An antibody specifically recognizing a surface antigen of toxoplasma and use thereof

Abstract

This invention discloses an antibody that specifically recognizes Toxoplasma gondii surface antigens and its applications. The invention utilizes hybridoma technology to establish a hybridoma cell line by fusing Balb / C mouse spleen cells with SP2 / 0 cells. Monoclonal antibodies were prepared from these cells using in vitro culture and in vivo ascites induction methods, yielding the monoclonal antibody SH12-S7. This invention provides clinical and research institutions with an antibody that specifically recognizes Toxoplasma gondii surface antigens. It can be used for ELISA assays to detect clinical samples, for immunohistochemical assays to diagnose pathological specimens, and provides a convenient research tool for toxoplasmosis research departments. It has broad application value in the diagnosis and detection of definitive host infections and provides technical support for epidemiological investigations and evaluation of control effects of Toxoplasma gondii.

Description

Technical Field

[0001] This invention relates to the field of bioquarantine technology, specifically to an antibody that specifically recognizes Toxoplasma gondii surface antigens and its application, and more specifically to an antibody that can specifically recognize recombinant proteins of Toxoplasma gondii surface antigens GRA1 and GRA7 and its application. Background Technology

[0002] Toxoplasma gondii parasitizes almost all nucleated cells, including those of humans and many other mammals. It is an opportunistic pathogen with a wide range of intermediate hosts, including mammals and birds. Felines are its definitive hosts. When infected, felines shed numerous highly infectious oocysts that persist in the environment. To develop a rapid diagnostic reagent for Toxoplasma gondii, screening of antigenic markers is necessary. Previously, researchers both domestically and internationally have used Toxoplasma gondii tachyzoites collected from infected animals or cultured from tissues and cells as antigens. While this method offers good specificity, it suffers from low antigen purity, complex composition, and high cost. In contrast, prokaryotic expression systems offer high yields of specific target proteins, are simple to operate, and are inexpensive, making them a popular research focus. In previous research, the inventors obtained a recombinant protein of Toxoplasma gondii surface antigens GRA1 and GRA7. By excluding the signal peptide and C-terminal hydrophobic sequence from GRA1 and GRA7, and extracting the B-cell antigenic epitope information from the soluble expression site, the recombinant gene expressing these two antigenic epitopes was reconstructed. This protein can be used to detect Toxoplasma gondii in specimens such as whole blood, serum, plasma, and cerebrospinal fluid (CN110423270B).

[0003] In clinical practice, PCR and ELISA are commonly used to detect Toxoplasma gondii. However, these methods are often cumbersome, time-consuming, and expensive, making them unsuitable for widespread use at the grassroots level. Therefore, finding a simple, rapid, and highly sensitive diagnostic method is imperative. The latex agglutination test (LAT), internationally recognized as the "gold standard" for Toxoplasma gondii detection, boasts high accuracy and specificity, but its sensitivity is relatively low. Therefore, there is an urgent need to establish a rapid and sensitive detection method for Toxoplasma gondii. Although several indirect ELISA detection methods exist, the lack of monoclonal antibodies targeting the antigen protein is a significant drawback. The preparation of monoclonal antibodies is fundamental to establishing a superior ELISA detection method; the absence of appropriate monoclonal antibodies leads to unsatisfactory accuracy and sensitivity in the established detection system. How to obtain monoclonal antibodies for Toxoplasma gondii infection detection and establish a detection system with high accuracy and sensitivity are pressing issues that need to be addressed in current technologies. Summary of the Invention

[0004] To address the aforementioned problems, this invention first provides a novel monoclonal antibody that specifically recognizes the recombinant proteins GRA1 and GRA7 surface antigens of Toxoplasma gondii. This monoclonal antibody is secreted by the hybridoma cell line SH12-S7 and can specifically, sensitively, and stably recognize Toxoplasma gondii.

[0005] The preparation process of the monoclonal antibody is as follows: Based on previous research results, the applicant excluded the signal peptide and C-terminal hydrophobic sequence from GRA1 and GRA7, extracted the B-cell antigenic epitope information from the soluble expression site, reconstructed the recombinant gene expressing these two gene antigenic epitopes, and then expressed it in E. coli. The rGRA recombinant protein was extracted and purified. Mice were immunized with the rGRA recombinant protein. After the antibody level rose, the obtained immunized mouse spleen lymphocytes were fused with mouse myeloma cells to obtain a hybridoma cell line. The rGRA recombinant protein was further used for screening to obtain hybridoma cells that could stably secrete high-affinity Toxoplasma gondii monoclonal antibodies. The applicant named this cell line hybridoma cell line SH12-S7, and the nucleotide sequence of the rGRA recombinant protein is shown in SEQ ID NO.1.

