Preparation method of monoclonal antibody for resisting treponema pallidum membrane protein TP17

Abstract

The invention belongs to the technical field of gene engineering, antibody engineering and immunodiagnosis, and discloses a preparation method of a monoclonal antibody for resisting treponema pallidum membrane protein TP17, which comprises the following steps: connecting a coding gene of the monoclonal antibody for resisting treponema pallidum membrane protein TP17 to an expression vector to construct a recombinant expression vector; transfecting the recombinant expression vector into a host cell, culturing, and finally collecting and separating a monoclonal antibody in a culture product; wherein the coding gene of the monoclonal antibody comprises a full-length coding gene sequence which is shown as SEQ ID NO.13 and is used for coding a light chain of the monoclonal antibody, and a coding gene which is shown as SEQ ID NO.14 and is used for coding the full length of a heavy chain of the monoclonal antibody. The yield and purity of the anti-treponema pallidum membrane protein TP17 monoclonal antibody prepared by the method are remarkably improved, the sensitivity and specificity of the antibody are high, and the monoclonal antibody can be applied to preparation of a treponema pallidum detection kit and detection of treponema pallidum.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of genetic engineering, antibody engineering and immunodiagnostic technology, and particularly relates to a preparation method of a monoclonal antibody against membrane protein TP17 of Treponema pallidum. BACKGROUND

[0002] Treponema pallidum, also known as Treponema pallidum (TP), is the pathogen of syphilis. It is also called Treponema pallidum because it is transparent and not easy to color. It was first discovered in 1905. It has 8 to 14 tight and orderly spiral structures, a slender body and straight ends, a dense and regular spiral, and active movement. The surface of the bacterial body has a capsular-like substance composed of lipopolysaccharide. The structure is similar to that of Leptospira. Treponema pallidum must grow and reproduce in living cells. It is extremely sensitive to temperature and dry environment, and has very weak resistance. Once separated from the body, it will die after 1 to 2 hours of drying or 5 minutes of heating at 50℃. The outer membrane of Treponema pallidum does not contain lipopolysaccharide, but contains abundant membrane proteins. It is speculated that TP has 22 membrane proteins, of which Tp17, Tp47, Tp44.5, Tp36, Tp35 and Tp15 are the most abundant. Among them, the outer membrane lipoprotein Tp17 and Tp15 have high immunogenicity and are also used as representative diagnostic antigens in the serological diagnosis of Treponema pallidum. Syphilis patients can have typical clinical manifestations of primary syphilis and secondary syphilis, and can also show latent syphilis. A small number of patients can also have tertiary syphilis. The clinical manifestations of syphilis are often complex and difficult to understand. Therefore, laboratory testing is very important for the diagnosis of syphilis.

[0003] In the traditional method, dark field examination is the gold standard for diagnosing Treponema pallidum, but this method has high technical requirements for the tester, so it is difficult to carry out in most hospitals. Other methods for detecting Treponema pallidum include serological tests for Treponema pallidum-specific antibodies and non-specific antibodies, as well as fluorescent quantitative polymerase chain reaction. Among them, serological testing is still the most important method for diagnosing syphilis at present, but the preparation of the current Treponema pallidum monoclonal antibody is high in cost and low in yield, and the purity needs to be improved.

[0004] Therefore, the prior art needs to be further improved. SUMMARY

[0005] In view of the above problems, the present application provides a preparation method of a monoclonal antibody against membrane protein TP17 of Treponema pallidum. The preparation method has high yield and purity of the prepared monoclonal antibody against membrane protein TP17 of Treponema pallidum, and the obtained monoclonal antibody has high sensitivity and specificity, and can be applied to industrial production.

