A specific antibody capable of identifying a cancer marker HPV E7 protein and application thereof

Abstract

The application provides a specific antibody capable of identifying cancer marker HPV E7 protein and application thereof. The application relates to a specific antibody capable of identifying cancer marker HPV E7 protein, which is used for detecting HPV E7 protein existing in pathological tissue paraffin sections by immunohistochemistry (IHC). The antibody can specifically combine with E7 proteins of HPV18, HPV31, HPV45, HPV51 and HPV56 types, effectively positions pathological tissue of cervical squamous cell carcinoma or adenocarcinoma, and improves the accuracy of pathological diagnosis. The antibody has high specificity and affinity, can provide precise detection for cervical cancer and other cancers or precancerous lesions caused by HPV infection, provides an effective detection technology for early diagnosis and targeted treatment of cancer, and is helpful for identification, grading and disease progression evaluation of tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and in vitro diagnosis, and particularly relates to a specific antibody capable of recognizing E7 protein marker in cervical cancer or other HPV-related cancer and application thereof. BACKGROUND

[0002] HPV is the main cause of cervical cancer, but HPV infection itself cannot directly cause cancer, only persistent high-risk HPV infection can cause precancerous lesions. More than 99% of cervical cancer cases worldwide are related to high-risk HPV. HPV is a widely existing virus that can infect the epidermis and mucosal squamous epithelium of the human body. It not only can survive on the skin, but also can cause infection on the mucosal tissues such as oral cavity, throat, genital organs, etc., and can cause various cancers such as cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer and oropharyngeal cancer. More than two hundred types of HPV have been identified, which are divided into high-risk and low-risk types according to the carcinogenic potential. The most common high-risk types include HPV16 / 18 / 31 / 33 / 35 / 45 / 52 / 58, etc., among which HPV16 and HPV18 types are particularly related to the development of cervical cancer.

[0003] Scientific research has proved the molecular mechanism of HPV virus carcinogenesis. High-risk HPV causes cancer in infected patients by integrating viral genes into human genes in the nucleus of the human body and producing E6 oncoprotein and E7 oncoprotein (oncoprotein, or simply "cancer protein"; "E6 or E7 oncoprotein", "E6 or E7 cancer protein", "E6 or E7 protein" in this patent have the same meaning, unless otherwise specified). The E6 and E7 proteins expressed in the host cell have transforming cell and carcinogenic activity. E6 and E7 proteins interfere with the normal regulatory mechanism of the cell, promoting the malignant transformation of the cell. E6 protein binds to the p53 protein of the host cell, leading to ubiquitination and degradation of p53, thereby inhibiting the tumor suppressor function of p53. p53 is usually activated when DNA is damaged, promoting cell cycle arrest or promoting apoptosis. The action of E6 protein eliminates the response of the cell to DNA damage, increasing the risk of genetic mutation and carcinogenesis. E7 protein binds to the pRB protein of the host cell, causing pRB to dissociate from the E2F transcription factor. pRB usually inhibits the activity of E2F, preventing the progress of the cell cycle.

[0004] E6 and E7 proteins are oncogenic factors driving the development and progression of cancer, and the amount of cells positively expressing them is positively correlated with the degree of lesions. E6 and E7 proteins cause cervical epithelial lesions by synergistic action. E7 protein acts as an early initiator, and its main function is to drive cell mutation, proliferation, and immortalization. E6 protein plays a more significant role in the later stages of lesions, and it interacts with intracellular proteins containing PDZ structures, further promoting the malignant development of lesion cells, and eventually evolving into invasive cancer. Therefore, detecting whether E6 or E7 protein is expressed in cervical cells may become an ideal cancer marker, providing effective clinical diagnosis and treatment monitoring.

