B cell epitope peptide based on human papilloma virus E6 protein and application of B cell epitope peptide in cancer diagnosis

Abstract

The invention discloses a B cell epitope peptide based on human papilloma virus E6 protein and application of the B cell epitope peptide in cancer diagnosis. According to the invention, the epitope peptide is displayed and expressed on the surface of an inert carrier bacterium S9H, so that an RI5 epitope peptide-antibody direct mediated agglutination test detection system based on thallus surface epitope display is successfully constructed. Compared with an immunoserological detection technology based on a complete protein antigen, the kit provided by the invention can be used for more specifically, sensitively, conveniently and quickly detecting the virus oncoprotein specific antibody in the body of an HPV persistent infection patient, and can be used for qualitatively and quantitatively determining the content of the antibody. The epitope peptide-antibody direct mediation agglutination test detection technology provides an efficient and reliable new method for accurate diagnosis of dynamic changes of virus oncoprotein antibodies in persistent infection and cancer stages of HPV patients, and has potential important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical and immunodiagnostic detection technology, specifically relating to human papillomavirus E6 protein B cell epitope peptide and its application in cancer diagnosis. Background Technology

[0002] Human papillomavirus (HPV) Human Papillomavirus, HPV (HPV) is a non-enveloped, double-stranded circular DNA virus with strict epithelial tropism. It infects the epithelial cells of human skin or mucous membranes, causing a variety of lesions, from minor warts to malignant tumors (such as cervical cancer). Currently, over 200 types have been identified, classified into high-risk and low-risk types based on their carcinogenic risk. Persistent infection with high-risk types (such as HPV-16 / 18 / 31 / 33) is the root cause of many cancers. 99.7% of cervical cancers are associated with persistent infection with subtypes 16 / 18. Other high-risk subtypes can also cause anal cancer, penile cancer, oropharyngeal cancer, etc. HPV infection is extremely common. Statistics show that approximately 80% of sexually active individuals worldwide will be infected with at least one HPV subtype in their lifetime. However, most infections are cleared by the immune system within 1-2 years, while persistent infection with high-risk types can lead to cancer. The core of its carcinogenic mechanism lies in the disruption of the host cell's regulatory system by the early viral proteins E6 and E7, with the E6 protein playing a particularly crucial role in this process.

[0003] E6 protein is the main oncogenic protein of high-risk HPV, and its most core function is to target and degrade the important tumor suppressor protein p53 in the host body. In the normal physiological state of cells, tumor suppressor protein p53 acts as a "genome guardian" and can initiate cell cycle arrest, promote DNA repair or induce programmed death when the cell DNA is damaged, thereby preventing the proliferation of abnormal cells. However, when high-risk HPV invades the body and infects cells, E6 protein begins to interfere with and destroy the normal function of p53. It recruits E3 ubiquitin ligase E6AP in the host cell and forms a unique E6-E6AP-p53 complex by closely combining with it. In this complex, E6 protein makes E6AP recognize and bind p53 protein through precise connection, resulting in p53 being ubiquitinated and degraded through the proteasome pathway. With the continuous degradation of p53 protein, its content in the cell drops sharply, causing its normal cell cycle arrest, DNA repair and apoptosis regulation function to be severely damaged. This process makes the infected cells unable to initiate the apoptosis program after DNA damage, and the genetic errors accumulate continuously, the genome stability decreases significantly, and the cell proliferation loses key control. At the same time, E7 protein forces the cell to enter the proliferation cycle abnormally by inhibiting another tumor suppressor protein pRb, and cooperates with E6 - E7 like "continuously stepping on the accelerator", and E6 is "destroying the brake system", both of which drive the cell to malignant transformation. In addition, after the virus integrates into the host genome, the expression of E6 / E7 is often further up-regulated, accelerating the carcinogenic process.

[0004] The diagnosis of HPV-related precancerous lesions and cervical cancer is a key step in the prevention and treatment of such diseases. Currently, the clinical screening method mainly uses thinprep cytology test (TCT) combined with HPV DNA detection, but both have obvious limitations. TCT cytology test is used to detect whether cervical cells are abnormal, with low sensitivity, and the results are affected by the slicing technology and the experience of the interpreter, which may lead to missed diagnosis. HPV DNA detection has high sensitivity but low specificity, and may have cross-reactions, causing false positives. The final diagnosis relies on cervical biopsy or conization under colposcopy, which is invasive and may cause tissue damage, bleeding, infection, and patient discomfort, significantly reducing the compliance of the examination, thereby affecting the timely detection and intervention of early cancer. E6 protein plays a key role in HPV infection and carcinogenesis. Detecting its B-cell epitopes and antibody responses can help understand the immune response mechanism against HPV infection and reveal the role and changes of the immune system in the process of fighting HPV infection. On the other hand, this detection method has higher specificity and sensitivity, which can accurately detect early lesions of HPV-related cancer and provide more reliable diagnostic evidence for clinicians. At the same time, in the aspect of disease progression monitoring, by dynamically observing the changes of antibody levels, the development trend of the disease can be understood in time, which provides strong support for the development of individualized treatment plans and the evaluation of treatment effect. Therefore, detecting the B-cell epitopes of E6 protein and its antibody response is not only helpful for understanding the immune response mechanism, but also has important application value for the early diagnosis and progression monitoring of HPV-related cancer.