[0006] To solve the above problems, the present application provides the following technical solutions: A preparation method of a monoclonal antibody against membrane protein TP17 of Treponema pallidum, comprising the following steps: The coding gene of the monoclonal antibody against membrane protein TP17 of Treponema pallidum is connected to an expression vector to construct a recombinant expression vector, the recombinant expression vector is transfected into host cells and cultured, and finally the recombinant humanized monoclonal antibody against membrane protein TP17 of Treponema pallidum is collected and separated from the culture product; The coding gene of the monoclonal antibody against membrane protein TP17 of Treponema pallidum includes 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.

[0007] The sequence of the light chain amino acid fragment of the recombinant humanized monoclonal antibody against membrane protein TP17 of Treponema pallidum is shown in SEQ ID NO. 11, and the sequence of the amino acid sequence from the N-terminal to the C-terminal region is in sequence: Signal-FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4-LH (hIgκC); wherein the light chain variable region amino acid sequence is shown in SEQ ID NO. 1.

[0008] The sequence of the light chain amino acid fragment of the recombinant humanized monoclonal antibody against membrane protein TP17 of Treponema pallidum is shown in SEQ ID NO. 11, and the sequence of the amino acid sequence from the N-terminal to the C-terminal region is in sequence: Signal-FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4-LH (hIgκC); wherein the light chain variable region amino acid sequence is shown in SEQ ID NO. 1.

[0009] The sequence of the light chain amino acid fragment of the recombinant humanized monoclonal antibody against membrane protein TP17 of Treponema pallidum is shown in SEQ ID NO. 11, and the sequence of the amino acid sequence from the N-terminal to the C-terminal region is in sequence: Signal-FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4-LH (hIgκC); wherein the light chain variable region amino acid sequence is shown in SEQ ID NO. 1.

[0010] The light chain variable region further includes four framework regions LFR1, LFR2, LFR3 and LFR4, and the sequences of LFR1, LFR2, LFR3 and LFR4 are shown in SEQ ID NO. 15-18.

[0011] The sequence fragments of LCDR1 and LCDR3 in the three light chain complementarity determining regions are shown in SEQ ID NO. 3-4, and the sequence of CDR2 is NAK. The signal peptide sequence of the light chain is shown in SEQ ID NO. 8; the light chain humanized constant region sequence (LH (hIgκC)) is shown in SEQ ID NO. 9. The sequence of the light chain amino acid fragment is shown in SEQ ID NO. 11.

[0012] The sequence of the heavy chain amino acid fragment of the recombinant monoclonal antibody is shown as SEQ ID NO. 12, and the amino acid sequence from the N-terminal to the C-terminal region is: Signal-FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4-CH (HIgG1).

[0013] The sequence of the heavy chain amino acid fragment of the recombinant monoclonal antibody is shown as SEQ ID NO. 12, and the amino acid sequence from the N-terminal to the C-terminal region is: Signal-FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4-CH (HIgG1).

[0014] Optionally, the preparation method of the monoclonal antibody specifically comprises the following steps: (1) The full-length coding gene sequence encoding the light chain of the monoclonal antibody or the full-length coding gene sequence encoding the heavy chain of the monoclonal antibody is respectively connected to a recombinant expression vector to construct a light chain recombinant vector and a heavy chain recombinant vector; (2) The light chain recombinant vector and the heavy chain recombinant vector are transfected into host cells for fermentation culture, so that the host expresses the monoclonal antibody; (3) The supernatant of the host cell culture solution is collected by centrifugation, and purified by an affinity chromatography column to obtain a purified recombinant monoclonal antibody.

[0015] Optionally, the recombinant expression vector is a pCDNA3.4 vector.

[0016] Optionally, the host cell is a HEK293 cell, i.e., a human kidney epithelial cell.

[0017] Optionally, in step (2), the fermentation culture method comprises the following steps: The light chain recombinant plasmid: heavy chain recombinant plasmid: transfection reagent (PEI) is added in an appropriate amount of suspension cell culture medium in a mass ratio of 2:1:3, mixed thoroughly, and then placed for 10-15 min; then the prepared host cells are counted, and the host cell density is adjusted to 3.0-3.5 x 10 6 cells / mL with the suspension cell culture medium prepared in the previous step; the transfected host cells are placed in a sterile flask (the filling volume is ≤30%), and the flask is placed in a carbon dioxide shaker for culture. When the culture reaches the 7th day, the cell viability is detected (the cell viability is ≥50%), and the culture supernatant is collected.

