A monoclonal antibody 5E10 for detecting oral streptococcal TMPC protein and its application

Abstract

The present invention belongs to the field of biological detection technology, and specifically relates to a monoclonal antibody 5E10 for detecting oral Streptococcus TMPC protein and its application. The monoclonal antibody has a clear heavy chain and light chain variable region structure: its heavy chain variable region contains three complementarity determining regions, as shown in SEQ ID NO.1 to SEQ ID NO.3, respectively; the light chain variable region also contains three complementarity determining regions, as shown in SEQ ID NO.4 to SEQ ID NO.6, respectively. The present invention successfully established a sensitive and reliable double-antibody sandwich ELISA detection method by preparing a high-affinity and high-specificity monoclonal antibody 5E10 targeting oral Streptococcus TMPC protein. This method can efficiently and accurately detect oral Streptococcus TMPC protein and can be applied to scientific research such as clinical detection or pathogenesis of oral Streptococcus, as well as the production of related detection products and tools.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to a monoclonal antibody 5E10 for detecting oral streptococcal TMPC protein and its application. Background Art

[0002] Streptococcus is the most common oral colonizing bacterium, comprising the largest proportion of the normal oral flora and can be isolated from all oral sites. With increasing research on oral Streptococci, its classification is becoming increasingly clear. Currently, oral Streptococci include the salivarius, anginosus, mutans, and pyogenes groups, as well as several unclassified species. As an opportunistic pathogen, oral Streptococcus not only colonizes the oral cavity but also exists in the throat, gastrointestinal tract, and genitourinary tract. Although part of the normal oral flora, it can cause a variety of infections under certain conditions, such as dental caries, periodontal disease, and infective endocarditis. Furthermore, researchers have found a correlation between anginosus Streptococcus and gastric cancer, garnering particular attention in the carcinogenic mechanisms of pathogens other than Helicobacter pylori (Hp).

[0003] TMPC protein is a surface protein of oral Streptococcus and is considered to be a virulence factor that can mediate the attachment and colonization of Streptococcus anginosus on the gastric mucosa, activate the mitogen-activated protein kinase (MAPK) signaling pathway, and thus promote the occurrence of gastric cancer.

[0004] Currently, there are no high-affinity, highly specific monoclonal antibodies targeting the oral Streptococcus TMPC protein, making it difficult to conduct accurate immunoassays. Furthermore, there is a lack of a sensitive and specific detection method to assess TMPC protein expression levels in clinical samples. Summary of the Invention

[0005] The present invention prepared oral Streptococcus TMPC recombinant protein and screened out high-affinity monoclonal antibodies. Based on this, a double-antibody sandwich ELISA detection method was established, which can effectively detect oral Streptococcus and oral Streptococcus TMPC recombinant protein. In the future, it can be applied to clinical detection of oral Streptococcus or related scientific research on the pathogenesis of oral Streptococcus.

[0006] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0007] A monoclonal antibody 5E10 for detecting oral Streptococcus TMPC protein, wherein the heavy chain variable region of the monoclonal antibody 5E10 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 1 to SEQ ID NO. 3, respectively;

[0008] The light chain variable region of the monoclonal antibody 5E10 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 4 to SEQ ID NO. 6, respectively.

[0009] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 5E10 is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of monoclonal antibody 5E10 is shown in SEQ ID NO.8.

[0010] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5E10 is shown in SEQ ID NO.9.

[0011] In some embodiments, the nucleotide sequence encoding the light chain variable region of monoclonal antibody 5E10 is shown in SEQ ID NO.10.

[0012] Based on the application of the above-mentioned monoclonal antibody 5E10 in the preparation of a tool for detecting oral Streptococcus TMPC protein.

[0013] In some embodiments, the tool is used to detect an S. oralis TMPC protein in a biological sample in vitro, and the in vitro detection is not used for diagnosis of a disease.

[0014] In some embodiments, the tools include reagents, kits, test strips, and antibody chips.

[0015] In some embodiments, the kit comprises a double antibody sandwich ELISA kit.

[0016] In some embodiments, the kit is coated and labeled with the monoclonal antibody 5E10.

[0017] In some embodiments, the monoclonal antibody 5E10 is used to construct an ELISA detection system, which is used to detect oral Streptococcus TMPC protein in a biological sample. The ELISA detection system is not used for disease diagnosis.