[0006] Furthermore, the light chain variable region of the monoclonal antibody SH12-S7 of the present invention comprises LCDR1-3, the amino acid sequence of LCDR1 is shown in SEQ ID NO:2, the amino acid sequence of LCDR2 is shown in SEQ ID NO:3, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:4; and the heavy chain variable region of the monoclonal antibody SH12-S7 comprises HCDR1-3, the amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:7.

[0007] Furthermore, the amino acid sequence of the light chain variable region of the monoclonal antibody SH12-S7 is shown in SEQ ID NO:8, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9.

[0008] Furthermore, this application provides one or more isolated nucleic acid molecules that encode the antibodies described in this application.

[0009] Furthermore, this application provides one or more vectors that contain one or more nucleic acid molecules described in this application.

[0010] Furthermore, this application provides one or more cells that contain one or more nucleic acid molecules or one or more vectors as described in this application.

[0011] Furthermore, this application provides a pharmaceutical composition comprising the antibody described in this application, the nucleic acid molecule described in this application, the carrier described in this application, the cell described in this application, and / or the fusion protein described in this application, and optionally a pharmaceutically acceptable adjuvant.

[0012] Furthermore, this application provides the use of the described antibody, and / or the described fusion protein, in the preparation of medicaments for diagnosing, preventing, or treating diseases or conditions. The disease described is a toxoplasmosis-related disease.

[0013] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] This invention involves excluding the signal peptides and C-terminal hydrophobic sequences from GRA1 and GRA7, extracting B-cell antigenic epitope information from the soluble expression sites, reconstructing recombinant genes expressing these two epitopes, and then expressing them in *E. coli*. The recombinant protein rGRA is extracted and purified, exhibiting immunogenicity. A monoclonal antibody against the recombinant protein rGRA is prepared. Western blot analysis confirms that the monoclonal antibody prepared in this invention specifically recognizes different stages of *Toxoplasma gondii* infection, while showing no response to infections from other microorganisms, demonstrating extremely high specificity. The monoclonal antibody prepared using this invention can be applied to an immunofluorescence assay, which can detect and diagnose the presence of *Toxoplasma gondii* in hosts or samples (ex vivo samples, water samples, food, contaminants, etc.). Attached Figure Description

[0016] Figure 1 Purification and identification of recombinant proteins, wherein: A: Recombinant protein solubility analysis; M: Protein markers; 1: Recombinant protein supernatant; 2: Recombinant protein precipitate; 3: Purified recombinant protein; B: Western blot detection of recombinant protein; M: Protein markers; 1: Detection of the binding ability of recombinant protein to mouse Toxoplasma gondii positive serum;

[0017] Figure 2 Affinity detection of monoclonal antibodies SH8-A12, SH10-D9 and SH12-S7;

[0018] Figure 3Western blot results of monoclonal antibody SH12-S7, where M: protein molecular weight standard; 1: negative control; 2: Toxoplasma gondii oocyst total protein; 3: Toxoplasma gondii adult worm total protein. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: Preparation of monoclonal antibodies against recombinant protein rGRA

[0021] Referring to the applicant's prior application CN110540602A, the recombinant protein rGRA was expressed and purified. SDS-PAGE electrophoresis results showed ( Figure 1 A), the expression product was purified, and the target protein with high purity was obtained at 50 kDa. Western blot results showed ( Figure 1 B) The recombinant protein can specifically bind to mouse Toxoplasma gondii-positive serum.

[0022] Six-week-old Babl / c female mice (Hunan Silex Biotechnology Co., Ltd.) were immunized with purified rGRA. The mice used were SPF grade (specific pathogen free). Immunization was performed via subcutaneous or intraperitoneal injection of recombinant protein. Mice were six weeks old after the first antigen infusion. For the first immunization, 50 μg of rGRA protein and Freund's complete adjuvant (purchased from Sigma) were thoroughly mixed at a v:v ratio of 1:1 and administered subcutaneously to the limbs. One week later, a second immunization was performed using 50 μg of rGRA and Freund's incomplete adjuvant (purchased from Sigma) at a v:v ratio of 1:1. One week later, a third immunization was administered intraperitoneally. Ramos cells were collected, washed twice with serum-free RPMI-1640 medium (purchased from Sigma), and diluted to a density of 10-1. 7 The antibody titer was measured at 1 / mL. Each mouse was then immunized subcutaneously. A booster immunization was administered one week later. One week after the fourth immunization, blood was collected from the eyeballs, and serum antibody titers were detected using ELISA. Antibody dilutions of not less than 10-1 were considered positive. 5 If the antibody dilution is greater than 10, then immunization is complete; 5The recombinant rGRA protein and Freund's incomplete adjuvant (purchased from Sigma) were thoroughly mixed at a ratio of v:v = 1:1 before subcutaneous immunization of the limbs, 50 μg per animal. The test was then repeated until the immunization was completed.