[0018] Further optionally, the fermentation culture method further comprises the following feeding steps: 24 hours after transfection, adding a feeding medium at 3-5% of the culture volume; adding an L-glutamine solution at 1% of the culture volume, and continuing to culture; on the third and fifth days after transfection, adding a feeding medium at 3-5% of the culture volume; adding an L-glutamine solution at 1% of the culture volume, and continuing to culture until the seventh day, when the cell viability is detected (cell viability ≥ 50%), and the culture supernatant is collected.

[0019] Optionally, the 293 suspension cell culture medium and the feeding medium are both purchased from Peking Biolabs Technology Co., Ltd.

[0020] Optionally, in the preparation method of the monoclonal antibody, in step (3), the centrifugation of the supernatant of the host cell culture solution is performed at 6-10°C and 8000-12000 rpm for 20-40 min, and the supernatant is collected for purification.

[0021] Optionally, in the preparation method of the monoclonal antibody, in step (3), the purification method of column affinity chromatography is: equilibrating the Protein-A chromatography column, and equilibrating the column with a loading buffer; loading the treated cell expression supernatant into the chromatography column, collecting the flow-through, and loading the flow-through again to collect the flow-through again; after equilibrating the column with the loading buffer, eluting with an elution buffer, collecting the eluate with a collection tube, and adding a neutralization buffer to the collection tube in advance, mixing the eluate and the neutralization buffer to obtain a mixed solution; dialyzing the collected mixed solution, and centrifuging the purified solution obtained after dialysis to collect the supernatant to obtain the recombinant humanized anti-Tp17 monoclonal antibody.

[0022] Preferably, in the purification of column affinity chromatography, the loading buffer is 10 times the volume of the equilibration buffer, and the loading buffer is a 20 mM PBS buffer at pH 7.4.

[0023] During the elution process, the elution buffer is 10 times the volume of the equilibration buffer, and the elution buffer is a 0.1 M Gly-HCl buffer at pH 2.5-3.0. The neutralization buffer is a 1 M Tris-HCl buffer at pH 9.0.

[0024] Preferably, the volume ratio of the neutralization buffer to the eluate is 3:40.

[0025] Optionally, the dialysis method is: the dialysis solution is 20 mM PBS (pH 7.4), 2 times / day, each time in the morning and evening, dialysis at 2-8°C for 3 days, at least 6 times; The centrifugal treatment condition after dialysis is: 7500-9000 rpm, 4-15 DEG C centrifugal 20-40 min, collecting supernatant, and obtaining the protein sample is the recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody.

[0026] Optionally, in the preparation method, the acquisition method of the full-length coding gene sequence encoding the light chain and the heavy chain of the monoclonal antibody is: (01) Hybridoma preparation: the purified Treponema pallidum membrane protein TP17 is used to immunize BALB / C mice to prepare hybridoma cells; (02) Sequencing of hybridoma cells: the prepared hybridoma cells are cultured, mRNA is extracted, cDNA is prepared by reverse transcription, the prepared cDNA is used as a template, amplification is carried out by light / heavy chain variable region primers, the amplification product is connected to a vector, and the constructed recombinant vector is sequenced; (03) Sequence analysis after sequencing and construction of light / heavy chain recombinant plasmid: the nucleotide sequences of the successfully sequenced light chain variable region are respectively connected with human light chain constant region sequences, the nucleotide sequences of the successfully sequenced heavy chain variable region are respectively connected with human heavy chain constant region nucleotide sequences, and then the connected sequences are subjected to codon optimization to obtain the monoclonal antibody light chain coding gene or the monoclonal antibody heavy chain coding gene.