[0018] Beneficial effects:

[0019] The present invention provides a monoclonal antibody 5E10 for detecting the oral Streptococcus TMPC protein. Monoclonal antibody 5E10 exhibits high specificity and good sensitivity. This monoclonal antibody has well-defined heavy and light chain variable region structures: the heavy chain variable region comprises three complementarity determining regions (CDRs), as shown in SEQ ID NOs. 1 to 3, respectively; the light chain variable region also comprises three complementarity determining regions, as shown in SEQ ID NOs. 4 to 6, respectively.

[0020] This study successfully established a sensitive and reliable double-antibody sandwich ELISA assay by preparing a high-affinity, highly specific monoclonal antibody, 5E10, targeting the oral Streptococcus TMPC protein. This method can efficiently and accurately detect the oral Streptococcus TMPC protein in biological samples such as saliva or oral swabs. The monoclonal antibody 5E10 of the present invention has a well-defined amino acid sequence and corresponding nucleotide sequence, laying a solid foundation for its large-scale production, stability control, and subsequent development of diverse detection tools based on this antibody, including test kits, test strips, and antibody chips. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0022] Figure 1 Figure 1 is the SDS-PAGE identification result of the purified recombinant TMPC protein;

[0023] Figure 2 The figure shows the reaction results of oral streptococcus culture with the screened monoclonal antibodies;

[0024] Figure 3 This is a sensitivity test diagram of double antibody sandwich ELISA;

[0025] Figure 4 This is a diagram showing the results of identifying the binding activity of monoclonal antibody 5E10 with the oral Streptococcus TMPC recombinant protein;

[0026] Figure 5 This is a graph showing the results of identifying the binding activity of monoclonal antibody 5E10 with whole oral Streptococcus. DETAILED DESCRIPTION

[0027] The following detailed description of the embodiments of the present application is provided in conjunction with the accompanying drawings and examples. The detailed description of the following examples and the accompanying drawings are intended to illustrate the principles of the present application, but are not intended to limit the scope of the present application. The present application may be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but rather includes all technical solutions within the scope of the claims.

[0028] The present application provides these embodiments to make this application thorough and complete, and to fully express the scope of this application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangement of parts and steps, the composition of materials, numerical expressions and numerical values ​​set forth in these embodiments should be interpreted as merely exemplary, and not as limiting.

[0029] Example 1

[0030] 1. Preparation of recombinant antigens

[0031] Primers were designed with reference to the TMPC protein of Streptococcus anginosus strain (ATCC 33397) in Genbank. The TMPC gene fragment of oral Streptococcus isolated from clinical patients was amplified using the gene as a template and cloned into the pET28a prokaryotic expression vector. The nucleotide sequence was sequenced (shown as SEQ ID NO. 11):

[0032]

[0033] Amino acid sequence (as shown in SEQ ID NO.12):

[0034] MNKKQWLGLGLVAVAAIGLAACGNRSSRNAASSSSDVKTKAAIVTDTGGVDDKSFNQSAWEGLQAWGKEHNLSKDKGYTYFQSTSEADYANNLQQAAGS-YNLIFGVGFALHNAVEEAAKEHTDLNYVLIDDVIKDQKNVASVTFADNEAAYLAGVAAAKTTKTKQIGFVGGMESE VISRFEAGFKAGVASVDSSIKVQVDYAGSFGDAAKGKTIAAAQYAAGADVVYQVAGGTGAGVFSEAKSLNESRPENEKVWVIGVDRDQVEEGKYTSKDGKESNFVLASTLKQVGTTVKDIANKTEKGEFPGGQVIVYSLKDKGVDLAITNLSEEGKKAVEDAKAKILDGSIKVPTE.

[0035] The nucleotide terminal is a TAA stop codon and is not expressed.

[0036] The TMPC gene was cloned into the pET28a vector by molecular cloning technology to obtain the recombinant expression plasmid pET28a-TMPC, which was confirmed to be correct by sequencing. The recombinant plasmid was transformed into BL21 (DE3) competent cells (Molecular Cloning) according to conventional methods, and the transformed bacteria were spread on LB agar plates (containing 50μg / mL kanamycin) and cultured at 37°C overnight. Pick a single colony and inoculate it into 5mL LB medium (containing 50μg / mL kanamycin) and culture it at 37°C and 220 rpm overnight. Inoculate 1% of the total volume of the culture medium into LB medium (containing 50μg / mL kanamycin) and culture it at 37°C and 220 rpm for about 3 hours until the OD 600 The concentration of IPTG was 0.6-0.9, and the final concentration was 0.1 mM IPTG. The cells were collected after induction at 30°C and 200 rpm for 4 hours.

