Monoclonal antibody of recombinant humanized anti-treponema pallidum membrane protein TP17 and application thereof

Abstract

The invention belongs to the technical field of gene engineering, antibody engineering and immunodiagnosis, and discloses a recombinant humanized monoclonal antibody for resisting treponema pallidum membrane protein TP17 and application of the recombinant humanized monoclonal antibody for resisting the treponema pallidum membrane protein TP17. The sequence of a light chain amino acid fragment of the monoclonal antibody is shown as SEQ ID NO.11, and the sequence of a light chain amino acid fragment of the monoclonal antibody is shown as SEQ ID NO.12. The sequence of a light chain amino acid fragment of the monoclonal antibody is shown as SEQ ID NO.13. The sequence of a heavy chain amino acid fragment of the monoclonal antibody is as shown in SEQ ID NO. 12. The invention further discloses the monoclonal antibody, nucleic acid encoded by the monoclonal antibody, a recombinant expression vector carrying the nucleic acid and a host cell of the recombinant expression vector. The monoclonal antibody has high specificity and high detection sensitivity, and can be applied to preparation of a treponema pallidum detection kit and detection of treponema pallidum.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering, antibody engineering, and immunodiagnostic technology, and particularly to a recombinant humanized monoclonal antibody against Treponema pallidum membrane protein TP17 and its applications. Background Technology

[0002] Syphilis is a chronic, systemic infectious disease caused by Treponema pallidum. Treponema pallidum can be found on the skin, secretions, and blood of patients with both overt and latent syphilis. Also known as Treponema pallidum (TP), it is the pathogen of the sexually transmitted disease syphilis. The TP gene is a 1,138,006 bp circular DNA molecule with 1,041 open reading frames (ORFs), 55% of which translate into functional proteins. The outer membrane of Treponema pallidum does not contain lipopolysaccharide but is rich in membrane proteins. It is estimated that TP possesses 22 membrane proteins, with Tp17, Tp47, Tp44.5, Tp36, Tp35, and Tp15 being among the most abundant. Among these, the outer membrane lipoproteins Tp17 and Tp15 are highly immunogenic and are used as representative diagnostic antigens in the serological diagnosis of Treponema pallidum.

[0003] Syphilis patients can present with typical clinical manifestations of primary and secondary syphilis, or latent syphilis, and in rare cases, tertiary syphilis. The clinical presentation of syphilis is often complex and difficult to discern. Therefore, laboratory testing is crucial for the diagnosis of syphilis.

[0004] Dark-field microscopy for Treponema pallidum has long been considered the gold standard for syphilis diagnosis; however, this method requires highly skilled personnel and is not widely available in most hospitals. Other methods for detecting Treponema pallidum include serological testing for specific and non-specific antibodies, as well as quantitative real-time polymerase chain reaction (qPCR). Serological testing remains the most important method for syphilis diagnosis; however, it has limitations such as prozone reaction and serum fixation, meaning antibodies may not be present in the early stages of infection, leading to delayed diagnosis and false positives in certain populations.

[0005] Therefore, it is essential to develop a syphilis diagnostic antibody with high sensitivity, affinity, and specificity. Current technology requires further improvement. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a recombinant humanized monoclonal antibody against Treponema pallidum membrane protein TP17 and its applications. The recombinant humanized monoclonal antibody against Treponema pallidum membrane protein TP17 has high specificity and high detection sensitivity, and can be used in the preparation of Treponema pallidum detection kits and in the detection of Treponema pallidum.

[0007] To address the above problems, the present invention provides the following technical solution: In one aspect, this application provides a recombinant humanized monoclonal antibody against Treponema pallidum membrane protein TP17, comprising a light chain variable region and a heavy chain variable region. The light chain variable region comprises three light chain complementarity-determining regions, LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5. The heavy chain variable region comprises heavy chain complementarity-determining regions, HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7.

[0008] Optionally, the C-terminus of the variable region of the light chain is connected to a human-derived light chain constant region LH, the sequence of which is shown in SEQ ID NO.9; the C-terminus of the variable region of the heavy chain is connected to a human-derived heavy chain constant region LH, the sequence of which is shown in SEQ ID NO.10. Optionally, a signal peptide sequence is added to the N-terminus of both the light chain variable region and the heavy chain variable region, as shown in SEQ ID NO.8.