[0005] Based on the carcinogenic properties of E6 protein, it is an ideal target for detecting the late stage of infection, which has the following advantages: (1) higher cancer-relatedness: E6 protein promotes cell malignant transformation by inhibiting tumor suppressor proteins such as p53, and its expression level is directly related to high-grade lesions and cancer progression; (2) lower risk of false positives: E6 protein is only stably expressed in persistent high-risk infection and carcinogenesis, which can effectively distinguish between transient infection and carcinogenic infection; (3) more accurate prognosis evaluation: the expression level of E6 protein can reflect the tumor burden, which is helpful for monitoring the treatment effect and predicting the risk of recurrence. Based on this, the detection of E6 protein can make up for the shortcomings of existing HPV-DNA detection, avoid misjudgment caused by the difference between the free state and integrated state of the viral genome, and provide more reliable molecular typing basis for clinical practice.

[0006] However, the current detection techniques for E6 protein still face many problems. On the one hand, due to the low expression level of E6 protein in the early infection stage, the sensitivity of the existing methods is limited, which easily leads to missed diagnosis of some early lesions; on the other hand, the cross-reaction phenomenon caused by limited specificity frequently occurs, which seriously affects the accuracy of the detection results. Therefore, it is urgent to develop an efficient detection technique for E6 protein. The ideal detection technique should have high specificity, be able to accurately identify E6 protein and avoid non-target interference; fast response, be able to output results in a short time to support clinical decision-making; simple operation, no need for complex equipment and advanced professional skills; low cost, to improve accessibility and reduce the economic burden of patients; at the same time, a non-invasive sampling method should be used, such as collecting cervical secretions, urine and other samples of patients for detection, to improve patient acceptance. The realization of such detection techniques will have great significance for the early warning, accurate diagnosis and individualized treatment of cervical cancer, and is expected to bring new breakthroughs in the prevention and treatment of cervical cancer. SUMMARY

[0007] The purpose of the present application is to provide a B cell conserved epitope peptide of human papillomavirus E6 protein.

[0008] The second purpose of the present application is to provide a nucleic acid molecule encoding the B cell epitope peptide of E6 protein.

[0009] The third purpose of the present application is to provide a recombinant gene fragment containing the nucleic acid molecule.

[0010] The fourth purpose of the present application is to provide an expression cassette, a recombinant expression vector, a recombinant cell or a recombinant strain containing the nucleic acid molecule or the recombinant gene fragment.

[0011] The fifth purpose of the present application is to provide a construction method of a recombinant expression vector or a recombinant strain.

[0012] The sixth purpose of the present application is to provide a human papillomavirus persistent infection and its later cancer stage antibody specificity detection system.

[0013] The seventh purpose of the present application is to provide the application of the B cell epitope peptide of E6 protein, the nucleic acid molecule of the B cell epitope peptide of E6 protein, the recombinant gene fragment, the expression cassette, the recombinant expression vector, the recombinant cell or the recombinant strain, the human papillomavirus persistent infection and its later cancer stage antibody specificity detection system in the preparation of a detection reagent or kit for human papillomavirus persistent infection, later cancer stage antibody detection, HPV type differentiation, cervical cancer grade diagnosis or prognosis evaluation.

[0014] The eighth purpose of the present application is to provide a human papillomavirus persistent infection and its later cancer stage antibody detection reagent or kit.

[0015] Technical solution: In order to solve the above technical problems, the present invention provides a human papillomavirus E6 protein B cell epitope peptide, wherein the amino acid sequence of the E6 protein B cell epitope peptide is RFHNI (SEQ ID NO.1).

[0016] The present invention provides a nucleic acid molecule encoding the B-cell epitope peptide of the E6 protein, wherein the DNA sequence of the nucleic acid molecule is AGATTCCATAATATA (SEQ ID NO.2).

[0017] The present invention provides a recombinant gene fragment, which is obtained by inserting the nucleic acid molecule into the coding gene of the Peg fimbriae operon, and the sequence of the recombinant gene DNA fragment is shown in SEQ ID NO.3.

[0018] The present invention provides expression cassettes, recombinant expression vectors, recombinant cells or recombinant strains, which contain the nucleic acid molecules or the recombinant gene fragments described herein.

[0019] The present invention provides a method for constructing a recombinant expression vector, comprising the following steps: inserting the nucleic acid molecule of the E6 protein B cell epitope peptide into the coding gene of the Peg fimbriae operon and introducing it into a vector, or introducing the recombinant gene fragment into a vector to construct a recombinant expression vector.