[0037] The oral Streptococcus TMPC recombinant protein is based on the gene sequence of the naturally occurring TMPC protein (a surface protein) in oral Streptococcus, expressed and purified through molecular cloning techniques. This recombinant protein retains the core structure and functional regions of the native TMPC protein and exhibits excellent biological activity. It can be used to immunize animals to generate monoclonal antibodies and serve as an antigen in subsequent detection experiments. Therefore, the recombinant TMPC protein mimics the properties of the native TMPC protein, making it an important tool for studying its immunoreactivity and establishing detection methods.

[0038] The monoclonal antibody 5E10 of the present invention can specifically recognize oral Streptococcus TMPC protein and oral Streptococcus TMPC recombinant protein.

[0039] 2. Purification of oral Streptococcus TMPC recombinant protein

[0040] Because the expressed oral Streptococcus TMPC recombinant protein has a histidine tag, the protein purification instrument and HisTrap TM Purification was performed using an HP affinity chromatography column. Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0, and buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The fermented bacterial broth was then centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in buffer A and sonicated in an ice-water bath for 30 min, followed by 5-second intervals and 5-second intervals. The precipitate was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a Jet Bio 0.22 μm filter, loaded onto the column, washed with buffer A, and finally eluted with a gradient of buffer B. Purification was observed by SDS-PAGE gel electrophoresis. Protein concentration was determined using a Thermo Nanodrop micro-spectrophotometer and stored at -20°C.

[0041] The purified oral Streptococcus TMPC recombinant protein was identified by SDS-PAGE to determine the molecular weight and purity of the target protein. Specifically: Protein sample pretreatment: add an equal volume of 2× SDS loading buffer to each sample, place it in a boiling water bath for 10 minutes, and centrifuge it at 12,000 rpm for 3 minutes. Dilute 5× glycine buffer to the working concentration, add it to the electrophoresis tank to the appropriate liquid level, and gently pull the comb out of the solidified gel. Add protein marker and 10ul of treated protein sample to the sample well. Turn on the power and adjust the voltage to 80V constant voltage electrophoresis to the separation gel, then change it to 120V until bromophenol blue reaches the bottom of the gel. Cut the gel from the glass plate, place it in Coomassie Brilliant Blue staining solution, shake and stain for 4 hours, and then decolorize it with destaining solution until the bands are clear.

[0042] See also Figure 1 , Figure 1 M in the middle represents a protein marker. Serial number 1 represents a recombinant protein from oral Streptococcus TMPC. The recombinant expression plasmid was induced and purified, resulting in soluble expression. SDS-PAGE analysis revealed a single band between 33 and 43 kDa, with a purity exceeding 85%. The size of the purified protein was consistent with the expected value (approximately 37 kDa), indicating successful expression of the recombinant protein.

[0043] 3. Mouse immunization

[0044] Six-week-old female BALB / c mice were immunized with purified oral Streptococcus TMPC recombinant protein mixed with an equal volume of Freund's complete adjuvant (200 μL) via multiple subcutaneous injections at multiple sites. Six-week-old female mice were then immunized with the same dose of 20 μg / mouse mixed with an equal volume of MF59 adjuvant via intramuscular injection at two and four weeks. At five weeks, sera were collected from the mice for antibody titer determination. Mice with the highest titer were selected for a booster immunization with 20 μg of oral Streptococcus TMPC recombinant protein via intraperitoneal bolus. Three days later, spleens were harvested from the mice for hybridoma cell production.

[0045] 4. Screening of hybridoma cell lines

[0046] All spleen cells from immunized mice were aseptically fused with SP2 / 0 myeloma cells in the logarithmic growth phase and then cultured in HAT medium for screening. When the fused cells reached half the bottom of the well, clones positive for the oral S. TMPC recombinant protein were screened using indirect ELISA. Because the immunogen is prokaryotically expressed and contains a His tag, background components must be screened to identify cell lines specific for the oral S. TMPC recombinant protein. Positive cells were then cloned to a monoclonal state by limiting dilution, expanded, and cryopreserved.