[0009] Preferably, the sequence of the light chain amino acid fragment of the monoclonal antibody is shown in SEQ ID NO.11, and the sequence of the heavy chain amino acid fragment of the monoclonal antibody is shown in SEQ ID NO.12.

[0010] This application selects Treponema pallidum membrane protein TP17 as the target antigen and uses hybridoma technology, hybridoma sequencing technology, and gene recombination technology to obtain a recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody.

[0011] Specifically, the variable region sequence of this recombinant monoclonal antibody was obtained by preparing hybridoma cells using recombinant TP17 immunization of mice and sequencing them using hybridoma gene sequencing technology. The light and heavy chain constant regions were then humanized. The full-length nucleotide sequences of the light and heavy chains were codon-optimized to obtain the coding gene of the improved recombinant monoclonal antibody. This gene was then constructed into the cell expression vector pCDNA3.4 and transfected into mammalian 293 suspension cells for recombinant expression. The expression product was extracted and purified to obtain the recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody, which was named R-Anti-TP17-Ab1.

[0012] Specifically, the sequence of the light chain amino acid fragment of the recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody is shown in SEQ ID NO.11, and the amino acid sequence from the N-terminus to the C-terminus is as follows: Signal-LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4-LH(hIgκC); wherein, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.1.

[0013] Among them, Signal is the signal peptide sequence added to the N-terminus of the variable region of the light and heavy chains, LCDR1, LCDR2, and LCDR3 are the complementarity-determining regions of the light chains, LFR1-LFR4 are the four backbone regions of the light chains, and LH (hIgκC) is the constant region of the human light chain.

[0014] The light chain variable region (VL) includes one signal peptide sequence, three light chain complementarity-determining regions (LCDR1, LCDR2, LCDR3), and four backbone regions (FR1-FR4).

[0015] The light chain variable region also includes four backbone regions: LFR1, LFR2, LFR3, and LFR4. The sequences of LFR1, LFR2, LFR3, and LFR4 are shown in SEQ ID NO.15~18.

[0016] The sequence fragments of LCDR1 and LCDR3 in the three light chain complementarity-determining regions are shown in SEQ ID NO. 3-4, respectively, and the sequence of CDR2 is NAK. The signal peptide sequence of the light chain is shown in SEQ ID NO. 8; its humanized constant region sequence (LH(hIgκC)) is shown in SEQ ID NO. 9. The sequence of the amino acid fragment of the light chain is shown in SEQ ID NO. 11.

[0017] The heavy chain amino acid fragment of the recombinant monoclonal antibody is shown in SEQ ID NO.12, and its amino acid sequence from the N-terminus to the C-terminus is as follows: Signal-HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4-CH (HIgG1).

[0018] The heavy chain variable region amino acid sequence is shown in SEQ ID NO.2, its heavy chain complementarity-determining region (HCDR1, HCDR2, HCDR3) sequence fragments are shown in SEQ ID NO.5-7 respectively, the heavy chain signal peptide sequence is shown in SEQ ID NO.8, and the heavy chain humanized constant region sequence (CH(HIgG1)) is shown in SEQ ID NO.10. The sequence of the heavy chain amino acid fragment is shown in SEQ ID NO.12. HFR1-HFR4 are the four backbone regions of the heavy chain, and their sequences are shown in SEQ ID NO.19-22.

[0019] Secondly, this application provides an isolated nucleic acid that encodes the aforementioned recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody.

[0020] Thirdly, this application also provides isolated nucleic acids comprising: a full-length coding gene sequence encoding the light chain of the monoclonal antibody as shown in SEQ ID NO.13, and a coding gene sequence encoding the full-length heavy chain of the monoclonal antibody as shown in SEQ ID NO.14.

[0021] After translating the light chain amino acid sequence (SEQ ID NO. 11) and heavy chain amino acid sequence (SEQ ID NO. 12) of the above recombinant monoclonal antibody into nucleotide sequences, codon optimization was performed to obtain the full-length light chain nucleotide sequence (SEQ ID NO. 13) and the full-length heavy chain nucleotide sequence (SEQ ID NO. 14). These sequences are preferred nucleotide sequences optimized for mammalian cell expression codons.