[0020] The present invention provides a method for constructing recombinant bacterial strains, the method comprising transforming the recombinant expression vector into a vector bacterium.

[0021] This invention provides an antibody-specific detection system for persistent human papillomavirus infection and its later stages of cancer, the detection system comprising the aforementioned expression cassette, recombinant vector, recombinant cells or recombinant strain.

[0022] This invention provides the application of the E6 protein B cell epitope peptide, the nucleic acid molecule of the E6 protein B cell epitope peptide, the recombinant gene fragment, the expression cassette, the recombinant expression vector, the recombinant cell or recombinant strain, and the antibody-specific detection system for persistent human papillomavirus infection in the late stage of cancer in the preparation of detection reagents or kits for persistent human papillomavirus infection, antibody detection in the late stage of cancer, HPV type differentiation, and diagnosis or prognostic assessment of cervical cancer.

[0023] This invention provides a reagent or kit for detecting antibodies against persistent human papillomavirus (HPV) infection and its later stages of cancer. The reagent or kit comprises the HPV E6 protein B-cell epitope peptide, the nucleic acid molecule, the recombinant gene fragment, the expression cassette, the recombinant expression vector, recombinant cells or recombinant strains, and the in vivo antibody-specific detection system for persistent HPV infection and its later stages of cancer.

[0024] The diagnosis of persistent human papillomavirus infection and its subsequent cancer includes diagnosing the grade of cervical cancer and / or the type of HPV.

[0025] Beneficial Effects: Compared with existing HPV detection technologies (such as Cobas 4800, HC2, E6 / E7 mRNA detection, etc.), this invention, based on the B-cell conserved epitope peptide (RI5) and inert vector bacterial surface presentation technology, achieves significant breakthroughs in specificity, sensitivity, ease of operation, and applicability. Specific advantages are as follows: The B-cell epitope peptide RI5 described in this invention originates from a highly conserved region of the HPV-E6 protein and binds only to antibodies produced during persistent HPV infection and carcinogenesis. It does not cross-react with transient infections or antibodies from other pathogens, significantly reducing the risk of false positives and exhibiting high specificity. In the detection and follow-up monitoring of positive serum samples from 12 patient volunteers with early-stage and cancer-stage HPV-related persistent infection, the antibody positive detection rate of the detection system in this invention reached 100% (12 / 12), significantly higher than the positive rate of HPV-DNA PCR detection (50%, 6 / 12). This invention boasts extremely high sensitivity, making it suitable for early cancer screening and late-stage infection monitoring. It utilizes an inert vector bacterial surface presentation technology to express the RI5 peptide on Peg pili, exponentially amplifying the number of epitopes on the inert vector bacterial surface and ensuring high efficiency and specificity of antibody binding. Compared to traditional serological methods, due to the efficient presentation of the RI5 peptide, it can detect HPV-E6 antibodies with significantly superior sensitivity compared to ELISA or Western blot techniques based on the intact E6 protein. This invention is simple to operate and inexpensive. Based on an agglutination assay, it requires no complex equipment; the detection process only requires sample and reagent mixing, and results can be visually interpreted within 2 minutes, far faster than PCR (several hours) or ELISA (2-3 hours). It is suitable for large-scale screening: no expensive instruments (such as qPCR instruments, chemiluminescence analyzers) or specialized technicians are needed, and consumables are inexpensive, allowing for widespread application in resource-constrained primary healthcare institutions. This invention has high clinical application value. Traditional HPV-DNA detection cannot distinguish whether the virus has integrated and the risk of cancer, while RI5 peptide detection can specifically identify E6 antibodies, indicating persistent high-risk infection and cancerous tendency. Furthermore, it can aid in prognostic assessment; quantitative changes in antibody levels can monitor treatment efficacy (e.g., a decrease in antibodies after surgery or radiotherapy indicates good efficacy) and provide early warning of recurrence risk. Therefore, this invention can provide strong support for early warning, accurate diagnosis, and primary care screening of HPV-related cancers. Attached Figure Description

[0026] Figure 1 Figure showing the results of bioinformatics analysis of conserved epitope peptides in HPV-E6 B cells.

[0027] Figure 2 A schematic diagram of the crystal structure of the conserved epitope peptide (RI5) of HPV-E6B cells.

[0028] Figure 3This is an electrophoresis image of Peg-E6-RI5 PCR amplified nucleic acid. Lane M is the Trans 2K Plus II DNA Marker, lane 1 is the PCR amplification product of the Peg fimbriae operon gene (positive control), and lane 2 is... Peg-E6-RI5 PCR amplification products.

[0029] Figure 4 This is an electrophoresis image of the pBR-Peg-E6-RI5 double enzyme digestion identification nucleic acid. Lane M is the Trans 2K Plus II DNA Marker, lane 1 is the pBR322 circular plasmid, lane 2 is the pBR-Peg expression vector, and lane 3 is the pBR-Peg expression vector. Nhe I and Bam The H1 double digestion product, lane 4 contains the pBR-Peg-E6-RI5 recombinant expression vector, and lane 5 contains the pBR-Peg-E6-RI5 recombinant expression vector. Nhe I and Bam H1 double digestion product.

