Specific antibody capable of recognizing cancer marker HPV E7 protein and application thereof

Abstract

The invention provides a specific antibody capable of recognizing a cancer marker HPV E7 protein and application of the specific antibody. The invention relates to a specific antibody capable of recognizing a cancer marker HPV (human papillomavirus) E7 protein, which is used for detecting the HPV E7 protein existing in a pathological tissue paraffin section by using an IHC (immunohistochemical) method. The antibody can be specifically combined with E7 proteins of HPV18, HPV31, HPV45, HPV51 and HPV56 types, pathological tissues of cervical squamous cell carcinoma or adenocarcinoma are effectively positioned, and the accuracy of pathological diagnosis is improved. The antibody has high specificity and affinity, can provide accurate detection for cancers or precancerous lesions caused by cervical cancer and other HPV infection, provides an effective detection technology for early diagnosis and targeted therapy of cancers, and is beneficial to identification and grading of tumors and evaluation of disease progression.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and in vitro diagnostic technology, and specifically relates to a specific antibody capable of identifying an E7 protein marker in cervical cancer or other HPV-related cancers and an application thereof. Background Art

[0003] 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 can not only survive on the skin, but also cause infection in mucosal tissues such as the mouth, throat, and genitals, and can cause cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, and oropharyngeal cancer. More than two hundred types of HPV have been identified, and they are divided into high-risk and low-risk types according to their carcinogenic potential. The most common high-risk types include HPV16 / 18 / 31 / 33 / 35 / 45 / 52 / 58, among which HPV16 and HPV18 are particularly related to the development of cervical cancer.

[0004] Scientific research has proven the molecular mechanism of HPV carcinogenesis. High-risk HPV causes cancer in infected patients by integrating viral genes into human genes in the nucleus of human cells and producing E6 oncoprotein and E7 oncoprotein (oncoprotein, or "cancer protein" for short; "E6 or E7 oncoprotein", "E6 or E7 oncoprotein", and "E6 or E7 protein" in this patent have the same meaning unless otherwise specified.) in infected human host cells. E6 and E7 proteins expressed in host cells have cell transformation and carcinogenic activities. E6 and E7 proteins promote malignant transformation of cells by interfering with the normal regulatory mechanism of cells. E6 protein binds to p53 protein of host cells, leading to ubiquitination and degradation of p53, thereby inhibiting the tumor suppressor function of p53. p53 is usually activated when DNA is damaged, causing cell cycle arrest or promoting cell apoptosis. This action of E6 protein eliminates the cell's response to DNA damage and increases the risk of gene mutation and cancer. The E7 protein binds to the host cell's pRB protein, causing pRB to dissociate from the E2F transcription factor. pRB usually inhibits the activity of E2F and prevents the cell cycle from progressing.

[0005] E6 and E7 proteins are carcinogenic factors that drive the occurrence and development of cancer, and the amount of cells expressing them positively correlates with the degree of lesions. E6 and E7 proteins cause cervical epithelial cell lesions through synergistic effects. As an early initiation factor, the main function of E7 protein is to drive cell mutation, proliferation and immortalization. The role of E6 protein is more significant in the later stage of lesions. It interacts with proteins containing PDZ structures in cells, further promoting the malignant development of diseased cells, and eventually evolving into invasive cancer. Therefore, detecting whether E6 or E7 proteins are expressed in cervical cells may become an ideal cancer marker, providing clinically effective diagnosis and treatment monitoring.

[0006] Compared with traditional pathology, the immunohistochemistry (IHC) test based on the E7 protein marker has multiple meanings: 1. As a specific biomarker for identifying cervical precancerous lesions, E7 protein has high specificity and sensitivity. 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 film reading work more efficient; 2. E7 protein is an oncoprotein expressed by transformed host cells after the HPV virus gene is integrated into the host genome. E7 protein expression and cell activity are prerequisites for cell transformation. Therefore, E7-IHC testing can identify and detect cervical cancer and precancerous lesions caused by HPV at an early stage, which is crucial for the early detection and accurate diagnosis of cervical cancer and other HPV-related cancers; 3. E7-IHC results are helpful in formulating personalized treatment plans. For patients with higher levels of E7 protein expression, it means that the risk of cancer development is high and more active treatment strategies are needed. Doctors can evaluate the treatment effect and provide personalized treatment plans by comparing the E7 protein expression levels of patients before and after treatment.