[0005] Compared with traditional pathology, E7 protein marker-based immunohistochemistry (IHC) detection has the following advantages: 1. E7 protein, as a specific biomarker for identifying cervical precancerous lesions, has high specificity and sensitivity, and its detection results do not completely depend on the morphological structure of tissues or cells, reducing the dependence on the experience of pathologists and making the reading work more efficient; 2. E7 protein is a cancer protein expressed by HPV virus gene-integrated host genome and transformed host cells, and E7 protein expression and cell activity are prerequisites for cell transformation. Therefore, E7-IHC detection can early identify and discover HPV-induced cervical cancer and precancerous lesions, which is crucial for the early discovery and accurate diagnosis of cervical cancer and other HPV-related cancers; 3. E7-IHC results help develop personalized treatment plans. For patients with high E7 protein expression levels, it indicates a high risk of cancer development, and more aggressive treatment strategies are needed. Doctors can evaluate the treatment effect by comparing the E7 protein expression levels before and after treatment, and provide individualized treatment plans.

[0006] Thinprep Cytologic Test (TCT) is a cervical cytology test technique commonly used for cervical cancer screening. It collects the exfoliated cells from the cervix, and performs cytological analysis to determine whether there is a lesion or cancer in the cervical area. Compared with traditional Pap smear, TCT technology improves the specimen satisfaction and the detection rate of cervical abnormal cells, and can detect some precancerous lesions and microbial infections such as mold, trichomonas, virus, chlamydia, etc. TCT report usually includes specimen satisfaction, cell count, microbiological test results and cytopathology report. The report results may include "no cervical intraepithelial lesion (NILM)", "atypical squamous cells of undetermined significance (ASC-US)", "low-grade squamous intraepithelial lesion (LSIL)", "high-grade squamous intraepithelial lesion (HSIL)", "squamous cell carcinoma (CC)", and "atypical glandular cells (AGC)", etc. These results help doctors judge the cervical health status and give further diagnosis and treatment recommendations. However, TCT as a primary screening test has certain limitations. First of all, it is subjective, influenced by the level and experience of the doctor, and different doctors' diagnosis results may differ, so it has certain requirements for the professional level and experience of the doctor. In addition, TCT examination can only reflect the current situation of cervical lesions, but cannot predict the future risk of canceration.

[0007] HPV molecular detection technology is usually used to detect the DNA of the pathogen HPV virus. The technical principle is generally based on PCR technology to amplify different types of HPV (i.e. HPV typing detection), or use Hybrid Capture technology. For example: HC2 detection, the second generation of Hybrid Capture technology, is a commonly used HPV virus DNA detection method for cervical cancer screening. It qualitatively detects the DNA sequence of HPV virus in cervical samples through signal amplification and chemiluminescence technology. HC2 technology detects whether there is HPV virus DNA in the sample by combining with specific DNA probes. Compared with cytology examination which depends on the subjective judgment of doctors, HC2 detection is performed by instruments, and the results are more objective and consistent. Although HC2 technology can detect HPV infection, it cannot determine the specific HPV type. For clinical management, it is very important to know the specific HPV type, because different types of HPV have different risks of cervical cancer. For example, HPV16 and HPV18 types have the highest risk of cervical cancer, while some other types, although also belong to high-risk types, may have lower risk. This limits the application and guiding significance of HC2 in clinical to some extent. Therefore, HC2 detection is usually used for preliminary screening, and if the result is positive, further detection may be needed to determine the specific HPV type (i.e. HPV typing detection). SUMMARY

[0008] The present application provides a specific antibody capable of recognizing cancer marker HPV E7 protein and application thereof to solve the above-mentioned defects in the prior screening and diagnostic detection technology.

[0009] The technical solutions of the present application are as follows:

[0010] The present application provides a specific antibody capable of recognizing cancer marker HPV E7 protein, which comprises a heavy chain variable region; the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 with the amino acid sequences shown in SEQ ID NO. 1-3.

[0011] Further, the heavy chain variable region further comprises a heavy chain framework region, and the structure of the antibody heavy chain is VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4, and the amino acid sequences of VHFR1, VHFR2, VHFR3 and VHFR4 are shown in SEQ ID NO. 4-7.