[0022] The heavy and light chains of the above-mentioned recombinant monoclonal antibodies can also be optimized differently based on different types of expression systems to better express them in the corresponding expression systems.

[0023] Fourthly, this application also provides a recombinant expression vector carrying the aforementioned isolated nucleic acid.

[0024] Optionally, the recombinant expression vector is a plasmid, a granule, or a viral vector.

[0025] The plasmids include: pCDNA3.4 vector or others.

[0026] Optionally, the recombinant expression vector is: inserting the aforementioned nucleotide sequences, such as those shown in SEQ ID NO.13 and SEQ ID NO.14, into the pCDNA3.4 vector to successfully obtain light chain recombinant plasmids and heavy chain recombinant plasmids, which are subsequently used for transfection of host cells.

[0027] Fifthly, this application also provides a host cell containing the aforementioned isolated nucleic acid or transformed with the aforementioned recombinant vector.

[0028] The host cells are prokaryotic or eukaryotic cells, such as Escherichia coli, Pichia pastoris, CHO cells, or HEK293 cells (human embryonic kidney cells 293).

[0029] Optionally, the host cell is a HEK293 cell.

[0030] The recombinant monoclonal antibody is expressed in mammalian cells using gene recombination technology, and has the characteristics of high yield and high purity. Furthermore, as a detection antibody, the recombinant monoclonal antibody exhibits the advantages of high sensitivity and high specificity.

[0031] Sixthly, this application also provides the use of the aforementioned monoclonal antibody, the aforementioned isolated nucleic acid, the aforementioned recombinant expression vector, or the aforementioned host cell in the preparation of a kit for enriching, isolating, and / or detecting Treponema pallidum.

[0032] Seventhly, this application also provides a Treponema pallidum detection kit, which includes the aforementioned monoclonal antibody.

[0033] Optionally, in the Treponema pallidum detection kit, the kit is a colloidal gold detection kit. It is a colloidal gold immunochromatographic test strip, which includes a recombinant humanized Treponema pallidum membrane protein TP17 monoclonal antibody. This monoclonal antibody subtype is human IgG1 / κ, and is named R-Anti-TP17-Ab1, capable of specifically recognizing Treponema pallidum membrane protein T17.

[0034] The present invention has the following beneficial effects: 1. This invention provides a recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody that selects TP17 as the target antigen and obtains it using hybridoma technology, hybridoma sequencing technology, and gene recombination technology. The successful acquisition of this recombinant monoclonal antibody lays the foundation for establishing a syphilis antigen detection and diagnosis method based on immunological technology.

[0035] 2. The recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody has high specificity and high detection sensitivity, and can be used in the preparation of Treponema pallidum detection kits and in the detection of Treponema pallidum, and can be used for the detection of Treponema pallidum in the early stage of infection.

[0036] 3. The preparation method of monoclonal antibody against Treponema pallidum membrane protein TP17 eliminates the ethical issues associated with the use of mice in the production process. At the same time, the humanization of the antibody constant region makes it more specific in application, solving the non-specificity problem often encountered in the application of mouse monoclonal antibodies. Moreover, this recombinant monoclonal antibody can have multiple uses, laying the foundation for clinical and laboratory research on syphilis antigen diagnosis, syphilis antibody diagnosis, and Treponema pallidum vaccine. Attached Figure Description

[0037] Figure 1 Electrophoresis diagram of light and heavy chain PCR products of mouse monoclonal antibody against IgG subtype of Treponema pallidum membrane protein TP17; Lane 1: DL2000, Lane 2: light chain variable region amplification product, Lane 3: heavy chain variable region amplification product. Figure 2 The results of SDS-PAGE electrophoresis of the purified target monoclonal antibody; Figure 3 The results are shown in the SEC-HPLC of the purified target monoclonal antibody. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0039] Example 1: Preparation of a monoclonal antibody against Treponema pallidum membrane protein TP17 1.1 Preparation of hybridoma cells resistant to Treponema pallidum membrane protein TP17 (1) Preparation of immunogen TP17: The sequence of Treponema pallidum membrane protein TP17 was analyzed using the NCBI database (refer to Genbank: WP_010881883.1). Recombinant TP17 protein was obtained through gene synthesis, plasmid construction (construction into pET28a vector), transformation and expression of Escherichia coli, and purification.

