Toxoplasma gondii autophagy-related protein 8 monoclonal antibody and preparation method and application thereof

Abstract

The application provides a Toxoplasma gondii autophagy-related protein 8 monoclonal antibody and a preparation method and application thereof, and comprises a first antigen binding region and a second antigen binding region, wherein the first antigen binding region comprises a first light chain variable region VL-1 and a first heavy chain variable region VH-1, and the second antigen binding region comprises a second light chain variable region VL-1 and a second heavy chain variable region VH-2; the mouse anti-TgAtg8 monoclonal antibody prepared in the application has less banding and lower fluorescence background compared with a rabbit polyclonal antibody, and is beneficial to subsequent in-depth research on the TgAtg8 protein.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody against Toxoplasma gondii autophagy-associated protein 8, its preparation method, and its application. Background Technology

[0002] Toxoplasma gondii ( Toxoplasma gondii Toxoplasmosis, commonly known as Toxoplasma gondii, is an obligate intracellular parasitic opportunistic protozoan found in warm-blooded animals, infecting approximately one-third of the world's population. Although most infections are asymptomatic, it poses a significant risk to immunocompromised individuals and newborns, causing clinical symptoms such as encephalitis, chorioretinitis, congenital infection, and neonatal death. Therefore, preventing and controlling toxoplasmosis in humans and other hosts is crucial. Current first-line treatments for toxoplasmosis have many limitations, including toxicity, drug resistance, and the inability to eradicate tissue cysts. This underscores the need to identify new drug targets to inhibit or treat toxoplasmosis.

[0003] Autophagy is a conserved intracellular degradation process in eukaryotes from yeast to mammals. One of the main functions of autophagy is to maintain basal cellular homeostasis under normal growth conditions. On the other hand, autophagy is crucial for stress management in almost all eukaryotes under different stress conditions. The formation of autophagosomes requires the function of autophagy-related proteins (Atg) and their complexes at different stages, and Atg8 plays a central role in the expansion, closure, and fusion with lysosomes to form autolysosomes. Current research shows that Toxoplasma gondii autophagy-related protein 8 (TgAtg8, TGGT1_254120) has at least two functions: (1) TgAtg8 can participate in autophagosome formation under starvation-induced conditions; (2) during normal cell division and proliferation, TgAtg8 can be localized to the outer membrane of the acroplast and participate in maintaining homeostasis within the acroplast. Therefore, new drug targets can be sought from the two pathways through which TgAtg8 exerts its function.

[0004] Currently, there are generally two approaches to studying the TgAtg8 protein. One approach involves constructing a eukaryotic expression vector with a tag protein linked to the N- or C-terminus of the TgAtg8 protein, transfecting it into *Toxoplasma gondii*, and using a tag antibody to indirectly identify the localization, protein expression, and protein-protein interactions of TgAtg8 in the parasite. The other approach involves preparing an anti-TgAtg8 antibody to directly recognize the endogenous TgAtg8 protein. The former allows for short-term protein studies but has some drawbacks. For example, the tag protein may affect the intracellular localization of TgAtg8 and protein-protein interactions. Furthermore, this overexpression method may, to some extent, affect the expression and function of the endogenous protein. The latter approach can overcome the shortcomings of the former.

[0005] The applicant's research group previously prepared a rabbit TgAtg8 polyclonal antibody {Preparation and preliminary application of polyclonal antibody against Toxoplasma gondii autophagy-associated protein 8 (TgAtg8) [J]. Chinese Journal of Parasitology and Parasitic Diseases, 2014, 32(2):9-130-134}, which had some shortcomings, such as poor antibody specificity—more stray bands and higher fluorescence background were observed when performing Western blotting (WB) and indirect immunofluorescence (IFA) experiments, respectively. Therefore, the inventors have been exploring improvement schemes in hopes of finding an antibody with fewer stray bands and lower fluorescence background. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology, and to provide a monoclonal antibody against Toxoplasma gondii autophagy-related protein 8, its preparation method, and its application. The technical solution adopted by this invention is as follows:

[0007] In a first aspect, a monoclonal antibody against Toxoplasma gondii autophagy-associated protein 8 is provided, comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region includes a first light chain variable region VL-1 and a first heavy chain variable region VH-1, and the second antigen-binding region includes a second light chain variable region VL-1 and a second heavy chain variable region VH-2.

[0008] The CDR1, CDR2, and CDR3 of the first light chain variable region VL-1 are shown in positions 27-37, 55-57, and 94-102 of SEQ ID NO: 1, respectively.

[0009] The CDR1, CDR2, and CDR3 of the first heavy chain variable region VH-1 are shown in positions 26-33, 51-58, and 97-106 of SEQ ID NO: 2, respectively.

[0010] The CDR1, CDR2, and CDR3 of the second light chain variable region VL-2 are shown in positions 27-36, 54-56, and 93-101 of SEQ ID NO: 3, respectively.

[0011] The CDR1, CDR2, and CDR3 of the second heavy chain variable region VH-2 are shown in positions 26-33, 51-58, and 97-108 of SEQ ID NO: 4, respectively.

[0012] The amino acid sequence of the first light chain variable region VL-1 of the first antigen-binding region is shown in SEQ ID NO: 1, and the amino acid sequence of the first heavy chain variable region VH-1 is shown in SEQ ID NO: 2;

[0013] The amino acid sequence of the second light chain variable region VL-2 of the second antigen-binding region is shown in SEQ ID NO: 3, and the amino acid sequence of the second heavy chain variable region VH-2 is shown in SEQ ID NO: 4.