[0047] 5. Screening of positive clones by indirect ELISA

[0048] The oral Streptococcus TMPC recombinant protein and the HPV16 / E7 recombinant protein expressed by the pET28a vector (with a His tag) were coated in a microplate respectively. The coating solution was carbonate buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L pure water, pH 9.6), the coating concentration was 1 μg / mL, and the coating was carried out overnight at 4°C. The next day, the coating solution was discarded and the plates were blocked with 1-2% BSA or gelatin, 150 μL per well, at 37°C for 2 hours, and the plates were washed once with washing solution and patted dry. 50 μL of cell culture supernatant was added and the plates were reacted at 37°C for 30 minutes. The liquid in the wells was shaken out, and the plate was washed 4 times with PBST solution. After patting dry, 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 in PBS) was added. The plate was reacted at 37°C for 30 min. The plate was washed 4 times again, and after patting dry, 50 μL / well of TMB color development solution was added to develop at room temperature for 10 min. Finally, 50 μL of TMB stop solution (acidic, Beijing Meikewande Biological, 1001SA) was added to terminate the reaction. The OD was measured using a microplate reader. 450 The positive cell lines that reacted only with the oral Streptococcus TMPC recombinant protein but not with the control antigen were selected for subsequent experiments.

[0049] The screening process is shown in Table 1.

[0050] Table 1: Indirect ELISA reaction results of hybridoma cell lines to oral Streptococcus TMPC recombinant protein and control antigen.

[0051]

[0052] Here, Ctrl is a negative control, which is the supernatant of normal SP2 / 0 myeloma cell culture, TMPC is the recombinant protein of oral Streptococcus TMPC, and HPV16 / E7 is the recombinant protein of HPV16 / E7.

[0053] 6. Preparation and purification of monoclonal antibody ascites

[0054] After the selected monoclonal cell lines were expanded and cultured, 0.2 mL (containing 2.5×10 6 Female BALB / c mice (100 cells) were pretreated with incomplete Freund's adjuvant. Approximately 10 days later, when the abdomen became noticeably swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged to collect the intermediate layer.

[0055] 7. Affinity chromatography purification of monoclonal antibodies

[0056] Ascites was centrifuged at 12,000 rpm for 5 minutes. The supernatant was diluted 10-fold with binding buffer (20 mM PBS, 150 mM NaCl, pH 7.4) and filtered through a 0.22 μm filter. The filtered sample was pumped at a low speed via a peristaltic pump onto a Protein L purification column equilibrated with binding buffer. The column was connected to a protein purifier and washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled out. The column was then eluted with elution buffer (0.1 M glycine, pH 2.7). The eluted peak was collected and adjusted to neutral with 1 M Tris-HCl, pH 9. The sample was placed in a dialysis bag (MW: 8,000-14,000) and dialyzed against 20 mM PBS, pH 7.4, at 2-8°C for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12,000 rpm for 5 minutes to remove precipitates and impurities. The supernatant was the purified monoclonal antibody. The concentration of the purified monoclonal antibody was measured using an ultra-micro spectrophotometer and stored in aliquots.

[0057] 8. Identification of Monoclonal Antibodies

[0058] 8.1 ELISA Identification

[0059] In order to identify the reaction between the monoclonal antibody and the oral Streptococcus TMPC recombinant protein, the recombinant oral Streptococcus TMPC recombinant protein (1µg / ml) was coated according to the above indirect ELISA method, and the purified monoclonal antibody was diluted to 10µg / ml, 1µg / ml, and 100ng / ml for detection. The OD 450 nm values ​​were used to evaluate the reactivity of the screened monoclonal antibodies with the oral Streptococcus TMPC recombinant protein. The screening process is shown in Table 2.

[0060] Table 2: OD values ​​of different monoclonal antibodies binding to oral Streptococcus TMPC recombinant protein at different concentrations 450 nm detection results.

[0061]

[0062] Ctrl is a negative control monoclonal antibody, which is a commercially available Candida albicans monoclonal antibody (Zhuhai Bomei).

[0063] 8.2 Western Blot Identification

[0064] In order to determine whether the monoclonal antibodies screened using oral Streptococcus TMPC recombinant protein could react with real oral Streptococcus samples, clinically isolated oral Streptococcus cultures (1×10 9CFU / mL) were separated by 12% SDS-PAGE electrophoresis according to SDS-PAGE operation requirements, and then transferred to 0.45 μm nitrocellulose membrane, blocked with 5% skim milk powder (diluted with TBST) at 4°C overnight, washed three times with TBST, and then added with the purified above-mentioned monoclonal antibody (1 μg / ml, diluted in blocking buffer). At the same time, a mouse monoclonal antibody (anti-Candida albicans monoclonal antibody, Zhuhai Bomei) of the same concentration was added as a control. The membrane was incubated at room temperature for 1 hour, washed three times with TBST, and then HRP-labeled goat anti-mouse IgG was added. The membrane was reacted in the dark for 1 hour at room temperature. After thorough washing, DAB (Beijing Solebao, DA1010) was added for color development.

