlapping peptide at 10 mu mol/L for primary screening of dominant linear B cell epitope of monoclonal antibody 69E2

Because the OD of the overlapping peptide was inhibited by mAb 69E2 at 1. mu. mol/L_450nmOD not reaching secondary antibody control_450nmTherefore, it is necessary to increase the concentration of the overlapping peptide to 10. mu. mol/L. One difference in the method from the indirect competitive ELISA method with overlapping peptides at 1. mu. mol/L is the addition of a mixture of primary antibody and overlapping peptide: monoclonal antibody 69E2 was formulated at 0.25. mu.g/ml, and several overlapping peptides that were inhibited by the monoclonal antibody at 1. mu. mol/L and 1-2 overlapping peptides that were not inhibited by the monoclonal antibody were selected as negative controls, and BSA as positive controls. Several overlapping peptides were made to 20. mu. mol/L, BSA was made to 20mg/ml, single antibody was mixed with overlapping peptide, BSA, PBST at 1:1 ratio, respectively, reacted with shaking at 25 ℃ for 30min and added to the plate, and the rest steps were the same as overlapping peptides at 1. mu. mol/L.

The indirect competition ELISA screens the peptide fragments containing the amino acid with strong hydrophilicity, and the results are shown as B in FIG. 12. The results of the indirect competitive ELISA method were determined by the inhibition rate method. Inhibition rate (OD of negative control BSA)_450nmvalue-OD of inhibited peptide_450nmValue) × 100%/OD of negative control BSA_450nmThe inhibition rate is more than or equal to 50 percent, and the inhibition rate is less than 50 percent, and the inhibition rate is negative. The result shows that the indirect competition ELISA method of the overlapping peptide at 10 mu mol/L shows that the monoclonal antibody 69E2 can inhibit the peptide fragment HPV16E7_67-84、HPV16E7_73-90And HPV16E7_85-98、HPV16E7_49-66There is a significant difference (P)<0.01)。

By combining the results of indirect ELISA and indirect competition ELISA, monoclonal antibody 69E2 and 4 overlapping peptide fragments HPV16E7 can be found_67-84、HPV16E7_73-90And HPV16E7_85-98、HPV16E7_49-66There is specific binding, but it is not clear which amino acids make up a particular epitope. Although the three-dimensional crystal structure of the HPV16E7 protein is not searched at present, the homology of the amino acid sequence of the HPV16E7 protein and the amino acid sequence of the HPV45E7 protein is 46 percent, the HPV45E7 protein has the three-dimensional crystal structure and is modeled by SWISS-MODEL homology,obtaining a three-dimensional crystal structure model diagram of the 46 th to 97 th amino acids of HPV16E7 by taking the HPV45 protein as a model. A three-dimensional crystal structure construction pattern of The HPV16E7 protein is presented by PyMOL (The PyMOL Molecular Graphics System2.2.0), only The 46 th-97 th aa structure of The HPV16E7 protein is analyzed at present, two monomers of The HPV16E7 protein are connected into a dimer through a zinc binding structural domain, The dimer forms a double-rotation symmetry axis, and The secondary structure is mainly alpha helix. The secondary structure of HPV16E7 protein was indeed alpha-helical as determined by circular dichroism.

2 peptide fragments HPV16E7 are found from a three-dimensional crystal structure construction model of HPV16E7 protein_67-72And HPV16E7_86-90Internal, 3 peptide fragments HPV16E7_49-66、HPV16E7_73-85And HPV16E7_91-97The exposed peptide segment is the position where the epitope is located when exposed, discontinuous and adjacent to the position, the 3 exposed peptide segments are discontinuous, which indicates that the epitope of the monoclonal antibody 69E2 is a conformational epitope, the titer of the monoclonal antibody 69E2 is high, the affinity is strong, and indicates that the epitope of the monoclonal antibody 69E2 is also a rigid epitope.

Marking peptides which are reacted by the monoclonal antibody in the indirect ELISA and peptides which are shown to be remarkably inhibited by the indirect competition ELISA in a three-dimensional crystal structure construction pattern of HPV16E7 protein, wherein peptide fragments marked as red are peptide fragment parts which are specifically combined by the monoclonal antibody 69E 2; mab 69E2 and mab 79a11 were paired, so the specific exposed epitope of paired mab 79a11 was also labeled with blue (C in fig. 12). The results of the pairing of the monoclonal antibody 69E2 and the monoclonal antibody 79A11 show that the monoclonal antibody 69E2 and the monoclonal antibody 79A11 respectively react with epitopes of two different monomers of the same HPV16E7 protein, so that the pairing can be carried out without conflict.