[0007] Thinprep Cytologic Test (TCT) is a cervical cytology examination technology, which is usually used for cervical cancer screening. It collects exfoliated cells from the cervical os and performs cytological analysis to determine whether there are lesions or cancer in the cervix. Compared with traditional Pap smears, TCT technology improves specimen satisfaction and the detection rate of abnormal cervical cells, and can detect some precancerous lesions and microbial infections in the early stages of cervical cancer, such as mold, Trichomonas, viruses, and Chlamydia. TCT reports usually include specimen satisfaction, cell count, microbiological examination results, and cytopathology reports. Report results may include "no cervical intraepithelial lesions (NILM)", "atypical squamous cells of undetermined significance (ASC-US)", "low-grade cervical intraepithelial lesions (LSIL)", "high-grade cervical intraepithelial lesions (HSIL)", "squamous cell carcinoma (CC)" and "atypical glandular cells (AGC)". These results help doctors judge the health of the cervix and give further diagnosis and treatment recommendations. However, TCT has certain limitations as a preliminary screening test. First, the results are subjective. The results are affected by the doctor's operation level and experience. Different doctors may have different diagnostic results. Therefore, there are certain requirements for the doctor's professional level and experience. In addition, TCT examination can only reflect whether there is a lesion in the cervix at present, but cannot predict the risk of future cancer.

[0008] 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 using hybrid capture technology. For example: HC2 test, i.e. second-generation hybrid capture technology (Hybrid Capture 2), is a HPV virus DNA detection method commonly used 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 HPV virus DNA is present in the sample by combining with specific DNA probes. Compared with cytology, which relies on the subjective judgment of doctors, HC2 testing 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 of infection. For clinical management, it is very important to know the specific HPV type because different types of HPV are associated with different risks of cervical cancer. For example, HPV16 and HPV18 are associated with the highest risk of cervical cancer, while some other types, although also high-risk, may have lower risks. This limits the clinical application and guiding significance of HC2 to a certain extent. Therefore, HC2 testing is usually used for preliminary screening. If the result is positive, further testing may be required to determine the specific HPV type (i.e., HPV typing test). Summary of the invention

[0009] In view of the above-mentioned defects in existing screening and diagnostic detection technologies, the present invention provides a specific antibody capable of recognizing the cancer marker HPV E7 protein and its application.

[0010] The technical solution of the present invention is as follows:

[0011] The present invention provides a specific antibody capable of recognizing HPV E7 protein, a cancer marker, wherein the antibody comprises a heavy chain variable region; the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 having amino acid sequences as shown in SEQ ID NO.1-3.

[0012] 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.

[0013] 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.

[0014] The DNA sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.17.

[0015] The present invention also provides a specific antibody capable of recognizing HPV E7 protein, a cancer marker, wherein the antibody comprises a light chain variable region; the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3 having amino acid sequences as shown in SEQ ID NO.9-11.

[0016] 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.

[0017] 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.

[0018] The DNA sequence of the light chain variable region of the antibody is shown in SEQ ID NO.18.

[0019] The present invention also provides an antibody detection reagent for HPV E7 protein, comprising the above-mentioned specific antibody capable of recognizing HPV E7 protein, a cancer marker.

[0020] Furthermore, the detection method of the antibody detection reagent is immunohistochemistry.

[0021] Furthermore, the antibody detection reagent is used in the preparation of a product for in vitro detection of HPV E7 protein.

[0022] The present invention also provides a use of the above-mentioned specific antibody capable of identifying the cancer marker HPV E7 protein in non-disease diagnosis by detecting HPV E7 protein.