[0030] Figure 5 This is a schematic diagram of the pBR-Peg-E6-RI5 recombinant expression vector.

[0031] Figure 6 pBR-Peg-E6-RI5 showed a significant agglutination reaction with HPV-positive serum (detection system), while no agglutination reaction was observed with the control system.

[0032] Figure 7 The graphs show the agglutination results of the S9H-Peg-E6-RI5 persistent infection late-stage cancer antibody-specific detection system with HPV positive / negative sera from different sources. A: Negative individual control, no agglutination reaction between the HPV infection late-stage cancer antibody-specific detection system and serum samples from healthy volunteers (not infected with human papillomavirus); B: Agglutination reaction between the HPV persistent infection and cancer stage antibody-specific detection system and serum samples from volunteers diagnosed with human papillomavirus infection; C: Negative control 1, no agglutination between the HPV persistent infection and cancer stage antibody-specific detection system and serum samples from healthy SPF mice; D: Negative control 2, no agglutination between the HPV persistent infection and cancer stage antibody-specific detection system and serum samples from clinical pigs; E: Negative control 3, no agglutination between the HPV persistent infection and cancer stage antibody-specific detection system and serum samples positive for Helicobacter pylori polyclonal antibodies; F: Negative control 4, no agglutination between the HPV persistent infection and cancer stage antibody-specific detection system and serum samples positive for avian leukemia. Detailed Implementation

[0033] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the embodiments, based on the knowledge of those skilled in the art and the description of the present invention, any prior art methods, devices, and materials similar to or equivalent to those described, used, and materials in the embodiments of the present invention can be used to implement the present invention.

[0034] Example 1: Bioinformatics Analysis for Predicting B-cell Epitope Peptides of Human Papillomavirus E6 Protein The amino acid sequences of the E6 protein of seven high-risk HPV subtypes (16, 18, 31, 33, 45, 52, and 58) closely associated with malignant tumors were retrieved from the NCBI (https: / / www.ncbi.nlm.nih.gov / ) Protein database. The seven amino acid sequences of HPV-E6 were aligned and analyzed using DNAStar MegAlign software. The B-cell epitope prediction tool from the immunological epitope database (Bepipred Linear Epitope Prediction 3.0, https: / / services.healthtech.dtu.dk / services / BepiPred-3.0 / ) was used to predict the B-cell linear epitope peptides contained in HPV-E6 (eptopes: RFHNI (SEQ ID NO.1), CVYCK (SEQ ID NO.4), FRDL (SEQ ID NO.5), VYRD (SEQ ID NO.6), YGTTLE (SEQ ID NO.7), CQKPL (SEQ ID NO.8), EKQRH (SEQ ID NO.9)). Based on the combined results of the two software analyses, the conserved B-cell epitope peptide RFHNI (RI5, SEQ ID NO.1) of the HPV-E6 protein was confirmed to be highly conserved within the HPV species and had a high prediction score (see Table 1). Figure 1 Furthermore, based on the pentamer crystal structure of HPV 58 / 33 / 52 E6 protein (PDB database index number, 6IGC), the spatial structure of this epitope (RI5) was analyzed using PyMol protein structure analysis software. The results showed that RI5 is located on the surface of the protein crystal structure. Figure 2Based on a comprehensive evaluation of conservation and immunogenicity prediction results, RI5 was ultimately selected as the preferred epitope peptide. This epitope can be used to construct detection methods applicable to antibodies against various HPV strains.

[0035] Table 1. Prediction and analysis of B-cell linear epitope peptides contained in HPV-E6

[0036] Note: Bold amino acids indicate residues that are inconsistent with the candidate epitope sequence; the homology rate is calculated based on the matching ratio between the corresponding polypeptide sequence and the candidate epitope sequence in different HPV E6 protein subtypes; the polypeptide score is based on the prediction results of the HPV16 E6 protein amino acid sequence on the BepiPred-3.0 platform, and the score of the amino acid sites covered by the candidate epitope is taken as the average.

[0037] Example 2: Construction and functional expression validation of an E6 protein antibody-specific detection system for late-stage cancer caused by persistent human papillomavirus infection. The specific implementation procedure is as follows: Nanjing Qingke Biotechnology Co., Ltd. synthesizes recombinant DNA fragments, which are replaced by the coding DNA sequence of E6-RI5. Peg The site of the operon gene sequence is AGATTCCATAATATA ( Peg The reference sequence is Salmonella enterica The recombinant DNA fragment obtained from the CYX strain (GenBank accession numbers CP113540.1, 1702724-1707552) was named... Peg-E6-RI5 , Peg-E6-RI5 The gene sequence is SEQ ID NO.3.