4. Conservation analysis of the specific epitope of mAb 69E2

The specific epitope of mab 69E2 is 3 exposed peptide fragments: HPV16E7_49-66、HPV16E7_73-85And HPV16E7_91-97. The amino acid sequences of these 3 exposed peptide stretches were aligned with the amino acid sequence of the HPV16E7 protein in an additional 30 HPV16 strains downloaded from NCBI using software MEGA 7.0. The 3 exposed peptide fragments were found by conservative alignment (HPV16E 7)_49-66、HPV16E7_73-85And HPV16E7_91-97) With another 30 downloaded in NCBIThe amino acid homology of HPV16E7 protein in the strain HPV16 strain is high, which indicates that the conservation is high, and the monoclonal antibody 69E2 has potential broad-spectrum application value (figure 13).

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

<110> Chongqing university of science and technology

<120> HPV16E7 resistant protein monoclonal antibody 69E2, hybridoma cell strain, and preparation method and application thereof

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Glu Gln Phe Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro

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Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe

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Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His

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Pro Gly Val Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe

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Thr Asp Tyr Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu

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Glu Trp Ile Gly Val Ile Ser Thr Tyr Asn Gly Asn Thr Asn Tyr Asn

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Gln Lys Phe Lys Gly Lys Ala Thr Val Thr Val Asp Lys Ser Ser Ser

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Thr Ala Tyr Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile

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Tyr Tyr Cys Ala Arg Arg Gly Phe Gly Asn Ser Trp Phe Ala Tyr Trp

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Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Ala Pro

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Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser

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Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr

165 170 175

Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val

195 200 205

Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His

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Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro

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Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu

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Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu

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Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser

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Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu

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Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr

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Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser

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Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro

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Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln

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Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val

370 375 380

Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val

385 390 395 400

Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu

405 410 415

Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg

420 425 430

Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val

435 440 445

Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg

450 455 460

Thr Pro Gly Lys

465

<210> 7

<211> 708

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt catgatgtcc 60

agaggagaaa atgttctcac ccagtctcca gcaatcatgt ctgcatctcc agggggaaag 120

gtcaccatga cctgcagtgc cagctcaggt gtgagttaca tgcactggta ccagcagaag 180

tcaagcacct cccccaaact ctggatttat gacacatcca acctggcttc tggagtccca 240

ggtcgcttca gtggcagtgg gtctggaaac tcttactctc tcacgatcag cagcatggag 300

gctgaagatg ttgccactta ttactgtttt caggggagtg ggtacccatt cacgttcggc 360

tcggggacaa agttggaaat tagacgggct gatgctgcac caactgtatc catcttccca 420

ccatccagtg agcagttaac atctggaggt gcctcagtcg tgtgcttctt gaacaacttc 480

taccccaaag acatcaatgt caagtggaag attgatggca gtgaacgaca aaatggcgtc 540

ctgaacagtt ggactgatca ggacagcaaa gacagcacct acagcatgag cagcaccctc 600

acgttgacca aggacgagta tgaacgacat aacagctata cctgtgaggc cactcacaag 660

acatcaactt cacccattgt caagagcttc aacaggaatg agtgttag 708

<210> 8

<211> 235

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Met Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Ala Ile

20 25 30

Met Ser Ala Ser Pro Gly Gly Lys Val Thr Met Thr Cys Ser Ala Ser

35 40 45

Ser Gly Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Ser Thr Ser

50 55 60

Pro Lys Leu Trp Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Val Pro

65 70 75 80

Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile

85 90 95

Ser Ser Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly

100 105 110

Ser Gly Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Arg

115 120 125

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

130 135 140

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

145 150 155 160

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

165 170 175

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

180 185 190

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

195 200 205

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

210 215 220

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210> 9

<211> 98

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln

1 5 10 15

Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser

20 25 30

Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp

35 40 45

Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr

50 55 60

Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu

65 70 75 80

Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln

85 90 95

Lys Pro

Claims

1. Monoclonal antibody 69E2 against HPV16E7 protein, characterized in that: the monoclonal antibody 69E2 comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6; the amino acid sequence of the light chain is shown as SEQ ID NO. 8.

2. Hybridoma cells secreting monoclonal antibody 69E2 against HPV16E7 protein, characterized in that: the hybridoma cells are preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2019181.

3. the use of the monoclonal antibody 69E2 against HPV16E7 protein according to claim 1 in the preparation of a kit for detecting HPV16E7 protein.

4. Use according to claim 3, characterized in that: the monoclonal antibody 69E2 in the kit is used as a detection antibody.

5. A kit comprising monoclonal antibody 69E2 against HPV16E7 protein of claim 1.

6. The kit of claim 5, wherein: the kit is an ELISA kit, an immunochemical kit, an immunofluorescence kit, a chemiluminescence detection kit or a Western Blot detection kit.