[0012] Further, the heavy chain variable region further comprises a heavy chain signal peptide, and the amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO. 8.

[0013] The DNA sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 17.

[0014] The present application also provides a specific antibody capable of recognizing cancer marker HPV E7 protein, which comprises a light chain variable region; the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3 with the amino acid sequences shown in SEQ ID NO. 9-11.

[0015] Further, the light chain variable region further comprises a light chain framework region, and the structure of the antibody light chain is VLFR1-VLCDR1-VLFR2-VLCDR2-VLFR3-VLCDR3-VLFR4, and the amino acid sequences of VLFR1, VLFR2, VLFR3 and VLFR4 are shown in SEQ ID NO. 12-15.

[0016] Further, the light chain variable region further comprises a light chain signal peptide, and the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO. 16.

[0017] The DNA sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 18.

[0018] The application also provides an antibody detection reagent for the HPV E7 protein, comprising the specific antibody that can recognize the cancer marker HPV E7 protein.

[0019] Further, the detection method of the antibody detection reagent is an immunohistochemical method.

[0020] Further, the antibody detection reagent is applied to the preparation of a product for detecting the HPV E7 protein in vitro.

[0021] The application also provides the application of the specific antibody that can recognize the cancer marker HPV E7 protein in the detection of the HPV E7 protein in non-disease diagnosis.

[0022] The application has the following prominent effects:

[0023] The application relates to a specific antibody that can recognize a cancer marker HPV E7 protein, which is used for detecting the HPV E7 protein existing in pathological tissue paraffin sections by an immunohistochemical method (IHC). The antibody can specifically combine with the E7 proteins of HPV18, HPV31, HPV45, HPV51 and HPV56 types, effectively locates the diseased tissue of cervical squamous cell carcinoma or adenocarcinoma, and improves the accuracy of pathological diagnosis. The antibody has high specificity and affinity, can provide precise detection for cervical cancer and other cancers or precancerous lesions caused by HPV infection, provides an effective detection technology for early diagnosis and targeted treatment of cancer, and is helpful for the identification, grading and disease progression evaluation of tumors.

[0024] The specific embodiment of the application is further described in detail below in combination with examples, so that the technical scheme of the application is easier to understand and master. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The figure is an ELISA detection result diagram of the B cell clone antibody of the embodiment 1 of the application combining different types of E7 proteins;

[0026] Figure 2 The figure is a staining mode result diagram of the antibody clone of the embodiment 2 of the application for IHC detection of HPV 16 and HPV 18 cervical cancer tissues;

[0027] Figures 3-23 The figure is an IHC detection result and staining mode diagram of the antibody clone of the embodiment 3 of the application for 21 samples of different HPV types (including cervical cancer and precancerous lesion tissues of cervical cancer). DETAILED DESCRIPTION

[0028] The present invention provides a specific antibody (antibody clone 35B10) that can recognize the cancer marker HPV E7 protein, wherein the antibody comprises a heavy chain variable region; the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO.1-3.

[0029] Furthermore, the heavy chain variable region also includes a heavy chain framework region, and the structure of the antibody heavy chain is: VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4, and the amino acid sequences of VHFR1, VHFR2, VHFR3 and VHFR4 are shown in SEQ ID NO.4-7.

[0030] Furthermore, the heavy chain variable region also includes a heavy chain signal peptide, and the amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO.8.

[0031] The DNA sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.17.

[0032] The present invention also provides a specific antibody (antibody clone 35B10) that can recognize the cancer marker HPV E7 protein, wherein the antibody comprises a light chain variable region; the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs. 9-11.

[0033] Furthermore, the light chain variable region also includes a light chain framework region, and the structure of the antibody light chain is: VLFR1-VLCDR1-VLFR2-VLCDR2-VLFR3-VLCDR3-VLFR4, and the amino acid sequences of VLFR1, VLFR2, VLFR3 and VLFR4 are shown in SEQ ID NO.12-15.