[0023] The outstanding effects of the present invention are:

[0024] The present invention relates to a specific antibody that can identify HPV E7 protein, a cancer marker, and uses immunohistochemistry (IHC) to detect HPV E7 protein present in paraffin sections of pathological tissue. The antibody can specifically bind to E7 proteins of HPV18, HPV31, HPV45, HPV51, and HPV56 types, effectively locate the pathological tissue of cervical squamous cell carcinoma or adenocarcinoma, and improve the accuracy of pathological diagnosis. The antibody has high specificity and affinity, and can provide accurate detection of cervical cancer and other cancers or precancerous lesions caused by HPV infection, providing an effective detection technology for early diagnosis and targeted treatment of cancer, and is helpful for tumor identification, grading, and evaluation of disease progression.

[0025] The specific implementation modes of the present invention are further described in detail below in conjunction with embodiments, so that the technical solutions of the present invention are easier to understand and grasp. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram showing the ELISA test results of the B cell clone antibody of Example 1 of the present invention binding to different types of E7 proteins;

[0027] Figure 2 This is a staining pattern result diagram of the antibody clone of Example 2 of the present invention for IHC detection of HPV 16 and HPV 18 cervical cancer tissues;

[0028] Figure 3-Figure 23 The IHC test results and staining pattern diagram of 21 samples of different HPV types (including cervical cancer and cervical precancerous lesion tissues) using the antibody clone of Example 3 of the present invention. DETAILED DESCRIPTION

[0029] 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 having amino acid sequences as shown in SEQ ID NO.1-3.

[0030] 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.

[0031] 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.

[0032] The DNA sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.17.

[0033] 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 having amino acid sequences as shown in SEQ ID NO.9-11.

[0034] 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.

[0035] 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.

[0036] The DNA sequence of the light chain variable region of the antibody is shown in SEQ ID NO.18.

[0037] The present invention also provides an antibody detection reagent for HPV E7 protein, comprising the above-mentioned specific antibody (antibody clone 35B10) capable of recognizing the cancer marker HPV E7 protein.

[0038] Furthermore, the detection method of the antibody detection reagent is immunohistochemistry.

[0039] Furthermore, the antibody detection reagent is used in the preparation of a product for in vitro detection of HPV E7 protein.

[0040] The present invention also provides a use of the above-mentioned specific antibody (antibody clone 35B10) capable of recognizing the cancer marker HPV E7 protein in non-disease diagnosis of detecting HPV E7 protein.

[0041] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0042] Example 1: Preparation of monoclonal antibodies

[0043] The monoclonal antibody (antibody clone 35B10) of the present invention can be prepared by a rabbit monoclonal antibody technology platform. Rabbits immunized with special antigen proteins are used to separate, culture, and screen the immune cells B cells produced in their bodies to identify B cell clones that secrete specific antibody proteins IgG. The specific experimental steps include: immunizing rabbits, extracting B cells from the peripheral blood of rabbits, and finally obtaining antibody clones with the best binding antigen affinity and specificity by screening a large number of B cell clones. Then, the gene sequences of the heavy chain and light chain of immunoglobulin IgG are obtained through gene cloning technology. Through gene sequence analysis, the DNA sequences of the variable regions (Complementarity Determining Region, CDR) of the heavy chain and light chain of the relevant antibodies are obtained. The antibody expression vector constructed by genetic engineering means is expressed by transiently transfecting HEK293 cells or constructing a CHO-S stable cell line, and then the antibodies secreted in the culture medium are purified by affinity chromatography columns (Protein A / G-Sephrose) to obtain immunoglobulin IgG protein reagents.

[0044] The preferred experimental technical route of the present invention: a rabbit immunization step is adopted, combined with single B cell separation and screening and molecular cloning technology, to obtain a rabbit monoclonal antibody with specific binding to E7 protein of HPV18, 31, 45, 51, 56 types. In a preferred embodiment of the present invention, the monoclonal antibody is prepared by the Single B Cell cloning method. B cells are obtained by collecting peripheral blood from rabbits for peripheral blood mononuclear cells (PBMC), isolating and amplifying B cells at the single cell level, and analyzing and screening the cell culture supernatant (containing secretory IgG), so as to accurately and efficiently screen out B cells that secrete target antibody molecules (A. Winters et al., 2019).