[0040] (2) Immunization of mice: Female 6-8 week old balb / c mice were immunized with recombinant protein TP17; the immunoglobulin was mixed with an equal volume of Freund's complete adjuvant at a dose of 75 μg / mouse, emulsified completely, and administered subcutaneously to multiple sites on the back of the mice; three weeks later, a booster immunization was performed, with the immunoglobulin mixed with an equal volume of incomplete Freund's adjuvant at a dose of 35 μg / mouse, emulsified completely, and administered subcutaneously to multiple sites on the back of the mice; one week later, blood was collected from the tails of the mice to detect serum titers. When the serum titer reached 100,000 or higher, fusion was prepared; three days before fusion, the mice were immunized by intraperitoneal injection without adjuvant, with an immunization dose of 100 μg / mouse. (3) Cell fusion: During fusion, the spleen of immunized mice was taken, the cells were ground and dispersed, and mouse B lymphocytes were collected after centrifugation. They were mixed at a ratio of 7:1 (B lymphocytes: SP2 / 0), centrifuged, and washed with serum-free DMEM medium to ensure that there was no fetal bovine serum in the mixed cells. Cell fusion was performed with PEG. After full fusion, fusion was terminated with DMEN medium. Finally, the fused cells were resuspended with HAT medium, placed in a 96-well cell culture plate, and cultured in a carbon dioxide incubator. (4) Hybridoma cell screening and subcloning: The medium was completely replaced with HAT medium on the 7th day after fusion, and the culture supernatant was detected by indirect ELISA on the 9th day.

[0041] The specific testing methods are as follows: Coating: Dilute the GST-tagged protein to 1 μg / mL with CBS buffer (pH 9.6), coat 100 μL / well into an ELISA plate, and incubate overnight at 4°C. Wash the plate 5 times with PBST buffer and blot dry. Block: Block the plate with PBST buffer containing 3% BSA, 200 μL / well, and incubate at 37°C for 2 h. Wash the plate 5 times with PBST buffer and blot dry. Detection: Add the cell supernatant to be tested to the plate, 100 μL / well, with mouse immune serum as a positive control and HAT medium as a blank control, and incubate at 37°C for 1 h. Wash the plate 5 times with PBST buffer and blot dry. Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-mouse antibody 20,000 times with PBST buffer and add 100 μL / well to the plate, incubate at 37°C for 1 h. Wash the plate 5 times with PBST buffer and blot dry. Color development: Develop the color with TMB chromogenic solution in the dark, 100 μL / well, and incubate at 37°C for 15 min. Terminate with 2M sulfuric acid stop solution, 50 μL / well, and detect at 450 nm. Select high valence (OD) 450Cells with a density ≥2.0 g / cm³ were subcloned using a serial dilution method. 200-500 cells were transferred to well A1 of a new 96-well cell culture plate and diluted 2-fold both longitudinally and laterally. After 7-9 days, the culture supernatant was tested using an indirect ELISA method. Wells with a single positive cell cluster were selected for subcloning until the positive rate of the 96-well plate reached 100%. Subcloning was repeated twice more until the positive rate reached 100% in both wells. Subcloning was then terminated to ensure the stability of the hybridoma cell line.

[0042] 1.2 Sequencing of hybridoma cells containing the anti-Treponema pallidum membrane protein TP17 (1) Hybridoma cell line culture and RNA extraction The prepared hybridoma cells were cultured in a large-scale manner, and the cultured cells were collected. RNA was extracted from the cells according to the instructions of Thermo's RNA extraction kit.

[0043] (2) Reverse transcription Following the instructions of Thermo's kit, the extracted RNA was immediately reverse transcribed to prepare cDNA.