[0034] Furthermore, the light chain variable region also includes a light chain signal peptide, and the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO.16.

[0035] The DNA sequence of the light chain variable region of the antibody is shown in SEQ ID NO.18.

[0036] The present invention also provides an antibody detection reagent for HPV E7 protein, comprising the above-mentioned specific antibody (antibody clone 35B10) that can recognize the cancer marker HPV E7 protein.

[0037] Furthermore, the detection method of the antibody detection reagent is immunohistochemistry.

[0038] Further, the application of the antibody detection reagent in the preparation of a product for detecting HPV E7 protein in vitro.

[0039] The application also provides a specific antibody (antibody clone 35B10) capable of recognizing the cancer marker HPV E7 protein in the detection of HPV E7 protein for non-disease diagnosis.

[0040] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments.

[0041] Embodiment 1: Preparation of a monoclonal antibody

[0042] The monoclonal antibody (antibody clone 35B10) of the application can be prepared by a rabbit-derived monoclonal antibody technology platform. Rabbits immunized with special antigen proteins produce immune cells B cells in vivo, which are subjected to cell separation, culture, screening, and identification of B cell clones secreting specific antibody protein IgG. The specific test steps include: immunizing rabbits, extracting B cells from the peripheral blood of the rabbits, and screening a large number of B cell clones to finally obtain an antibody clone with the best binding antigen affinity and specificity. Then, through gene cloning technology, the gene sequences of the heavy chain and light chain of the immunoglobulin IgG are obtained. Through gene sequence analysis, the DNA sequences of the antibody heavy chain and light chain variable region (Complementarity Determining Region, CDR) are obtained. An antibody expression vector constructed by genetic engineering is used to express the antibody by transfecting HEK293 cells or to construct a CHO-S stable cell line, and the antibody secreted in the culture medium is purified by affinity chromatography column (Protein A / G-Sephrose) to obtain immunoglobulin IgG protein reagent.

[0043] The preferred experimental technical route of the application is as follows: a rabbit-derived monoclonal antibody with specific binding to E7 proteins of HPV types 18, 31, 45, 51, and 56 is obtained by using the rabbit immunization step, combining single B cell separation and screening, and molecular cloning technology. In a preferred scheme of the application, the monoclonal antibody is prepared by the Single B Cell cloning method. The B cells are obtained by collecting peripheral blood of rabbits to separate peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC), and the B cells are subjected to single cell level separation and amplification culture, and the cell culture supernatant (containing secreted IgG) is analyzed and screened, so as to accurately and efficiently screen out B cells (A. Winters et al., 2019) secreting target antibody molecules.

[0044] 1.1 Antigen design

[0045] HPV16 / 18 E7 full-length sequences were inserted into prokaryotic expression vector pGEX-4T-1 by genetic engineering, so that HPV16 / 18 E7 protein was constructed into fusion protein with 6His, MBP (Maltose binding protein), GST (Glutathione S-transferase), SUMO (Small Ubiquitin-like Modifier), or Flag tag. After activation of the plasmid, the prokaryotic E. coli was transformed and expressed after low-temperature induction by IPTG. The bacteria solution was ultrasonically lysed, and the protein was purified and identified for concentration and purity.

[0046] 1.2 Animal immunization

[0047] Immunization: The rabbits were immunized with purified recombinant protein HPV16 E7 or HPV18 E7. The dose of antigen protein and interval time for each injection are shown in Table 1:

[0048] Table 1 Schedule of animal immunization

[0049]

[0050] 1.3 B cell separation and clone screening

[0051] 15ml to 30ml of fresh peripheral blood was collected from 2 rabbits using a sterile tube. The antigen GST-E7 fusion protein was used to coat the plate, and the specific B cells were enriched by binding of the B cells expressing membrane antibodies to the E7 antigen protein. The enriched cells were plated into a 96-well cell culture plate with feeder cells at a certain cell density. The plate was incubated at 37°C in 5% CO2. After 6 days of incubation, the fresh culture medium was replaced, and the supernatant was collected after overnight culture on the 7th day. The ELISA binding described below was used to test the presence of antibodies against E7 protein.