[0023] Serum from immunized mice was tested using an ELISA method as described by Fishwild et al. (1996). In summary: serum antibody was obtained after immunization with recombinant RGRA protein at a dilution of 10-1. 5 The mice were euthanized by cervical dislocation, and their spleens were removed. Single B cells were isolated by crushing the spleen through a 70 μm corning sieve. These B cells were then mixed with SP2 / 0 cells at a 1:10 ratio and fused into hybridoma cells using PEG (from Sigma). The fused cells were aliquoted into 30 96-well cell culture plates (200 μL per well). The 96-well plates were cultured at 37°C in a 5% CO2 incubator. ELISA was then used to screen for fusion cell lines secreting highly active antibodies. ELISA plates were coated with 100 μL of purified rGRA protein dissolved in PBS (1 ng / μL per well). After incubation at 37°C for 2 hours, the plates were blocked with 280 μL of 5% chicken serum dissolved in PBS / Tween (0.05%), and then incubated at 37°C for 2 hours before use. Add 100 μL of supernatant from hybridoma fusion cells to each well and incubate at 37°C for 30 minutes. Wash the plates five times with PBS / Tween, then add 100 μL of goat-anti-human IgGFc polyclonal antibody conjugated with horseradish peroxidase (HRP) to each well and incubate at 37°C for 30 minutes. After washing five times with PBS / Tween, add 100 μL of TMB chromogenic buffer (Shandong Zibo Yunqiao Biotechnology Co., Ltd.) to each well and incubate at 37°C for 5 minutes. Terminate the reaction with 50 μL of 2M sulfuric acid and read the OD value using a microplate reader (wavelength 450 nm). Hybridoma fusion cell lines with OD values ​​higher than 1.0 are selected, and single-clonal cell lines that specifically bind to rGRA protein are screened using a limiting dilution method.

[0024] Three anti-RGRA murine antibodies with different affinities and binding epitopes were screened using hybridoma fusion technology and named SH8-A12, SH10-D9, and SH12-S7, respectively. The affinity of these three antibodies was detected using ELISA.

[0025] Dissolve 100 μg of rGRA protein in 300 μL of ultrapure water (ultrapure water system purchased from PALL) to obtain a 0.33 μg / μL rGRA protein dilution. Coat a 96-well detection plate with 100 ng of dilution per well.

[0026] Dilute the rGRA protein to 1 μg / μL with PBS buffer, and take 40 mL of the solution. Add 120 μL of the rGRA protein dissolved in ultrapure water to another 40 mL of PBS buffer. Mix thoroughly and add 100 μL per well to a 96-well plate. Incubate at 37°C for 2 hours. After incubation, remove any remaining protein solution from the 96-well plate. Add 280 μL of blocking buffer (1% BSA + PBS) to each well, incubate at 37°C for 2 hours, and store at 4°C for later use.

[0027] The monoclonal antibody prepared in Example 1 was used for detection, with no primary or secondary antibody added as a negative control. The antibody to be tested was uniformly diluted to a concentration of 0.1 mg / mL with PBS. 100 μL of PBS was added to the test plate according to the serial dilution method. 100 μL of the diluted 0.1 mg / mL antibody solution was added to the first test well, mixed thoroughly, and then 100 μL was added to the next well. This was repeated for 11 serial dilutions, with each sample being tested once. The diluted test plate was incubated at 37°C for 30 minutes.

[0028] After incubation, wash the plate 5 times with 280 μL each time. After washing, gently tap the plate a few times to remove any remaining washing buffer. Then, add 100 μL of the prepared goat anti-human secondary antibody dilution buffer (goat anti-human secondary antibody purchased from Abcam, diluted with PBS at a ratio of 1:10000) to each well, incubate at 37°C for 30 minutes, and wash again. After washing, add 100 μL of TMB chromogenic solution to each well, incubate at 37°C for 5 minutes, and then add 50 μL of 2M sulfuric acid stop solution to each well to terminate the reaction. Read the OD value at 450 nm using an MD-M2E (Molecular Instruments, Inc.) microplate reader and process the data using MD-M2E software. Results are as follows: Figure 1 As shown in the figure. The results showed that SH12-S7 had the strongest affinity for rGRA protein, and still had a strong binding ability even at low concentrations. The detection results for SH8-A12, SH10-D9 and others were similar.