[0027] Optionally, the Treponema pallidum membrane protein TP17 is obtained by cloning the TP17 gene sequence into an expression vector, expressing in BL21 (DE3) induced by isopropyl thiogalactoside, and purifying.

[0028] The present application has the following beneficial effects: 1. The present application provides a preparation method of an anti-Treponema pallidum membrane protein TP17 monoclonal antibody, which selects the Treponema pallidum membrane protein TP17 as a target antigen, immunizes mice with the TP17 antigen, prepares hybridoma cells, obtains the nucleotide sequence of the anti-TP17 monoclonal antibody through sequencing technology of the hybridoma cells, analyzes and modifies the nucleotide sequence, modifies the constant region into a humanized sequence, optimizes the light / heavy chain sequences through codon optimization, constructs the light / heavy chain sequences into a pCDNA3.4 vector, transfects HEK293 suspension cells with the obtained recombinant plasmid at a certain ratio, obtains the TP17 monoclonal antibody through cell secretion expression and purification, realizes efficient expression in a mammalian cell expression system and purification, significantly improves the yield and purity of the anti-Treponema pallidum membrane protein TP17 monoclonal antibody, and the preparation method is suitable for large-scale production.

[0029] 2. The monoclonal antibody against Treponema pallidum membrane protein TP17 prepared by the method provided in this invention has high specificity and high detection sensitivity. It 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.

[0030] 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

[0031] 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

[0032] 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.

[0033] 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.

[0034] (2) Mouse immunization: 6-8 weeks old female balb / c mice were immunized with recombinant protein TP17; the immunization protein was mixed with an equal volume of Freund's complete adjuvant, emulsified completely, and immunized the mice subcutaneously at multiple points on the back; three weeks later, the mice were boosted, the immunization protein was mixed with an equal volume of incomplete Freund's adjuvant, emulsified completely, and immunized the mice subcutaneously at multiple points on the back; one week later, the mice were tail-bled to detect the serum titer. When the serum titer reached more than 100,000, the mice were prepared for fusion; three days before fusion, the mice were intraperitoneally injected with immunization without adjuvant, and the immunization dose was 100 μg per mouse. (3) Cell fusion: when the fusion was performed, the spleen of the immunized mouse was ground and dispersed, and the mouse B lymphocytes were collected after centrifugation, mixed at a ratio of 7:1 (B lymphocytes: SP2 / 0), 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, and after sufficient fusion, DMEN medium was used to terminate the fusion, and finally HAT medium was used to resuspend the fused cells, which were placed in a 96-well cell culture plate and cultured in a carbon dioxide incubator. (4) Hybridoma cell screening and subcloning: HAT medium was completely replaced on the 7th day after fusion, and the culture supernatant was detected by indirect ELISA on the 9th day. The specific detection method is as follows: Coating: dilute the GST-tagged protein to 1 μg / mL with CBS buffer (pH 9.6), 100 μL / well coated in the enzyme-labeled plate, and 4°C coated overnight. Wash the plate 5 times with PBST buffer and pat dry the enzyme-labeled plate; blocking: block the enzyme-labeled plate with PBST buffer containing 3% BSA, 200 μL / well, 37°C incubation for 2 h; wash the plate 5 times with PBST buffer and pat dry the enzyme-labeled plate; detection: add the cell supernatant to be detected to the enzyme-labeled plate, 100 μL / well, and at the same time, use mouse immune serum as a positive control and HAT medium as a blank control, 37°C incubation for 1 h; wash the plate 5 times with PBST buffer and pat dry the enzyme-labeled plate; add enzyme-labeled secondary antibody: dilute the HRP-labeled goat anti-mouse antibody 20,000 times with PBST buffer, add to the enzyme-labeled plate, 100 μL / well, 37°C incubation for 1 h; wash the plate 5 times with PBST buffer and pat dry the enzyme-labeled plate; color development: color development with TMB color developing liquid in the dark, 100 μL / well, 37°C incubation for 15 min; terminate with 2M sulfuric acid, 50 μL / well, and detect at 450 nm wavelength; Select high titer (OD 450Cells with a density ≥2.0 g / L 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 was 100% in both wells. Subcloning was then terminated to ensure the stability of the hybridoma cell line.