[0045] 1.1 Antigen design

[0046] The full-length sequence of HPV16 / 18 E7 was inserted into the prokaryotic expression vector pGEX-4T-1 by genetic engineering, so that the HPV16 / 18 E7 protein was constructed into a fusion protein with 6His, MBP (Maltose binding protein), GST (Glutathione S-transferase), SUMO (Small Ubiquitin-like Modifier), or Flag tag. After plasmid activation, prokaryotic Escherichia coli was transformed for expression. After low-temperature induction with IPTG, the bacterial liquid was ultrasonically lysed, the protein was purified, and the concentration and purity were identified.

[0047] 1.2 Animal immunization

[0048] Immunization: Rabbits were immunized with purified recombinant protein HPV16 E7 or HPV18 E7. The dosage and interval of each injection of antigen protein are shown in Table 1:

[0049] Table 1 Animal immunization schedule

[0050]

[0051] 1.3 B cell isolation and clone screening

[0052] 15ml to 30ml of fresh peripheral blood was collected from 2 rabbits using sterile tubes, and then the plate was coated with antigen GST-E7 fusion protein. The specific B cells were enriched by binding of B cells expressing extracellular antibodies to E7 antigen protein. The enriched cells were plated into a 96-well cell culture plate pre-plated with feeder cells according to a certain cell density. The plate was incubated at 37°C in 5% CO2. After 6 days of incubation, fresh culture medium was replaced, and the supernatant of overnight culture was collected on the 7th day, and the presence of antibodies against E7 protein was tested using the ELISA combination described below.

[0053] 1.4 ELISA detection of B cell supernatant clones

[0054] The antigens were selected as HIS-HPV16 E7 and MBP-HPV18 E7 recombinant proteins, which were coated separately, and a blank control (coated antigen, added with blank antibody diluent) was set up at the same time. The plate was coated with 1 μg / ml, overnight at 4°C, washed with PBST and patted dry, and then 5% skim milk powder was added for blocking at room temperature for 2 hours. After washing with PBST and patting dry, B cell supernatants (diluted at 1:4) were added respectively, reacted at 37°C for 1 hour, washed with PBST and patted dry, and then goat anti-rabbit HRP secondary antibodies (Sigma A0545) (1:2000) were added respectively, reacted at 37°C for 30 minutes, washed with PBST and patted dry, and then TMB solution was added. After reacting at 37°C for 15 minutes, 2M H2SO4 was used to stop the color development, and the reading was read at OD450nm of the microplate reader. The antibody clones of the present invention detect the supernatant secreted by B cells by the ELISA method to screen B cell clones with specific binding activity to the E7 antigen protein. ELISA plate antigens include: HPV16 E7 (with His-tag), HPV18 E7 (with MBP tag), HPV18 E7 (with SUMO tag). Figure 1 As shown, by screening thousands of B cell clones, at least 15 candidate clones were found to bind to HPV18 E7 protein in ELISA detection.

[0055] 1.5 Monoclonal antibody variable region sequencing

[0056] RNA extraction reagent (Novozyme, R701-01 / 02) was used to extract RNA from total cells in wells with ELISA detection OD values ​​greater than 1.0, and reverse transcribed into cDNA using universal primers (Prime ScriptTM1stStrand cDNA Synthesis Kit, Takara). Subsequently, rabbit immunoglobulin heavy and light chain V-region fragments were amplified by antibody signal peptide and constant region specific primers, and the resulting PCR fragments were homologously recombined into the pCDNA3.4 vector, and the insert fragments were sequenced using vector-specific primers. Finally, the cloned unique V-region protein amino acid sequence and plasmid were obtained.

[0057] 1.6 Expression and purification of recombinant monoclonal antibody protein

[0058] The variable region V region sequence of the immunoglobulin gene was cloned into the mammalian expression vector of the IgG heavy chain HC and light chain framework for expression, and then scaled up for transient transfection into CHO cells (Expi-Fectamine CHO Transfection Kit, Gibco). After 48 hours of cell culture, the supernatant containing secretory immunoglobulin IgG was collected. The antibody was purified by Protein A / G affinity chromatography, and the protein peak effluent was collected. After dialyzed with phosphate buffered saline (PBS), the antibody protein concentration was determined by UV spectrophotometer OD260 / OD280, and the antibody titer was detected by indirect ELISA.