[0044] (3) PCR amplification and recovery Using universal primers for the variable region of the IgG subtype mouse monoclonal antibody, and with the above-mentioned cDNA as a template, PCR amplification was performed on the variable regions of the light and heavy chains. The amplification products were identified by DNA gel extraction, and the PCR products were recovered by gel cutting using a TIANGEN kit. The light and heavy chain PCR products are shown below. Figure 1 .

[0045] (4) The above light chain and heavy chain PCR products were identified by vector construction and expression, positive clones were selected and sent to a sequencing company for sequencing.

[0046] 1.3 Analysis of variable region sequences, humanization modification, and construction of recombinant plasmids (1) Sequencing results were analyzed using the IMGT antibody variable region analysis tool in the database. The amino acid sequence of the light chain variable region (VL) of the anti-Treponema pallidum membrane protein TP17 monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO.2. The sequencing results of the light and heavy chain variable regions include one signal peptide sequence, three complementarity-determining regions, and four backbone regions (FR1-FR4).

[0047] Analysis revealed that the light chain complementarity determination regions LCDR1 and LCDR3 sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, while the heavy chain complementarity determination regions HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7.

[0048] Adding the same signal peptide sequence to the N-terminus of the variable region of the light and heavy chains, as shown in SEQ ID NO.8, can promote efficient secretory expression of the protein. At the same time, the constant region of the antibody light and heavy chains is humanized to reduce interference from anti-mouse antibody (HAMA), avoid false positives, and improve specificity.

[0049] A human light chain constant region (hIgκC) sequence was added to the C-terminus of the light chain variable region: the sequence is referenced in Genbank: P01834.2, as shown in SEQ ID NO.9; a human heavy chain constant region (hIgG1-CH) was added to the C-terminus of the heavy chain variable region: the sequence is referenced in Genbank: AFR78282.1, as shown in SEQ ID NO.10; the full-length amino acid sequences of the humanized light and heavy chains were obtained: the sequences are shown in SEQ ID NO.11 and SEQ ID NO.12, respectively; and the translation codons were optimized, the optimized full-length base sequence of the light chain is shown in SEQ ID NO.13; the full-length base sequence of the heavy chain is shown in SEQ ID NO.14.

[0050] The optimized light and heavy chain base sequences were given to a gene synthesis company (Qingke Biotechnology Co., Ltd.) and synthesized into the cell expression vector pCDNA3.4. The light chain recombinant plasmid was named pCDNA3.4-hL01; the heavy chain recombinant plasmid was named pCDNA3.4-hH01.

[0051] 1.4 Expression and purification of recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody (1) Cell resuscitation A. Remove one self-acclimated HEK293 suspension cell (human embryonic kidney cell 293) from the liquid nitrogen tank. Immediately place the cryovial into a water bath for rapid thawing, no more than 1 minute. The facility temperature is 37°C; ensure the temperature remains stable at 37°C during thawing. When placing the cryovial in the water bath, ensure the liquid level does not exceed 2 / 3 of the tube's height to avoid water contact with the tube opening and contamination.

[0052] B. In a clean bench, transfer 293 cells to a centrifuge tube containing 10 mL of culture medium, centrifuge at 1000 rpm for 10 min, collect the cell pellet, resuspend in culture medium, count the cells, and adjust the cell density to 0.4-0.6 × 10⁻⁶. 6 cells / mL.

[0053] C. Transfer the resuspended cells to a shake flask, ensuring the culture volume does not exceed 25% of the total shake flask volume. Place the shake flask in a CO2 shaker and culture at 37°C, 5% CO2, and 110 rpm for approximately 3 days, then observe and passage the cells.

[0054] (2) Cell passage A. After approximately 3 days of cell resuscitation, the cells are in the logarithmic growth phase. Cells are then harvested for cell density determination (×10⁻⁶). 6 Cell counts (cells / mL) and viability (%) were measured, and cells were passaged after the tests.

[0055] B. Calculate cell density ≥2×10 6 When the cell count / mL is above 90%, use 0.8-1.2 × 10⁻⁶ cells / mL. 6 The cells were passaged at a density of cells / mL and then transferred to shake flasks for culture.

[0056] C. Continue with 1-2 more passages. Before transient transfection, cells need to be stably passaged at least 3 times. If the cell condition is poor or cell acclimatization is required during passage, ensure stable passage 3 times before transfection after the cell condition is stable or acclimatization is complete.