[0038] SEQ ID NO.3 ( Peg-E6-RI5 ): Nanjing Qingke Biotechnology Co., Ltd. Synthetic peg Operator amplification primers, SP Peg Up: 5'-CGC GCTAGC ATGAAACGTTCACTTATTGCTGCT-3'; SP Peg Down: 5'-CGT GGATCC TTAATTATAAGATACCACGATTAATGC-3', the underscore sequence represents respectively... Nhe I and Bam HI restriction enzyme sites. Using the above-mentioned chimeric genes... Peg-E6-RI5 Using SEQ ID NO.3 (1 μL, containing 1 ng of chimeric gene) as a template, and a positive control (Genome of Salmonella pullorum standard strain CVCC526, purchased from the China Institute of Veterinary Drug Control and the China Veterinary Microbial Culture Collection Center), high-fidelity PCR was employed for amplification. The PCR reaction system (50 μL) contained: 2 μL pfu high-fidelity DNA polymerase (Beijing TransGen, catalog number AP221-01), 10 μL 5×pfu DNA polymerase buffer, 5 μL dNTPs, 2 μL each of forward and reverse primers (10 mM), 2 μL template DNA (250 ng / μL), and 27 μL ultrapure water. The amplification program was set as follows: 94℃ pre-denaturation for 5 min; 30 cycles of 94℃ denaturation for 30 s, 52℃ annealing for 30 s, and 72℃ extension for 5 min; and a final extension at 72℃ for 10 min, followed by storage at 12℃. Prepare a 1.5% agarose gel, electrophoresis at 100 V for 50 min, stain with ethidium bromide, and image under a UV imager. PCR results are as follows. Figure 3 As shown, the amplification size of the positive control was 4850 bp; Peg-E6-RI5 The PCR product was 4847 bp. It was recovered using a universal DNA purification kit (Tiangen Biotech (Beijing) Co., Ltd.). Peg-E6-RI5 The PCR amplification product (4847 bp).

[0039] use Nhe I and Bam Double digestion with HI restriction endonuclease (NEB) Peg-E6-RI5 The PCR amplification product and pBR322 plasmid were ligated using T4 DNA ligase (NEB) in a 16°C metal bath overnight. The ligation system was as follows: 2 μL pBR322 plasmid (20.4 ng / μL). Peg-E6-RI5A ligation system consisting of 6 μL of DNA fragment (35.4 ng / μL), 1 μL of T4 DNA ligase (NEB, 400 U / μL), 2.5 μL of 10×T4 DNA Ligase Reaction Buffer, and 13.5 μL of ddH2O, totaling 25 μL, was prepared. The ligation product was transformed into S9H competent cells the following day (S9H inert vector bacteria are derived from Chinese invention patent ZL202010427735.8, "A Universal Inert Vector Salmonella and Its Potential Applications"). Resistance was screened by plating on solid medium containing 100 μg / mL ampicillin. Single colonies from the plates were inoculated into LB liquid medium containing 100 μg / mL ampicillin and grown to the plateau phase. The plasmid was then recovered and... Nhe I, Bam Double digestion with two restriction endonucleases (HI) was performed. A 1.0% agarose gel was prepared, electrophoresed at 100 V for 50 min, stained with ethidium bromide, and then imaged under UV light. The results are shown below. Figure 4 As shown, the recombinant plasmid pBR-Peg-E6-RI5 Nhe I and Bam The HI double digestion product contains a 4215 bp linear vector and a 4835 bp [unclear text - possibly a typo]. Peg-E6-RI5 The linear DNA fragment is consistent with the expected size. A schematic diagram of the expression vector pBR-Peg-E6-RI5 plasmid is shown below. Figure 5 As shown.

[0040] S9H engineered bacteria carrying the recombinant plasmid pBR-Peg-E6-RI5 were grown to the plateau phase in LB broth containing 100 μg / mL ampicillin. After centrifugation at 4000 rpm for 5 min, the supernatant was discarded, and the bacteria were resuspended in an equal volume of sterile physiological saline. This process was repeated twice to prepare a bacterial suspension (concentration 1×10⁻⁶). 10 CFU / mL). A bacterial suspension of recombinant S9H engineered bacteria carrying the above recombinant plasmid (10 μL, concentration 1×10⁻⁶ CFU / mL) was prepared. 10CFU / mL) were separately subjected to agglutination tests with equal volumes of Peg antibody-positive serum (10 samples, obtained by immunizing mice with rPegA fimbriae protein purified in the laboratory in the previous stage, the article has been published (Yang Weifeng. Development and preliminary clinical application of anti-Salmonella PEG fimbriae monoclonal antibody [D]. Yangzhou University, 2016., preserved in this experiment), HPV infection antibody-negative serum (10 samples of serum from healthy volunteers, provided by Xishan People's Hospital of Wuxi City), and HPV persistent infection and cancer stage antibody-positive serum (10 samples of serum from HPV-infected volunteers, all of which were in the stage of cervical and endometrial malignant tumors after infection, provided by Xishan People's Hospital of Wuxi City). The bacterial suspension containing pBR-Peg S9H engineered bacteria (the pBR-Peg S9H engineered bacteria is derived from Chinese invention patent ZL202211488632.8, the target antigen of the novel coronavirus receptor binding domain B cell epitope and its expression and application in Peg fimbriae) as negative controls. The experimental results (Table 2) showed that both bacterial suspensions exhibited specific agglutination reactions with Peg antibody-positive serum, indicating that the functional Peg protein was successfully expressed on the surface of the vector bacteria. Furthermore, neither strain showed cross-reactivity with HPV infection antibody-negative serum. When the S9H-Peg-E6-RI5 engineered bacteria carrying the recombinant plasmid pBR-Peg-E6-RI5 reacted with HPV-positive serum exhibiting persistent infection and cancer-stage antibody-positive status, significant agglutination was observed, characterized by large agglutination particles and a clear background. However, no agglutination reaction was observed in the S9H-Peg control strain carrying only pBR-Peg, and its reaction system remained uniformly turbid. Figure 6 This result indicates that the B-cell epitope RI5 derived from the HPV-E6 protein can be functionally presented and expressed on the cell surface via Peg pili, and can specifically detect antibodies against persistent HPV infection and cancer stages.