[0052] 1.4 ELISA detection of B cell supernatant clones

[0053] The antigens are respectively selected from HIS-HPV16 E7 and MBP-HPV18 E7 two kinds of recombinant proteins, and a blank control (coated with the antigen, and added with a blank antibody diluent) is set. 1 μg / ml is coated, and is left overnight at 4°C. After being washed with PBST and dried, 5% skimmed milk powder is added for blocking, and is left for 2 hours at room temperature. After being washed with PBST and dried, B cell supernatant (diluted according to 1:4) is added, and is left for 1 hour at 37°C. After being washed with PBST and dried, goat anti-rabbit HRP secondary antibody (Sigma A0545) (1:2000) is added, and is left for 30 minutes at 37°C. After being washed with PBST and dried, TMB solution is added, and is left for 15 minutes at 37°C. After being terminated by 2M H2SO4, the color is read at an enzyme label OD450nm. The antibody clone of the application is used to detect the supernatant secreted by the B cells, and the B cell clone with the specific binding E7 antigen protein activity is screened. The ELISA plate coating antigens include: HPV16 E7 (containing His-tag), HPV18 E7 (containing MBP tag), and HPV18 E7 (containing SUMO tag). For example, as shown in Table 1, by screening thousands of B cell clones, at least 15 candidate clones are found to bind to HPV18 E7 protein in the ELISA detection. Figure 1

[0054] 1.5 Sequencing of the variable region of the monoclonal antibody

[0055] The RNA of the total cells in the well with an ELISA detection OD value greater than 1.0 is extracted by using an RNA extraction reagent (Novagen, R701-01 / 02), and is reversely transcribed into cDNA by using a universal primer (Prime ScriptTM1stStrand cDNA Synthesis Kit, Takara). Subsequently, the rabbit immunoglobulin heavy chain and light chain V-region fragments are amplified by using antibody signal peptide and constant region specific primers, and the obtained PCR fragments are homologously recombined into a pCDNA3.4 vector, and the inserted fragments are sequenced by using vector specific primers. Finally, the unique V-region protein amino acid sequence of the clone and the plasmid are obtained.

[0056] 1.6 Expression and purification of the recombinant monoclonal antibody protein

[0057] ​The immunoglobulin gene variable region V region sequence is cloned into a mammalian expression vector of IgG heavy chain HC and light chain framework for expression, and then large-scale transient transfection CHO cells (Expi-Fectamine CHO Transfection Kit, Gibco) are carried out. After 48 h of cell culture, the supernatant culture medium containing secreted immunoglobulin IgG is collected. The antibody is purified by Protein A / G affinity chromatography, the protein peak effluent is collected, the antibody protein concentration is determined by ultraviolet spectrophotometer OD260 / OD280 after dialysis with phosphate buffer (PBS), and the antibody titer is detected by indirect ELISA.

[0058] Example 2: Monoclonal antibody IHC identification

[0059] The performance of the antibody reagent is identified by immunohistochemical staining method, and the specific test steps include: the pathological paraffin section is soaked in xylene twice, each time for 10 min; then sequentially soaked in 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, each for 5 min, and finally washed with deionized water twice; the tissue section is placed in a boiling EDTA buffer solution (pH 9.0) for 20 min; cooled at room temperature for 30 min, washed with PBST three times, each for 3 min; in order to inactivate endogenous peroxidase, 3% H2O2 is added and treated at room temperature for 10 min; washed with PBST for 3 times, each for 3 min; the above-mentioned 7 antibodies are added respectively, and incubated at room temperature for 1 h; after washing thoroughly, the immunochromatographic reagent is added dropwise, and incubated at room temperature for 30 min; after washing thoroughly, DAB staining solution is developed for 6 min (observed under a microscope), and immersed in pure water for 3 min; stained with hematoxylin for 1 min, and then washed with tap water and returned to blue; dehydrated step by step, sequentially immersed in 70% ethanol, 80% ethanol, 95% ethanol and anhydrous ethanol for 5 min each time; finally immersed in xylene twice, each time for 5 min; sealed with neutral gum; and finally observed under a microscope and photographed.