[0035] 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.

[0036] (2) Reverse transcription Following the instructions of Thermo's kit, the extracted RNA was immediately reverse transcribed to prepare cDNA.

[0037] (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 .

[0038] (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.

[0039] 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 (CDR1, CDR2, CDR3), and four backbone regions (FR1-FR4).

[0040] Analysis revealed that the light chain complementarity determination regions CDR1 and CDR3 sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, while the heavy chain complementarity determination regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7.

[0041] The same signal peptide sequence was added to the N-terminus of the variable region of the light and heavy chains, as shown in SEQ ID NO. 8. This signal peptide sequence can promote efficient secretory expression of the protein. At the same time, the constant regions of the antibody light and heavy chains were humanized to reduce interference from anti-mouse antibody (HAMA), avoid false positives, and improve specificity. A human light chain constant region (hIgκC) sequence was added to the C-terminus of the variable region of the light chain: the sequence reference is 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 variable region of the heavy chain, the sequence reference is 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. The translation codons were optimized, and 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.

[0042] The 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.

[0043] 1.4 Expression and purification of monoclonal antibody against Treponema pallidum membrane protein TP17 (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.

[0044] 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.

[0045] 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.

[0046] (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.

[0047] 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.

[0048] 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.

[0049] (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.

[0050] 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.

[0051] C. Load the transfected cells into sterile shake flasks (volume ≤30%) and place the shake flasks in a carbon dioxide shaker for incubation.

[0052] D. 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.

[0053] (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.

[0054] B. Protein A-column affinity chromatography purification method (the ambient temperature is controlled at 20-25℃ during the purification process). 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%.

[0055] 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.

[0056] 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). Elution: 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.

[0057] Equilibration: Equilibrate with 10 column volumes of loading buffer. Use pH test paper to check the pH of the outlet liquid. When the pH matches that of the loading buffer, the equilibration is complete. Rinse the column with 20% ethanol and store it in 20% ethanol at 4°C.

[0058] Dialysis: Place the collected protein in a dialysis bag (specifications: MWCO=8-14kDa; diameter=8mm), using 20mM PBS (pH 7.4) as the dialysate, twice a day, changing the medium twice daily (morning and evening), at 2-8℃ for 3 days (at least 6 times). After dialysis, remove the protein and place it in a 50 ml centrifuge tube. Centrifuge at 7500-9000 rpm, 4-15℃ for 30 min, collect the supernatant, and determine the concentration. The obtained protein sample is the recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody (R-Anti-TP17-Ab01).

[0059] The purified recombinant monoclonal antibody R-Anti-TP17-Ab01 was analyzed by SDS-PAGE gel imaging and SEC-HPLC.

[0060] Yield detection: The OD280 of the purified product was detected by ultraviolet spectrophotometry. 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 calculated by multiplying the concentration and volume.

[0061] 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.

[0062] The results are as follows Figure 3 The SEC-HPLC results show that the recombinant monoclonal antibody has a single peak at 7.892 min, and the peak area accounts for as high as 98%, indicating that the recombinant monoclonal antibody can form the correct dimer and the dimer purity can reach 98%.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] ② Wash the plate three times with PBST, dry it, add 200ul of blocking buffer per well, and block for 2 hours.

[0067] ③Wash the plate three times with PBST, dry it, and set aside.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] ⑧ Determine the sensitivity of recombinant monoclonal antibody R-Anti-TP17-Ab01 in detecting TP17.

[0073] 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.

[0074] Table 1. ELISA detection of recombinant monoclonal antibody R-Anti-TP17-Ab01

[0075] 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.

[0076] 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 M K2CO3 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 PB, 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.

[0077] (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.

[0078] (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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] (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.