[0057] (3) Instantaneous transfection A. Plasmid and PEI pretreatment: On the day of transfection, add light chain recombinant plasmid: heavy chain recombinant plasmid: transfection reagent (PEI) at a mass ratio of 2:1:3 (for example, to transfect 1L of suspension cells, add light chain recombinant plasmid: heavy chain recombinant plasmid: transfection reagent (PEI) at masses of 2mg, 1mg, and 3mg respectively). Add to an appropriate amount of 293 suspension cell culture medium, mix thoroughly, and let stand for 10-15 minutes.

[0058] B. Transfection: Count the prepared cells and adjust the cell density to 3.0-3.5 × 10⁻⁶ using the 293 suspension cell culture medium prepared in step (2). 6 cells / mL.

[0059] C. Load the transfected cells into sterile shake flasks (volume ≤30%) and place the shake flasks in a carbon dioxide shaker for incubation.

[0060] D. Feeding: 24 hours after transfection, add feed medium at 3-5% of the culture volume; add L-glutamine solution at 1% of the culture volume, and continue culturing. On days 3 and 5 post-transfection (day 0 is the day of transfection), calculate cell viability (3-day cell viability ≥90%; 5-day cell viability ≥75%), and add feed medium at 3-5% of the culture volume; add L-glutamine solution at 1% of the culture volume, and continue culturing. On day 7, test cell viability (cell viability ≥50%) and collect the culture supernatant. The 293 suspension cell culture medium and feed medium mentioned above were purchased from Peiding Biotechnology Co., Ltd.

[0061] (5) Collection and purification of expression supernatant ① Experimental method: A. Supernatant collection: The culture product is placed in a centrifuge bottle, balanced, and centrifuged at 8℃ and 9000rpm for 30min. The supernatant is collected for purification.

[0062] B. Protein A-column affinity chromatography purification method (during purification, the ambient temperature is controlled at 20-25℃). Equilibration: Connect the Protein-A chromatography column to a nucleic acid computer UV detector for purification. Equilibrate the column with 10 volumes of loading buffer (20mM PBS, pH 7.4) and adjust the absorbance (OD). 280 The concentration was increased to 0.10, and the flow rate was controlled at 3 mL / min ± 10%.

[0063] Sample loading: Add the processed cell expression supernatant to the chromatography column and collect the flow-through using a centrifuge tube; after all the sample has passed through the column, load the flow-through again and collect the flow-through at the same time, and label the flow-through. Washing: After loading the sample, wash with approximately 10 column volumes of loading buffer; stop washing when the absorbance stabilizes at 0.10-0.20.

[0064] Elution: Add 30 μl of neutralization buffer (1 M Tris-HCl, pH 9.0) to a 600 μL LEP tube beforehand, and then elute with 10 column volumes of elution buffer (0.1 M Gly-HCl, pH 2.5-3.0). During elution, when the absorbance rises to 0.20-0.50 (the absorbance rises very quickly; be ready to collect protein as the absorbance starts to rise from the baseline to avoid protein loss due to insufficient preparation), start collecting the eluent (target protein). Collect 400 μL of eluent per tube and mix it thoroughly. Stop collecting when the absorbance drops to 0.20-0.50.

[0065] Equilibration: Equilibrate with 10 column volumes of the loading buffer. Detect the pH of the outlet liquid using pH test paper. End equilibration when the pH matches that of the loading buffer. Rinse the column with 20% ethanol and store in 20% ethanol at 4°C.

[0066] Dialysis: Place the collected protein in a dialysis bag (specifications: MWCO=8-14kDa; diameter=8mm), use 20mMPBS (pH 7.4) as the dialysate, twice a day, changing the solution once in the morning and once in the evening, and dialyze at 2-8℃ for 3 days (at least 6 times).

[0067] After dialysis, the protein was removed and placed in a 50 ml centrifuge tube. The protein was centrifuged at 7500-9000 rpm and 4-15℃ for 30 min. The supernatant was collected and the concentration was measured. The obtained protein sample is the recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody (R-Anti-TP17-Ab01).