[0065] The culture of oral streptococci was separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membrane, and its reaction with the screened monoclonal antibodies was identified by Western blotting. Figure 2 M: protein marker; numbers 1-8 are TMPC monoclonal antibodies: 5H6, 2C10, 2C7, 1A5, 3C7, 5E10, 3G5, and 4H10; number 9 is a commercially available Candida albicans monoclonal antibody (Zhuhai Bomei). All concentrations were 1µg / ml. Results showed that all of these TMPC monoclonal antibodies reacted specifically with oral S. oralis cultures, with a single band observed around 33-43 kDa, consistent with the expected size. Control monoclonal antibodies were all negative, indicating that the monoclonal antibodies screened and immunized with oral S. oralis TMPC recombinant protein react with authentic whole-cell S. oralis samples. This indirectly demonstrates that the prepared oral S. oralis TMPC recombinant protein has excellent biological activity and that the screened monoclonal antibodies are specific for oral S. oralis and can be used in subsequent paired detection studies for oral S. oralis.

[0066] 8.3. Double Antibody Sandwich ELISA Pairing

[0067] HRP labeling of antibodies:

[0068] Specifically, dilute the labeled antibody to a final concentration of 2 mg / mL in carbonate coupling buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L pure water, pH 9.6). Dissolve 2 mg HRP in 0.5 mL ultrapure water and mix thoroughly with 0.5 mL of 0.06 M sodium periodate solution. Add 1 mg of the diluted antibody solution to the HRP tube and pipette to mix thoroughly. Incubate at room temperature for 1 hour, mixing regularly during the incubation period. Terminate the labeling reaction by adding 50 μL of 5 mg / mL sodium borohydride and mixing for 15 minutes. Finally, dialyze the labeled antibody overnight against 0.01 M PBS, pH 7.4. Add glycerol in a 1:1 ratio and store in aliquots at -20°C.

[0069] Establishment of double antibody sandwich method:

[0070] Screening of paired antibodies: Purified monoclonal antibodies were coated at a concentration of 1 μg / mL with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L pure water, pH 9.6) at 50 μL / well for overnight coating at 4°C. The next day, the coating solution was discarded and the cells were blocked with 1-2% BSA or gelatin at 150 μL / well and incubated at 37°C for 2 h. The blocking solution was discarded and oral streptococcal culture was diluted 10 times (1×10 8 CFU / mL) were added to the ELISA plate at 50 μL / well, and Escherichia coli culture (DH5α) (1×10 8 CFU / mL) were incubated at 37°C for 35 min, washed 4 times with PBST, and HRP-labeled monoclonal antibody diluted 1000 times with PBS was added at 50 μL / well. The plate was incubated at 37°C for 35 min, washed 4 times again, patted dry, and TMB color development solution was added at 50 μL / well. The color was developed at room temperature for 10 min, and finally 50 μL TMB stop solution (acidic, Beijing Meikewande Biological, 1001SA) was added to terminate the reaction. The OD was measured using a microplate reader. 450 Select the paired mAb with the largest P / N value.

[0071] Table 3: OD of oral Streptococcus detected by different monoclonal antibody combinations 450 Comparison of nm value and P / N value.

[0072]

[0073] In Table 3, 1000* indicates 1000-fold dilution.

[0074] In order to screen the paired antibody combination that can be used to detect oral streptococci, the candidate monoclonal antibodies were used as coating antibodies and HRP-labeled antibodies (diluted 1000 times) respectively, and double antibody sandwich ELISA cross-matching experiments were performed to evaluate their specific recognition ability for oral streptococci and exclude cross-reactions with Escherichia coli. 450 The results showed that when both the coating antibody and the HRP-labeled antibody were both at 5E10, the detection signal was the strongest and the specificity was good, so this was determined to be the best antibody pairing.

[0075] 8.4. Optimization of Double Antibody Sandwich Method

[0076] After determining the coating and labeling antibody combination, the optimal monoclonal antibody coating concentration and HRP-labeled monoclonal antibody dilution were further determined.