[0083] 3. Test Results and Analysis The sensitivity test results are shown in Table 2. These results indicate that, at the optimal labeling concentration, the experimental group's test strips can detect 10 ng / mL of Treponema pallidum membrane protein TP17, with higher sensitivity than the control group. Simultaneously, 20 negative samples were tested; the control group showed false positives, while the monoclonal antibody prepared in this patent produced negative results, demonstrating the high specificity of the test strips prepared using the monoclonal antibody in this patent.

[0084] Table 2 Positive control and negative sample testing

[0085] 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 method for preparing a monoclonal antibody against Treponema pallidum membrane protein TP17, characterized in that, Includes the following steps: The encoding gene of the monoclonal antibody against Treponema pallidum membrane protein TP17 was ligated into an expression vector to construct a recombinant expression vector. The recombinant expression vector was then transfected into host cells and cultured. Finally, the monoclonal antibody against Treponema pallidum membrane protein TP17 in the culture product was collected and isolated. The encoding gene of the monoclonal antibody against Treponema pallidum membrane protein TP17 includes: a full-length encoding gene sequence of the light chain of the monoclonal antibody as shown in SEQ ID NO. 13, and a encoding gene sequence of the heavy chain of the monoclonal antibody as shown in SEQ ID NO.

14.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: (1) The full-length coding gene sequence of the light chain of the monoclonal antibody or the full-length coding gene of the heavy chain of the monoclonal antibody is respectively ligated into a recombinant expression vector to construct a light chain recombinant vector and a heavy chain recombinant vector; (2) The light chain recombinant vector and the heavy chain recombinant vector were transfected into the host cells respectively, and fermented to induce the host cells to express the monoclonal antibody; (3) Collect the supernatant of the host cell culture medium by centrifugation, and purify it using an affinity chromatography column to obtain purified recombinant monoclonal antibody.

3. The preparation method according to claim 2, characterized in that, The recombinant expression vector is the pCDNA3.4 vector.

4. The preparation method according to claim 2, characterized in that, The host cell was HEK293 cell.

5. The preparation method according to claim 2, characterized in that, Step (3) is as follows: The centrifugation conditions are: 6~10℃, 8000~12000rpm for 20~40min, collect the supernatant for purification.

6. The preparation method according to claim 2, characterized in that, In step (3), the purification method using column affinity chromatography is as follows: The Protein-A chromatography column was equilibrated with loading buffer. The processed cell expression supernatant was added to the column, the flow-through was collected, and the flow-through was loaded again and collected again. After equilibrating the column with loading buffer, elution was performed with elution buffer, and the eluent was collected in a collection tube pre-filled with neutralization buffer. The eluent and neutralization buffer were mixed to obtain a mixture. The collected mixture was then dialyzed, and the purified solution was centrifuged after dialysis. The supernatant was collected to obtain the recombinant humanized anti-Treponema pallidum membrane protein TP17 monoclonal antibody.

7. The preparation method according to claim 1, characterized in that, The method for obtaining the full-length coding gene sequences of the light and heavy chains of the monoclonal antibody is as follows: (01) Hybridoma preparation: BALB / c mice were immunized with purified Treponema pallidum membrane protein TP17 to prepare hybridoma cells; (02) Sequencing of hybridoma cells: After the hybridoma cells are cultured, their mRNA is extracted and cDNA is prepared by reverse transcription. The prepared cDNA is used as a template and amplified by primers for the light and heavy chain variable regions. The amplified product is ligated into a vector and the constructed recombinant vector is sent for sequencing. (03) Sequencing sequence analysis and construction of light and heavy chain recombinant plasmids: The nucleotide sequence of the successfully sequenced light chain variable region is ligated to the human light chain constant region sequence, and the nucleotide sequence of the successfully sequenced heavy chain variable region is ligated to the human heavy chain constant region sequence. The codons of the ligated sequences are then optimized to obtain the monoclonal antibody light chain encoding gene or the monoclonal antibody heavy chain encoding gene.

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