[0068] The purified recombinant monoclonal antibody R-Anti-TP17-Ab01 was analyzed by SDS-PAGE gel imaging and SEC-HPLC.

[0069] The purified product was analyzed by UV spectrophotometry to detect OD280. The concentration of the recombinant monoclonal antibody was calculated using the formula: concentration (mg / ml) = OD280 / 1.43. The yield of the recombinant monoclonal antibody was then calculated by multiplying the concentration and volume.

[0070] ②Experimental Results and Analysis The results are as follows Figure 2 The SDS-PAGE gel results show that the light and heavy chains of the recombinant monoclonal antibody are correctly expressed, and their sizes are consistent with the theoretical values ​​of 25 kDa for the light chain and 50 kDa for the heavy chain.

[0071] Figure 3 The results from SEC-HPLC analysis show that the recombinant monoclonal antibody has a single peak at 7.892 min with a peak area of ​​up to 98%, indicating that the recombinant monoclonal antibody can form the correct dimer and the dimer purity can reach 98%.

[0072] The signal peptide sequence selected for the monoclonal antibody in this invention, as well as the optimization of the translation codon, transfection conditions, and purification methods, can all improve protein yield. Calculations show that the yield of this recombinant monoclonal antibody can reach 800 mg / L, which is high.

[0073] Example 2: Detection of the sensitivity of recombinant monoclonal antibody R-Anti-TP17-Ab01 The recombinant monoclonal antibody R-Anti-TP17-Ab01 prepared in Example 1 of this invention can be used to quantitatively detect Treponema pallidum membrane protein Tp17, and can be detected indirectly by ELISA.

[0074] 2.1 Detection Method ① Dilute the monoclonal antibody at 0.5 μg / mL in 0.01 M pH 9.6 carbonate buffer coating solution, 100 μL / well, and coat overnight at 4°C.

[0075] ② Wash the plate three times with PBST, dry it, add 200ul of blocking buffer per well, and block for 2 hours.

[0076] ③Wash the plate three times with PBST, dry it, and set aside.

[0077] ④ Sample addition: Dilute the recombinant TP17 protein to final concentrations of 250 ng / mL, 125 ng / mL, 60 ng / mL, 30 ng / mL, 15 ng / mL, and 6 ng / mL, respectively. Add 100 μL of each to a 96-well microplate and allow to react at room temperature for 2 hours.

[0078] ⑤ Wash the plate three times with PBST and dry it. Add 0.1 mL of His-HRP antibody diluted 1:10000 with PBST to each well and incubate at room temperature for 1 hour.

[0079] ⑥ Wash the plate five times with PBST and dry it. Add 100 μL of TMB colorimetric solution and react at room temperature in the dark for 10 minutes.

[0080] ⑦ Add 50 μL of stop solution to terminate the reaction; measure the OD value at 450 nm, and consider P / N ≥ 2.1 as positive.

[0081] ⑧ Determine the sensitivity of recombinant monoclonal antibody R-Anti-TP17-Ab01 in detecting TP17.

[0082] 2.2 Test Results and Analysis The results are shown in Table 1. The lowest concentration of TP17 detected was 15 ng / ml, with an OD value of 0.642, indicating high sensitivity.

[0083] Table 1. ELISA detection of recombinant monoclonal antibody R-Anti-TP17-Ab01

[0084] Example 3: Application of recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody (R-Anti-TP17-Ab01) in gold-labeled chromatographic Treponema pallidum antigen detection reagent This embodiment provides an application of a gold-labeled chromatographic detection kit for Treponema pallidum antigen prepared using the recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody prepared in Example 1. In this embodiment, the kit is a Treponema pallidum antigen detection test strip.

[0085] 1. Preparation of a gold-labeled chromatographic Treponema pallidum antigen detection kit (1) Colloidal gold labeling Take 1 mL of colloidal gold and place it in a small glass beaker. Add an appropriate amount of 0.2 Mk2CO3 to adjust the pH to 7.6 and stir for 1 min. Add 20 μg of recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody (R-Anti-TP17-Ab01) and stir for 1 min. Add 100 μL of 1% PEG20000 and stir for 1 min. Centrifuge at 5000g for 10 minutes, discard the supernatant, and resuspend the precipitate in 100 μL of colloidal gold dilution buffer (20 mM PPB, 150 mM NaCl, 0.1% PEG20000, 0.1% Triton X-100, 2% Sucrose, 0.01% NaN3). Mix thoroughly and store at 4℃. Dilute the above gold-labeled complex 10 times with colloidal gold dilution buffer and coat it evenly on glass fiber. Dry at 37℃ to prepare a gold-labeled pad.