[0077] 5E10 monoclonal antibody was coated at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL using coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L pure water, pH 9.6) at 50 μL / well for overnight coating at 4°C. The next day, the coating solution was discarded and the plates were blocked with 2% BSA at 150 μL / well and incubated at 37°C for 2 h. The blocking solution was discarded and oral Streptococcus culture and Escherichia coli culture were diluted 10-fold with PBS and added to the ELISA plate at 50 μL / well. The plates were incubated at 37°C for 35 min and washed four times with PBST. HRP-labeled 5E10 monoclonal antibody diluted 1000, 2000, and 4000 times with PBS was added at 50 μL / well and incubated at 37°C for 35 min. The plate was then washed 4 times, patted dry, and TMB color development solution was added at 50 μL / well. The color was developed at room temperature for 10 min. Finally, 50 μL TMB stop solution (acidic, Beijing Meikewande Biological, 1001SA) was added to terminate the reaction. The OD was measured using a microplate reader. 450 nm values. The conditions that maximized the ratio of positive readouts to negative control values ​​(P / N) were considered the optimal experimental conditions for the double-antibody sandwich ELISA. The screening process is shown in Table 4.

[0078] Table 4: Optimization of the best coating and labeling conditions for double antibody sandwich ELISA.

[0079]

[0080] As shown in Table 4, the coating concentration and dilution of the HRP-labeled antibody were optimized, and the optimal reaction conditions for the double-antibody sandwich ELISA were determined as follows: monoclonal antibody 5E10 was used as the coating antibody at a coating concentration of 1 μg / mL; HRP-labeled monoclonal antibody 5E10 was used as the labeling antibody and diluted 2000 times before use.

[0081] 8.5. Detection Sensitivity of Double Antibody Sandwich ELISA for Oral Streptococcus TMPC Recombinant Protein

[0082] The detection sensitivity of this method was evaluated under this optimal condition. Specifically, the 5E10 monoclonal antibody was coated at a concentration of 1µg / mL, 50ul / well at 4°C overnight using the above-mentioned coating buffer, the coating solution was discarded, 150ul of 2% BSA was added to each well and incubated at 37°C for 2h for blocking, and a gradient dilution of oral Streptococcus TMPC recombinant protein of 1μg / mL, 100ng / mL, 10ng / mL, 1ng / mL, 100pg / mL, 50μL / well was added. After incubation, the plate was washed 4 times, and HRP-labeled 5E10 monoclonal antibody was added at a dilution factor of 2000 times. At the same time, HPV16 / E7 recombinant protein was used as a negative control, and 50μL was added to each well at the same concentration for detection. The incubation conditions for each step were 37°C for 35min. Finally, TMB was added for color development at room temperature for 10min, and 50μL of stop solution was added to terminate the reaction. At OD 450 The detection value was read at a wavelength of 1.5 nm to determine the detection sensitivity and specificity of the detection system for the oral Streptococcus TMPC recombinant protein.

[0083] according to Figure 3 Available, Figure 3 TMPC represents oral Streptococcus TMPC recombinant protein, HPV16 / E7 represents HPV16 / E7 recombinant protein, and mAb on the horizontal axis is the abbreviation of "monoclonal antibody", which is translated into "monoclonal antibody" in Chinese. Figure 3 In the assay, mAb Conc. represents the concentration of the monoclonal antibody. This double-antibody sandwich ELISA, comprised of paired antibodies, showed a positive reaction even at a dilution of 1 ng / ml against the oral Streptococcus TMPC recombinant protein and showed no reaction with unrelated antigens, demonstrating excellent sensitivity and specificity.

[0084] The HPV16 / E7 gene was downloaded from NCBI, synthesized by Anhui General Biotechnology Co., Ltd., and cloned into the pET28a expression vector.

[0085] Nucleotide sequence (SEQ ID NO.13): ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACA GAGCCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA.

[0086] Amino acid sequence (SEQ ID NO. 14): MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP.

[0087] The nucleotide terminal is a TAA stop codon and is not expressed.

[0088] The recombinant plasmid pET28a-HPV16 / E7 was transformed into BL21 (DE3) competent cells (Molecular Cloning, 3rd edition, Science Press) according to conventional methods. The transformed bacteria were spread on LB agar plates (containing 50 μg / mL kanamycin) and cultured at 37°C overnight. A single colony was picked and inoculated into 5 mL LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C, 220 rpm, and shaken overnight. 1% of the total volume of the culture medium was inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C, 220 rpm, and shaken for about 3 hours until the OD 600 The concentration of the protein was 0.6-0.9, and IPTG was added to a final concentration of 0.1 mM. After induction at 30°C and 200 rpm for 4 hours, the bacteria were collected to obtain the HPV16 / E7 recombinant protein.