[0029] The amino acid sequence of its light chain variable region was obtained by sequencing SH12-S7, which has the highest affinity, using Sanger high-throughput sequencing technology.

[0030] QIALTASPAIPASLAVSLGQRATTMTCRASSSFAGKSYMYWYAQKKPGQSPKAWIYHTS NLASGVPARFSGSGAGTSASLTISRVEEADAAATYYCQQWNGVPFTFGGGTKKLALKR (SEQ ID NO:8), wherein the amino acid sequences of LCDR1-3 are: RASSSFAGKSYMY (SEQ ID NO:2), HTSNLAS (SEQ ID NO:3), and QQWNGVPFT (SEQ ID NO:4), respectively;

[0031] The amino acid sequence of the heavy chain variable region is as follows:

[0032] QAKQQPGAELVKAGASVKASGYTFIDWMNAMHWAKQTQGLEGLEWIGGIDPANGSY TNYAQKQKFAGKATLTADKASSTASTAYMQLSSLTSSAAYYCARYYDDSANWGGLPYWTT VIAVTV (SEQ ID NO:9), wherein the amino acid sequences of HCDR1-3 are: WMNAMH (SEQ ID NO:5), GIDPANGSYTNYAQKQKFAG (SEQ ID NO:6), YYDDSANWGGLPY (SEQ ID NO:7). Example 2: Characterization of the monoclonal antibody SH12-S7

[0033] The monoclonal antibody SH12-S7 was diluted 1:100 in ascites fluid with PBS buffer, and then serially diluted. 100 μL / well was added to an ELISA plate coated with recombinant rGRA protein and incubated at 37°C for 90 min. The plate was washed three times with PBST for 5 min each time, and the plate was blotted dry after the last wash. 100 μL / well of HRP-labeled goat anti-mouse IgG (1:2000 dilution) was added and incubated at 37°C for 60 min. The plate was washed five times with PBST for 5 min each time, and the plate was blotted dry after the last wash. 100 μL / well of TMB-H2O2 substrate was added and the plate was incubated at room temperature in the dark for 10 min. The reaction was terminated by adding 50 μL of 2mol / L sulfuric acid to each well. The OD450 value was measured using an ELISA reader. A negative OD450 value less than 0.2 and a ratio of the test well to the negative OD450 value greater than 2.1 were considered positive. The highest dilution of the positive well was used as the ascites fluid titer of the monoclonal antibody. The results showed that the indirect ELISA titer of the monoclonal antibody SH12-S7 was 1:327600.

[0034] The reactivity and specificity of the obtained monoclonal antibodies were identified by Western blotting (Feng Renqing, Guo Zhenquan. Modern Antibody Technology and Its Application (1st Edition) [M]. Beijing: Peking University Press, 2006: 199-206.). The purified adult Toxoplasma gondii protein and oocyst protein were subjected to SDS-PAGE electrophoresis on a 12% separating gel and a 5% stacking gel. The gel was then transferred to a nitrocellulose membrane (NC membrane) in a Tris-Gly buffer system at 0.65 mA / cm². 2 The sample was transferred for 100 minutes, blocked with 5% skim milk, and developed using a monoclonal antibody as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody, followed by a DAB chromogenic assay kit. Results showed that the monoclonal antibody specifically bound to both the adult Toxoplasma gondii protein and the oocyst protein, exhibiting significant positive reactions in both cases, demonstrating that the monoclonal antibody SH12-S7 can specifically recognize Toxoplasma gondii.

[0035] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A monoclonal antibody which can specifically recognize a surface antigen of Toxoplasma gondii, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:8, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

9.

2. A nucleic acid molecule, characterized in that... The nucleic acid molecule encodes the antibody of claim 1.

3. A carrier, characterized in that... The carrier comprises the nucleic acid molecule as described in claim 2.

4. A cell, characterized in that... The cell contains the nucleic acid molecule of claim 2 or the vector of claim 3.

5. A pharmaceutical composition, characterized in that... It comprises the antibody of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, and / or the cell of claim 4, and a pharmaceutically acceptable adjuvant.

6. Use of the antibody of claim 1, the nucleic acid molecule of claim 2, the carrier of claim 3, the cell of claim 4, and / or the pharmaceutical composition of claim 5 in the preparation of a medicament for diagnosing diseases related to toxoplasmosis infection.

Images