[0041] In this antibody detection system, S9H-Peg bacterial suspension is used as a control system. The S9H-Peg-E6-RI5 antibody detection system adds only the RI5 epitope peptide to the control system, enabling it to specifically recognize and bind to HPV-specific antibodies, excluding non-specific reactions, thereby ensuring accurate individual diagnosis. S9H-Peg-E6-RI5 bacterial suspension serves as a specific detection system for antibodies against persistent HPV infection and cancer stages, and can specifically recognize and bind to antibodies against persistent HPV infection and cancer stages.

[0042] Table 2. Functional validation of Peg-E6-RI5 expression on the surface of the S9H vector bacteria.

[0043] Note: "-" indicates a negative agglutination reaction and all samples tested negative; "+" indicates a positive agglutination reaction and all samples tested positive.

[0044] Example 3: Specificity and sensitivity testing of an in vivo antibody-specific detection system for persistent HPV infection and cancer stages. Based on the functional validation of Peg-E6-RI5 expressed on the surface of the S9H vector bacteria, the specificity and sensitivity of the antibody-specific detection system for persistent HPV infection and cancer stages were further tested. The specific implementation procedure is as follows: Following the same method as in Example 2, the specificity of the detection system was verified through an agglutination experiment. Two bacterial suspensions were prepared: a control system S9H-peg bacterial suspension and an HPV persistent infection and cancer stage antibody specific detection system S9H-Peg-E6-RI5 bacterial suspension. The two bacterial suspensions (bacterial concentration 1×10⁻⁶) were then... 10 The bacterial suspension (CFU / mL) was subjected to agglutination tests with sera from different background sources to verify the specificity of the agglutination antibody detection system. Both the bacterial suspension and the test serum were 10 μL in volume. The sera used in the tests included: 54 serum samples from healthy volunteers (not infected with HPV) (provided by Xishan People's Hospital, Wuxi), 37 serum samples from volunteers diagnosed with HPV infection (provided by Xishan People's Hospital, Wuxi), 7 serum samples from healthy SPF mice (provided by Professor Zhu Guoqiang's research group at the Comparative Medicine Center of Yangzhou University), 15 serum samples from mice positive for Helicobacter pylori antibodies (provided by Professor Zhu Guoqiang's research group at the Comparative Medicine Center of Yangzhou University), 51 SPF pig serum samples (provided by the Animal Hospital of Zhejiang Agriculture and Forestry University), and 30 serum samples from chickens positive for avian leukosis virus antibodies (from a chicken farm in Xixiang, Nanning, Guangxi). The test results are shown in Table 3. Serum samples from 37 HPV-confirmed patients did not agglutinate with the control system, but only with the detection system. Furthermore, the above system did not agglutinate with serum from all healthy volunteers (not infected with HPV), healthy SPF mice, Helicobacter pylori antibody-positive serum, SPF pig serum, or avian leukosis virus antibody-positive chicken serum. These results demonstrate the good specificity of this invention. Agglutination patterns of serum samples are shown in Table 3. Figure 7 As shown.

[0045] Table 3. Specificity verification results of S9H-Peg-E6-RI5

[0046] Note: "-" indicates a negative agglutination reaction and all sample test results are negative; "+" indicates a positive agglutination reaction and all sample test results are positive.