[0059] Example 2: IHC identification of monoclonal antibodies

[0060] The performance of the antibody reagent was identified by immunohistochemical staining. The specific test steps included: soaking the pathological paraffin sections in xylene twice, each time for 10 minutes; then soaking them in 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol in turn, each for 5 minutes, and finally washing them with deionized water twice; placing the tissue sections in a boiling EDTA buffer solution (pH 9.0) and bathing them for 20 minutes; cooling at room temperature for 30 minutes, and washing them with PBST three times, each for 3 minutes; in order to inactivate endogenous peroxidase, adding 3% H2O2 to treat at room temperature 10min; wash with PBST 3 times, 3min each time; add the above 7 antibodies respectively, incubate at room temperature for 1h; after sufficient washing, add immunochromogenic reagent dropwise, incubate at room temperature for 30min; after sufficient washing, use DAB staining solution for color development for 6min (observe under a microscope), and immerse in pure water for 3min; counterstain with hematoxylin for 1min, rinse with tap water and return to blue; dehydrate in steps, immerse in 70% ethanol, 80% ethanol, 95% ethanol, and anhydrous ethanol for 5min each; finally immerse in xylene twice, 5min each time; seal the slides with neutral gum; finally observe and take pictures under a microscope.

[0061] After IHC testing and identification, among the 10 18-type E7 antibody clones with ELISA activity, the antibody clones 7C4 and 35B10 of the present invention have the best performance in pathological histochemical staining, and can specifically stain cervical epithelial cancer lesions, but do not stain normal tissues and chronic inflammatory tissues. Figure 2 As shown, antibody clone 35B10 can specifically recognize E7 protein in HPV18-positive cervical cancer tumors, with little non-specific staining. Antibody clone 35B10 did not stain HPV16 tumor tissues, showing the specificity of the antibody for HPV18 E7 protein.

[0062] Example 3: IHC detection of tissue samples

[0063] To further analyze the antibody clone binding to E7 proteins of other HPV types, with the approval of the hospital ethics committee, the antibody clone was tested for paraffin sections of tumors positive for different HPV types by IHC test. The experimental specimens were derived from paraffin sections of clinical retrospective samples, including cervical cancer pathological sections and cervical precancerous lesion biopsy tissue pathological sections. The method of using antibody clone 35B10 to detect pathological sections was consistent with that described in Example 2. The experimental results showed that antibody clone 35B10 can specifically bind to HPV18, HPV31, HPV45, HPV51, and HPV56-positive tumors (Table 2, Table 3, Figure 3-Figure 19 , Figure 21-23 Results for HPV18, HPV45, HPV51, and HPV56 are shown; Fig. 20 Results for HPV31 are shown). The gene sequences of these types have a high degree of homology (similar protein structure) with HPV18.

[0064] Table 2 summarizes representative IHC test data. Antibody staining shows that E7 protein expression varies in different biopsy site tissues and samples of different HPV types, which may be related to the nature of the lesion (chronic inflammation vs HPV transfection leading to carcinogenesis), the integration of HPV oncogene E7 into host cells, and the amount of protein expression. For example, in samples B165, B436, and B311, E7 protein expression was positive at most sites; while in other samples, such as B068, B231, B381, and B196, all sites were negative. This difference may indicate different degrees of lesions or have progressed to the stage of cancer. E7 antibody IHC testing can provide additional information about the nature and risk of lesions, help to more accurately evaluate lesions, and further understand the lesions or cancer progression caused by E7 protein in different HPV types, thereby providing a scientific basis for clinical diagnosis and treatment.