[0060] Among the 10 clones of the 18 type E7 antibody with ELISA activity identified by IHC detection, the antibody clones 7C4 and 35B10 of the present application have the best performance on pathological group staining, and can specifically stain cervical epithelial cancer lesion tissues, and do not show staining on normal tissues and chronic inflammation tissues. As shown in Figure 2 , the antibody clone 35B10 can specifically recognize E7 protein in HPV18 type positive cervical cancer tumor, and has little non-specific staining. The antibody clone 35B10 does not stain HPV16 type tumor tissues, which shows the specificity of the antibody to HPV18 type E7 protein.

[0061] Example 3: IHC detection of tissue samples

[0062] To further analyze the antibody clone's ability to bind to the E7 protein of other HPV types, with the approval of the hospital's ethics committee, the antibody clone was tested on paraffin sections of tumors positive for different HPV types by IHC testing. The experimental specimens were derived from paraffin sections of clinical retrospective samples, including cervical cancer pathology sections and cervical precancerous lesion biopsy tissue pathology sections. The method for detecting pathological sections using antibody clone 35B10 was consistent with that described in Example 2. The experimental results showed that antibody clone 35B10 could specifically bind to tumors positive for HPV18, HPV31, HPV45, HPV51, and HPV56 (Tables 2 and 3, Figures 3-19 、 Figures 21-23 Results for HPV18, HPV45, HPV51, and HPV56 are shown; Figure 20 Results for HPV31 are shown.) These types have high homology (similar protein structures) to HPV18 in their gene sequences.

[0063] Table 2 summarizes representative IHC data. Antibody staining reveals that E7 protein expression varies across biopsy sites and HPV types. This may be related to the nature of the lesion (chronic inflammation vs. HPV-transfected carcinoma), the integration of the HPV oncogene E7 into host cells, and the amount of protein expression. For example, samples B165, B436, and B311 showed positive E7 protein expression at most sites, while other samples, such as B068, B231, B381, and B196, showed negative expression at all sites. This discrepancy may indicate varying lesion severity or progression to cancer. E7 antibody IHC testing can provide additional information on lesion nature and risk, enabling more accurate lesion assessment and a deeper understanding of the role of E7 protein in lesions or cancer progression caused by different HPV types, thus providing a scientific basis for clinical diagnosis and treatment.

[0064] Table 2 IHC test sample results

[0065]

[0066] As shown in Table 3, the results of histopathological histochemistry ("E7-IHC") analysis using antibody clone 35B10 do not fully align with those of traditional pathology. For example, samples B165 and B433 had negative / normal pathology results but positive E7-IHC results, suggesting a tendency toward HPV-induced carcinogenesis and progression. Scientific research has demonstrated that E7 protein expression is essential for cancer development and progression. Similarly, tissue sections from samples B231, B381, and B196 showed CIN1 pathology results, but negative E7-IHC results. These data demonstrate that IHC analysis of E7 protein staining can provide information not available through traditional pathology testing, helping to identify samples with ambiguous pathology or requiring further evaluation, thereby more accurately assessing the nature and risk of lesions.