[0047] We analyzed 91 serum samples from volunteers provided by the Department of Obstetrics and Gynecology of Xishan People's Hospital in Wuxi City. Combined with the HPV-DNA (PCR) test results provided by the hospital, we selected 12 serum samples confirmed by pathological conization to be early-stage or at different stages of cancer (see pathological stages in Table 4) for focused analysis. The testing method is as follows: The above 12 serum samples were subjected to 2... n For serial dilution, add 10 μL of sterile saline to each well of a 96-well plate. Then, add 10 μL of serum to the first well of each column, mix thoroughly with the sterile saline, and then add 10 μL of diluted serum to the next well. Repeat this process to dilute to 2 μL. 4 Serum at different dilutions was mixed with S9H-Peg-E6-RI5 bacterial suspension (10 μL, initial bacterial concentration 1×10⁻⁶). 10 Agglutination tests were performed using CFU / mL, with S9H-peg as the control system (10 μL, initial concentration of control bacterial culture was 1×10⁻⁶). 10 The serum antibody titer was determined by the dilution at which the last agglutinated particles appeared (CFU / mL). The results showed that the positive detection rate of this system reached 100% (12 / 12), significantly higher than the 50% (6 / 12) positive rate of traditional HPV-DNA (PCR) testing. Furthermore, this system can quantitatively detect antibody titers (up to a maximum titer of 1:16), whereas HPV-DNA testing can only qualitatively determine whether an infection has occurred.

[0048] Table 4. Sensitivity Verification Results of S9H-Peg-E6-RI5

[0049] Note: "-" indicates a negative agglutination reaction; "+" indicates a positive agglutination reaction; information in parentheses indicates antibody agglutination titer.

[0050] In summary, the S9H-Peg-E6-RI5 detection system of this invention exhibits excellent specificity and sensitivity. Compared with traditional HPV-DNA (PCR) detection, this system not only significantly improves detection sensitivity in cases of persistent HPV infection and cancer stages, but also quantitatively detects the titer of specific agglutinating antibodies, which is of great significance for clinical detection and prognostic monitoring.

[0051] Example 4: Repeatability and stability testing of the HPV persistent infection and cancer stage antibody specific detection system Based on the functional validation of Peg-E6-RI5 expressed on the surface of the S9H vector bacteria, the repeatability and stability of the antibody-specific detection system for persistent HPV infection and cancer stages were further tested. The specific implementation procedure is as follows: Following the same method as in Example 2, bacterial suspensions of the control system S9H-peg and the HPV persistent infection and cancer stage antibody specific detection system S9H-Peg-E6-RI5 were prepared, with an initial bacterial concentration of 1×10⁻⁶. 10 CFU / mL was added to 10 μL of each. Serum samples were subjected to agglutination detection using the control system S9H-peg bacterial suspension stored at 4℃ for more than one year and the HPV persistent infection and cancer stage antibody specific detection system S9H-Peg-E6-RI5 bacterial suspension to verify the stability of the agglutination antibody detection system.

[0052] The same batch of serum samples was tested three times using the two bacterial suspensions prepared in the same batch (detection system batch number: 20230526) to verify the intra-batch reproducibility of the agglutination antibody detection system. The same batch of serum samples was tested three times at different times using the detection system S9H-Peg-E6-RI5 and the control system prepared in different batches (the three batches of detection system batch numbers are: 20230526, 20231217, and 202404211, respectively) to verify the inter-batch reproducibility of the agglutination antibody detection system.

[0053] The two detection systems prepared in our laboratory exhibited stable performance. The serum samples used in the tests included: 50 serum samples from healthy volunteers (not infected with HPV) and 50 serum samples from patients diagnosed with HPV infection. The results showed that storage at 4℃ for more than one year did not reduce the detection performance of the antibody-specific detection system for persistent HPV infection and cancer stages; the results were completely consistent, indicating that the detection system and control system prepared in our laboratory are stable, as shown in Table 5. The same batch of detection system and control system (detection system batch number: 20230526) were used to perform three repeated tests on the same batch of samples, and the results were consistent, as shown in Table 6, indicating that the agglutination antibody detection system has good intra-batch repeatability. Different batches of detection systems were used to perform three tests on the same batch of samples at different times, and the results were consistent, as shown in Table 7, indicating that the agglutination antibody detection system has good inter-batch repeatability.

[0054] Table 5. Stability test results of the HPV persistent infection and cancer stage antibody specific detection system.

[0055] Note: Control system: S9H-Peg, detection system: S9H-Peg-E6-RI5; "-" indicates negative agglutination reaction and all sample test results are negative; "+" indicates positive agglutination reaction and all sample test results are positive.

[0056] Table 6. Intra-batch repeatability test results of the HPV persistent infection and cancer stage antibody specific detection system.

[0057] Note: Control system: S9H-Peg, detection system: S9H-Peg-E6-RI5; "-" indicates negative agglutination reaction and all sample test results are negative; "+" indicates positive agglutination reaction and all sample test results are positive.

[0058] Table 7. Results of inter-batch repeatability experiments of the HPV persistent infection and cancer stage antibody-specific detection system. Note: Control system: S9H-Peg, detection system: S9H-Peg-E6-RI5; "-" indicates negative agglutination reaction and all sample test results are negative; "+" indicates positive agglutination reaction and all sample test results are positive.