[0086] (2) Coating with nitrocellulose membrane (NC membrane) The coating diluent formulation is as follows: 0.01M PB, pH 7.4, with a mass percentage concentration of 0.05% trehalose. The test line (coated with another recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody (R-Anti-TP17-Ab02)) and the control line are sequentially coated onto the NC membrane from bottom to top, and then dried at 37°C for 30 min.

[0087] (4) Assembly Assemble the solidified NC membrane with the sample pad, S pad, gold label pad, PVC base plate, absorbent paper, and Mark sticker, and cut it into 3-4 mm test strips using a strip cutter for later use.

[0088] 2. Detection of the sensitivity and specificity of the kit (1) Sensitivity detection method: Experimental group: Treponema pallidum membrane protein TP17 was diluted to 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 0.5 μg / mL, 50 ng / mL, 25 ng / mL and 10 ng / mL, respectively, as positive control samples. The Treponema pallidum antigen test strip prepared in the above example was used to test these positive control samples to determine the sensitivity of the colloidal gold test strip in detecting Treponema pallidum membrane protein TP17.

[0089] Control group 1: Recombinant anti-TP17 monoclonal antibody expressed in mammalian HKE293 cells without humanization of the constant region was also used; the kit preparation method was the same as that of the experimental group.

[0090] Control group 2: Mouse anti-TP17 monoclonal antibody prepared by injecting ascites fluid into hybridoma cell lines. The kit preparation method was the same as that of the experimental group.

[0091] (2) Specific detection method: The prepared test strips were used to test the negative samples provided by 20 volunteers, and the color development was observed.

[0092] 3. Test Results and Analysis A. The sensitivity test results are shown in Table 2. These results indicate that, at the optimal labeling concentration, the experimental group's reagent strips could detect 10 ng / mL of Treponema pallidum membrane protein TP17, with a higher sensitivity than the control group.

[0093] B. When testing 20 negative samples, the control group showed false positives, while the monoclonal antibody prepared in this patent showed negative results, indicating that the test strip prepared using the monoclonal antibody in this patent has good specificity.

[0094] Table 2 Positive control and negative sample testing

[0095] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A recombinant humanized monoclonal antibody against Treponema pallidum membrane protein TP17, characterized in that, The sequence of the light chain amino acid fragment of the monoclonal antibody is shown as SEQ ID NO. 11, and the sequence of the heavy chain amino acid fragment of the monoclonal antibody is shown as SEQ ID NO.

12.

2. An isolated nucleic acid, comprising, The recombinant humanized monoclonal antibody against Treponema pallidum membrane protein TP17 according to claim 1.

3. The nucleic acid of claim 2, wherein Comprise: The full-length coding gene sequence encoding the light chain of the monoclonal antibody is shown as SEQ ID NO. 13, and the full-length coding gene sequence encoding the heavy chain of the monoclonal antibody is shown as SEQ ID NO.

14.

4. A recombinant expression vector, characterized in that, Carrying the isolated nucleic acid according to claim 2 or 3.

5. A host cell characterized in that, Containing the isolated nucleic acid according to claim 2 or 3 or transformed with the recombinant vector according to claim 4.

6. The host cell of claim 5, wherein, The host cell is a HEK293 cell.

7. Use of the monoclonal antibody according to claim 1, the isolated nucleic acid according to claim 2 or 3, the recombinant expression vector according to claim 4 or the host cell according to claim 5 in the preparation of a kit for enriching, isolating and / or detecting Treponema pallidum.

8. A Treponema pallidum detection kit, characterized by, Comprise the monoclonal antibody according to claim 1.

9. The kit of claim 8, wherein The kit is a colloidal gold immunochromatographic test strip.

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