[0067] Table 3 Comparison between E7 antibody immunostaining and traditional pathology

[0068]

[0069] like Figures 3-20 As shown in the figure, antibody clone 35B10 can specifically bind to the E7 protein in cervical squamous cell carcinoma tumor tissue and show a brown-yellow color. It has less nonspecific staining on non-tumor cells and areas in the sections and can effectively identify cervical squamous cell carcinoma caused by HPV18, 31, 45, 51, and 56. The test results are basically consistent with the pathological diagnosis results, such as Figure 4 、 5 As shown in Figure 2, strong E7-IHC staining was observed at points B436-4 and B436-10 in CIN2 grade samples; Figure 11 As shown in Figure 2, strong E7-IHC staining was observed in sample 201918067 C at the CIN3 level; Figures 12-20 As shown in the figure, all 9 cervical squamous cell carcinoma samples showed squamous-specific brown staining of cancerous cells.

[0070] Cervical adenocarcinoma has the characteristics of deep hidden lesions, multifocality and skipping growth, such as Figures 21-23 As shown in the figure, all the cancerous cells in the cervical adenocarcinoma samples stained with antibody 35B10 showed round or oval-shaped adenocarcinoma-specific staining characteristics.

[0071] However, there are also cases where the E7-IHC test results are inconsistent with the traditional pathological diagnosis results, such as Figure 3 、 6, 7, 8, 9, 10, strong E7-IHC staining is observed at B165-10, B234-8, B311-4, B311-8, B311-12, B299-12 points of sample B at CIN1 level, which indicates that even if the pathological morphology is diagnosed as "polyp" or low-grade lesion CIN1 level, the E7 protein still has strong expression, which may indicate that the lesion of the case is in an active state, and the patient's cancer development risk is increased. Therefore, the E7-IHC using antibody clone 35B10 can obtain more accurate pathological diagnosis results, and can be used as an important indicator for evaluating the risk of cervical cancer. According to the principle of the cervical cancer diagnosis and treatment guidelines in China, low-grade lesions (CIN1) do not need treatment in principle, and clinical observation can be performed. For high-grade lesions (CIN2 or CIN3), active treatment intervention should be taken to avoid progression to invasive or advanced cervical cancer. The E7-IHC detection result using the antibody clone 35B10 of the application can prevent over-diagnosis and treatment of patients and effectively reduce the missed detection rate of high-risk patients.

[0072] The above is only the preferred embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can make equivalent replacement or change according to the technical scheme and the inventive concept of the present application within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A specific antibody that can recognize the cancer marker HPV E7 protein, characterized by: The antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes VHCDR1, VHCDR2 and VHCDR3 as shown in the amino acid sequences of SEQ ID NOs.1-3; the light chain variable region includes VLCDR1, VLCDR2 and VLCDR3 as shown in the amino acid sequences of SEQ ID NOs.9-11.

2. The antibody according to claim 1, characterized in that: The heavy chain variable region also includes a heavy chain framework region. The structure of the antibody heavy chain is: VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4. The amino acid sequences of VHFR1, VHFR2, VHFR3 and VHFR4 are shown in SEQ ID NOs. 4-7.

3. The antibody according to claim 2, characterized in that: The heavy chain variable region further includes a heavy chain signal peptide, and the amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO.

8.

4. The antibody according to claim 1, wherein: The light chain variable region also includes a light chain framework region. The structure of the antibody light chain is: VLFR1-VLCDR1-VLFR2-VLCDR2-VLFR3-VLCDR3-VLFR4, and the amino acid sequences of VLFR1, VLFR2, VLFR3 and VLFR4 are shown in SEQ ID NO.12-15.

5. The antibody according to claim 4, characterized in that: The light chain variable region further includes a light chain signal peptide, and the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO.

16.

6. An antibody detection reagent for HPV E7 protein, characterized in that: The invention comprises the specific antibody capable of recognizing the cancer marker HPV E7 protein according to any one of items 1 to 5.

7. The antibody detection reagent according to claim 6, characterized in that: The detection method of the antibody detection reagent is immunohistochemistry.

8. The antibody detection reagent according to claim 6, characterized in that: The antibody detection reagent is used in preparing a product for in vitro detection of HPV E7 protein.

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