[0059] Example 5: Precise Diagnostic Test for Early Cervical Cancer Caused by HPV Infection Using an HPV Persistent Infection and Cancer Stage Antibody Specific Detection System. Following the same method as in Example 2, bacterial suspensions of the control system S9H-peg and the HPV persistent infection and cancer stage antibody specific detection system S9H-Peg-E6-RI5 were prepared. The concentration of the prepared bacterial suspensions was 1×10⁻⁶. 10 CFU / mL.

[0060] Thirteen serum samples from patients with clinical HPV infection were provided by Xishan People's Hospital of Wuxi City (No. 1128, Dacheng Road, Anzhen Street, Xishan District, Wuxi City; sample collector: Wang Haojue). According to the pathological diagnosis results of conization, these patients included 7 patients with low-grade cervical intraepithelial neoplasia (CIN I), 3 patients with intermediate-grade cervical intraepithelial neoplasia (CIN II), and 3 patients with high-grade cervical intraepithelial neoplasia (CIN III).

[0061] The serum was tested using the aforementioned HPV persistent infection and cancer stage antibody-specific detection system, with bacterial culture (1×10⁻⁶) as the sample. 10 The serum of all patients with persistent HPV infection and early cancer stage (cervical intraepithelial neoplasia CIN I-III) was positive for the S9H-Peg-E6-RI5 antibody-specific detection system for persistent HPV infection and cancer stage, while it was negative for the control system S9H-Peg which lacks the E6-RI5 epitope, as shown in Table 8.

[0062] Table 8. Accurate Diagnostic Results of HPV-Infected Early Cervical Cancer Caused by the HPV Persistent Infection and Cancer Stage Antibody Specific Detection System

[0063] Note: Control system: S9H-Peg, detection system: S9H-Peg-E6-RI5; "-" indicates negative agglutination reaction and all sample test results are negative; "+" indicates positive agglutination reaction; the number in "()" represents the antibody titer of quantitative detection.

[0064] The results above show that the S9H-Peg-E6-RI5 detection system of the present invention can not only detect multiple types of HPV infection, but also diagnose cervical intraepithelial neoplasia (CIN) grades I-III with a diagnostic accuracy of 100%.

Claims

1. Based on the human papillomavirus E6 protein B cell epitope peptide, characterized in that, The amino acid sequence of the B-cell epitope peptide of the E6 protein is RFHNI.

2. A nucleic acid molecule encoding the E6 protein B-cell epitope peptide of claim 1, characterized in that, The DNA sequence of the nucleic acid molecule is AGATTCCATAATATA.

3. A recombinant gene fragment, characterized in that, The recombinant gene fragment is obtained by inserting the nucleic acid molecule of claim 2 into the coding gene of the Peg fimbriae operon, and the sequence of the recombinant gene fragment is shown in SEQ ID NO.

3.

4. An expression cassette, a recombinant expression vector, a recombinant cell or a recombinant bacterial strain, characterized in that, It comprises the nucleic acid molecule of claim 2 or the recombinant gene fragment of claim 3.

5. The method for constructing the recombinant expression vector according to claim 4, characterized in that, The process includes the following steps: inserting the nucleic acid molecule of the E6 protein B cell epitope peptide as described in claim 2 into the coding gene of the Peg fimbriae operon and introducing it into a vector, or introducing the recombinant gene fragment as described in claim 3 into a vector to construct a recombinant expression vector.

6. The method for constructing the recombinant strain according to claim 4, characterized in that, The method comprises converting the recombinant expression vector of claim 4 into a vector bacterium.

7. A system for the specific detection of antibodies against persistent human papillomavirus infection and its later stages of cancer, characterized in that, The detection system includes the expression cassette, recombinant vector, recombinant cell or recombinant strain as described in claim 4.

8. The application of the E6 protein B cell epitope peptide of claim 1, the nucleic acid molecule of the E6 protein B cell epitope peptide of claim 2, the recombinant gene fragment of claim 3, the expression cassette, recombinant expression vector, recombinant cell or recombinant strain of claim 4, and the antibody-specific detection system for persistent human papillomavirus infection in the late stage of cancer of claim 7 in the preparation of detection reagents or kits for persistent human papillomavirus infection, antibody detection in the late stage of cancer, HPV type differentiation, cervical cancer grade diagnosis or prognostic assessment.

9. A reagent or kit for detecting antibodies against persistent human papillomavirus infection or its later stages of cancer, characterized in that, The reagents or kits include the human papillomavirus E6 protein B-cell epitope peptide of claim 1, the nucleic acid molecule of claim 2, the recombinant gene fragment of claim 3, the expression cassette, recombinant expression vector, recombinant cells or recombinant strains of claim 4, and the in vivo antibody-specific detection system for persistent human papillomavirus infection and its later cancer stages of claim 7.

10. The detection reagent or kit according to claim 9, characterized in that, The diagnosis of persistent human papillomavirus infection and its subsequent cancer includes diagnosis of HPV type and / or cervical cancer grade.

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