[0089] 9. Identification of the binding activity of monoclonal antibodies to oral Streptococcus TMPC recombinant protein and whole oral Streptococcus

[0090] Activity identification of paired monoclonal antibodies.

[0091] According to the above-mentioned indirect ELISA method, oral Streptococcus TMPC recombinant protein (1 μg / ml) was coated, and the cultured oral Streptococcus (1×10 9CFU / mL) by centrifugation at 8000 r / min. The collected bacterial pellet was resuspended in coating solution, frozen and thawed three times, and then centrifuged at 12000 r / min for 10 minutes. The supernatant was collected for coating. The screened purified monoclonal antibody was diluted in a gradient of 10µg / ml, 1µg / ml, 100ng / ml, 10ng / ml, and 1ng / ml. A commercially available Candida albicans monoclonal antibody (Zhuhai Bomei) was used as a negative control to detect the binding activity of the screened monoclonal antibody 5E10.

[0092] See also Figure 4 and Figure 5 , 5E10 represents the monoclonal antibody 5E10, Ctrl represents the commercially available Candida albicans monoclonal antibody (Zhuhai Bomei), and the horizontal axis mAb is the abbreviation of "monoclonal antibody", which is translated into Chinese as "monoclonal antibody". Figure 4 and attached Figure 5 In the table, mAb Conc. indicates the concentration of the monoclonal antibody. Indirect ELISA results showed that monoclonal antibody 5E10 reacted positively with oral S. TMPC recombinant protein at 10 ng / ml, demonstrating strong binding activity. However, the lowest mAb concentration for positive results on the oral S. TMPC-coated plate was 100 ng / ml, and negative results were observed when the mAb was diluted to 10 ng / ml. This may be due to the low proportion of TMPC protein in the total bacterial load, which reduced the effective coating content on the ELISA plate and resulted in lower test readings.

[0093] In summary, the present invention successfully expressed and purified the oral Streptococcus TMPC recombinant protein using a prokaryotic expression system, screened for the monoclonal antibody 5E10 that reacts with whole oral Streptococcus, and established a double-antibody sandwich ELISA assay based on this. This assay can effectively detect oral Streptococcus and the oral Streptococcus TMPC recombinant protein, and is expected to be used in clinical testing of oral Streptococcus or related scientific research. This application further detects the presence of oral Streptococcus and its expression level in samples by preparing and using the monoclonal antibody 5E10 specific for the oral Streptococcus TMPC protein.

[0094] 10. Monoclonal antibody variable region gene cloning and sequencing

[0095] Total RNA from hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers. Universal primers for the mouse antibody variable regions were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the third-round PCR primers. The PCR products were gel-cleaved and purified, then ligated into the pUC19 vector and transformed into the TOP10 strain. After incubation at 37°C for 14 hours, single colonies were picked and sequenced to obtain the gene sequences of the monoclonal antibody light and heavy chains.

[0096] Monoclonal antibody 5E10 variable region sequence

[0097] Heavy chain:

[0098] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5E10 is shown in SEQ ID NO.9:

[0099] CAGATCCAGCTGCAGCAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCATCACCTGCACCGTGAGCGGCTTCAGCCTGACCAGGTACAGCGTGCACTGGGTGAGGCAGCCCCCCGGCAAGGACCTGGAGTGGCTGGGCCACATCTGGGCCCAGCAGCTGACCGCCTACAACAGCCC CCTGATGAAGAGGCTGAGCATCAGCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGGTGAACAGCCTGCACACCGACGACACCGCCATGTACTTCTGCGCCAGGGGCTACCTGCACGTGGTGGGCGAGAGCTACATCAGCATGGACCAGTGGGGCCAGGGCACCAGCGTGACCGTGAGCGCC.

[0100] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5E10 is shown in SEQ ID NO.7:

[0101] QIQLQQSGPGLVAPSQSLSITCTVSGFSLTRYSVHWVRQPPGKDLEWLGHIWAQQLTAYNSPLMKRLSISKDNSKSQVFLKVNSLHTDDTAMYFCARGYLHVVGESYISMDQWGQGTSVTVSA.