[0065] Table 2 IHC test sample results

[0066]

[0067] As can be seen from Table 3, the results of pathological histochemistry testing ("E7-IHC") of antibody clone 35B10 are not completely consistent with the results of traditional pathological testing. For example, the pathological results of samples B165 and B433 were negative / normal, but the E7-IHC results were positive, indicating the presence of HPV-induced carcinogenesis and deterioration trends, because scientific research has proven that the expression of E7 protein is a necessary condition for the occurrence and development of cancer. For another example, the pathological results of tissue sections of samples B231, B381, and B196 were CIN1, but the E7-IHC results were negative. These data show that IHC analysis of E7 protein marker staining can provide information that traditional pathological testing cannot provide, and can help identify samples with unclear pathological results or those that require further evaluation, thereby more accurately assessing the nature and risk of lesions.

[0068] Table 3 Comparison between E7 antibody immunostaining and traditional pathology

[0069]

[0070] like Figure 3-Figure 20 As shown in the figure, antibody clone 35B10 can present a brown-yellow color by specifically binding to the E7 protein in cervical squamous cell carcinoma tumor tissue, with less nonspecific staining of non-tumor cells and areas in the sections, and can effectively identify cervical squamous cell carcinoma caused by HPV18, 31, 45, 51, and 56 viruses. The test results are basically consistent with the pathological diagnosis results, such as Figure 4 , 5 As shown, strong E7-IHC staining was observed at points B436-4 and B436-10 in the CIN2 grade sample; Fig.11 As shown, stronger E7-IHC staining was observed in sample 201918067 C at the CIN3 level; Figure 12-Figure 20 As shown in the figure, cancerous cells in all 9 cervical squamous cell carcinoma samples showed squamous-specific brown staining.

[0071] Cervical adenocarcinoma has the characteristics of deep hidden lesions, multifocality and leapfrog growth, such as Figure 21-23 As shown, in the cervical adenocarcinoma samples stained with antibody 35B10, cancerous cells were observed to show round or oval-shaped specific adenocarcinoma staining characteristics.

[0072] However, there are also cases where the E7-IHC test results are inconsistent with the traditional pathological diagnosis results, such as Figure 3 , 6As shown in Figures 7, 8, 9, and 10, strong E7-IHC staining was observed at B165-10, B234-8, B311-4, B311-8, B311-12, and B299-12 in CIN1-level samples, indicating that even if the pathological morphology is diagnosed as "polyp" or low-grade lesion CIN1 level, E7 protein is still strongly expressed, which may indicate that the lesion in this case is in an active state and the patient's risk of cancer development is increased. Therefore, E7-IHC using antibody clone 35B10 can obtain more accurate pathological diagnosis results and can be used as an important indicator for assessing the risk of cervical cancer. According to the principles of the diagnosis and treatment guidelines for cervical cancer in my country, in principle, low-grade lesions (CIN1) do not require treatment, and clinical observation is sufficient. For high-grade lesions (CIN2 or CIN3), active therapeutic intervention should be taken to avoid progression to invasive or advanced cervical cancer. The E7-IHC test results using the antibody clone 35B10 of the present invention can prevent overdiagnosis and treatment of patients and effectively reduce the missed detection rate of high-risk patients.

[0073] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A specific antibody capable of recognizing the cancer marker HPV E7 protein, characterized in that: 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.

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 NO.4-7.

3. The antibody according to claim 2, characterized in that: 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.

4. A specific antibody capable of recognizing HPV E7 protein, a cancer marker, characterized in that: The antibody comprises a light chain variable region; the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3 whose amino acid sequences are shown in SEQ ID NO.9-11.

5. The antibody according to claim 4, characterized in that: 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. The amino acid sequences of VLFR1, VLFR2, VLFR3 and VLFR4 are shown in SEQ ID NO.12-15.

6. The antibody according to claim 5, characterized in that: 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.

7. 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 as described in any one of items 1-6.

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

9. The antibody detection reagent according to claim 7, characterized in that: The antibody detection reagent is used in preparing a product for in vitro detection of HPV E7 protein.

10. Use of the specific antibody capable of recognizing the cancer marker HPV E7 protein according to any one of claims 1 to 6 in non-disease diagnosis by detecting HPV E7 protein.

Images