[0102] CDR region annotation:

[0103] The amino acid sequences of the complementarity determining region CDR-H1 of the heavy chain variable region of the monoclonal antibody 5E10 are shown in SEQ ID NO. 1: RYSVH;

[0104] The amino acid sequences of the complementarity determining region CDR-H2 of the heavy chain variable region of the monoclonal antibody 5E10 are shown in SEQ ID NO. 2: HIWAQQLTAYNSPLMK;

[0105] The amino acid sequence of the complementarity determining region CDR-H3 of the heavy chain variable region of the monoclonal antibody 5E10 is shown in SEQ ID NO. 3: GYLHVVGESYISMDQ.

[0106] Light chain:

[0107] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 5E10 is shown in SEQ ID NO.10:

[0108] GACATCCAGATGACCCAGAGCCCCGCCAGCCTGAGCGCCAGCGTGGGCGAGACCGTGACCATCACCTGCGGCGCCAGCACCGACATCTACAGCGCCCTGATGTGGTACCAGAGGAAGCAGGGCAAGAGCCCCCAGCTGCTGATCTACGGCGCCATCAACCTGGCC AGGGGCATGAGCAGCAGGTTCAGCGGCAGCGGCAGCGGCAGGCAGTACAGCCTGAAGATCAGCAGCCTGCACCCCGACGACGTGGCCACCTTCTACTGCCAGAACAAGCTGCAGCAGCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGCTGAAGAGGACCGTG.

[0109] The amino acid sequence of the light chain variable region of monoclonal antibody 5E10 is shown in SEQ ID NO.8:

[0110] DIQMTQSPASLSSASVGETVTITCGASTDIYSALMWYQRKQGKSPQLLIYGAINLARGMSSRFSGSGSGRQYSLKISSLHPDDVATFYCQNKLQQPYTFGGGTKLELKRTV.

[0111] CDR region annotation:

[0112] The amino acid sequences of the complementarity determining region CDR-L1 of the light chain variable region of the monoclonal antibody 5E10 are shown in SEQ ID NO. 4: GASTDIYSALM;

[0113] The amino acid sequences of the complementarity determining region CDR-L2 of the light chain variable region of the monoclonal antibody 5E10 are shown in SEQ ID NO. 5: GAINLAR;

[0114] The amino acid sequence of the complementarity determining region CDR-L3 of the light chain variable region of the monoclonal antibody 5E10 is shown in SEQ ID NO. 6: QNKLQQPYT.

[0115] So far, the various embodiments of the present application have been described in detail. To avoid obscuring the concept of the present application, some details well known in the art have not been described. Based on the above description, those skilled in the art can fully understand how to implement the technical solutions disclosed herein.

[0116] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art will understand that the above examples are for illustration only and are not intended to limit the scope of the present application. Those skilled in the art will understand that the above embodiments may be modified or some technical features may be replaced with equivalents without departing from the scope and spirit of the present application.

Claims

1. A monoclonal antibody 5E10 for detecting oral Streptococcus TMPC protein, characterized in that: The heavy chain variable region of the monoclonal antibody 5E10 includes three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3). The amino acid sequence of CDR-H1 is shown in SEQ ID NO. 1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO. 2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

3. The light chain variable region of the monoclonal antibody 5E10 includes three complementarity determining regions CDR-L1, CDR-L2 and CDR-L3. The amino acid sequence of the CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO.5, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO.

6.

2. The monoclonal antibody 5E10 for detecting oral Streptococcus TMPC protein according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5E10 is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of the monoclonal antibody 5E10 is shown in SEQ ID NO.

8.

3. The monoclonal antibody 5E10 for detecting oral Streptococcus TMPC protein according to claim 2, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5E10 is shown in SEQ ID NO.

9.

4. The monoclonal antibody 5E10 for detecting oral Streptococcus TMPC protein according to claim 3, characterized in that The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5E10 is shown in SEQ ID NO.

10.

5. Use of the monoclonal antibody 5E10 according to claim 1 in the preparation of a tool for detecting oral Streptococcus TMPC protein.

6. The use according to claim 5, characterized in that The tool is used for in vitro detection of oral streptococcal TMPC protein in biological samples, and the in vitro detection is not used for diagnosis of diseases.

7. The use according to claim 6, characterized in that The tools include reagents, test kits, test strips and antibody chips.

8. The use according to claim 7, characterized in that The kit includes a double antibody sandwich ELISA kit.

9. The use according to claim 8, characterized in that The kit is coated and labeled with the monoclonal antibody 5E10.

10. The use according to claim 9, characterized in that The monoclonal antibody 5E10 is used to construct an ELISA detection system, which is used to detect oral streptococcal TMPC protein in biological samples. The ELISA detection system is not used for disease diagnosis.

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