[0014] Secondly, in the above-mentioned method for preparing Toxoplasma gondii autophagy-related protein 8 monoclonal antibody, cells from mice with high serum titers and good specificity after immunization are fused with myeloma cells, and positive hybridoma cells are obtained through culture and screening.

[0015] Thirdly, it also includes the application of the aforementioned Toxoplasma gondii autophagy-associated protein 8 monoclonal antibody in the preparation of detection kits, especially in kits for detecting Toxoplasma gondii autophagy-associated protein 8.

[0016] The beneficial effects of this invention are as follows: This invention prepares a mouse anti-TgAtg8 monoclonal antibody, which has fewer impurities and lower fluorescence background compared with rabbit polyclonal antibodies, which is beneficial for subsequent in-depth research on TgAtg8 protein. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0018] Figure 1 The results show the induced expression, purification, and identification of the His-TgAtg8 fusion protein. A represents the SDS-PAGE verification result of the prokaryotically expressed His-TgAtg8 fusion protein; B represents the WB verification result of the His antibody recognizing the prokaryotically expressed His-TgAtg8 fusion protein; and C represents the SDS-PAGE verification result of the purified His-TgAtg8 fusion protein.

[0019] Figure 2 For the detection of antibody titer and identification of monoclonal cell supernatant, where A is the result of antibody titer detection in monoclonal cell supernatant; and B is the result of antibody subtype identification in monoclonal cell culture supernatant.

[0020] Figure 3The mouse monoclonal antibody TgAtg8 was used to recognize endogenous TgAtg8 protein. A represents the Western blot (WB) result of the monoclonal antibody recognizing His-TgAtg8; B represents the WB result of the monoclonal antibody recognizing endogenous TgAtg8 protein; C represents the in vitro anatomical (IFA) result of the monoclonal antibody recognizing intracellular TgAtg8 protein; and D represents the IFA result of the monoclonal antibody recognizing extracellular TgAtg8 protein. The scale bar is 5 μm. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0022] In this invention, BALB / c mice were immunized with His-TgAtg8 protein expressed and purified in prokaryotes. After four immunizations, mouse serum was collected, and antibody titers and specificities were monitored by indirect ELISA and Western blot (WB), respectively. Mice with high antibody titers and good specificity were selected, and spleen cells were fused with sp2 / 0 myeloma cells. Positive hybridoma cells obtained by screening in HAT medium were subcloned using limiting dilution. The titers, subtypes, specificities, and localizations of the purified monoclonal antibodies were detected by indirect ELISA, Western blot (WB), and indirect immunofluorescence assay (IFA). The hybridoma cell lines corresponding to the purified monoclonal antibodies were subjected to antibody sequencing.

[0023] Implementation method introduction:

[0024] 1. Induction, purification and identification of recombinant protein His-TgAtg8

[0025] pCOLDⅢ-TgAtg8 bacterial culture was cultured overnight at 37℃. It was then re-inoculated into 100 mL of new LB liquid culture medium at a ratio of 1:100 for expansion culture. 1 mM IPTG inducer was added and induced at low temperature (16℃) for 20-24 h. The bacterial cells were collected, and 100 mL of bacterial culture was resuspended in 10 mL of pre-cooled PBS buffer. The bacterial culture was lysed by sonication in an ice bath, and the supernatant was collected by centrifugation at 11,000 rpm and 4℃ for 15 min.

[0026] Screening and identification of mouse immune and hybridoma cells

[0027] Five BALB / c mice were used. Purified His-TgAtg8 protein (100 μl) and immunoadjuvant (100 μl) were emulsified in a 1:1 ratio and injected at two points on the back and one point on the abdomen, each with 1 / 3 of the volume (avoiding injection into the left spleen). The initial immunization dose was 0.1 mg / mouse, followed by 0.05 mg / mouse every two weeks for a total of four immunizations. Serum from immunized mice was collected for antibody titer and specificity testing (Western Blot analysis to determine if the immunized serum recognized Toxoplasma gondii endogenous TgAtg8 protein). Mice with high serum titers and good specificity were selected and given a booster immunization via intraperitoneal injection of 0.015 mg His-TgAtg8 protein three days before cell fusion. After euthanasia of mice, spleen cells were isolated from the spleen and fused with myeloma cells SP2 / 0. The cells were then fused with 50% polyethylene glycol and screened for positive hybridoma cells using a medium containing hypoxanthine-aminopterin-thymidine (HAT). These positive hybridoma cells were then subcloned using a limiting dilution method to obtain monoclonal cell lines. During each subcloning process, the cell supernatant was collected as a primary antibody and subjected to 2-3 indirect ELISA screenings to obtain positive hybridoma cell lines that specifically recognize the His-TgAtg8 protein.

[0028] Monoclonal antibody titer detection, subtype identification and purification

[0029] (1) Antibody titer detection

[0030] Culture supernatants of positive hybridoma cell lines were collected, with SP2 / 0 cell culture supernatants used as a negative control. A was detected by indirect ELISA. 450 Value, based on the supernatant A of monoclonal hybridoma cell lines 450 Value compared to SP2 / 0 cell supernatant A 450 The highest dilution factor with a value ≥ 2.1 is taken as the highest titer of the monoclonal antibody.

[0031] (2) Antibody subtype identification

[0032] Indirect ELISA method was used to identify monoclonal antibody subtypes.

[0033] (3) Antibody purification

[0034] (4) Antibody sequencing

[0035] 4. Monoclonal antibodies recognize endogenous TgAtg8

[0036] (1) Western blot detection of antibody specificity

[0037] Total protein extracted from the parasites was subjected to SDS-PAGE electrophoresis. Pre-immunization mouse serum and purified mouse anti-TgAtg8 monoclonal antibody (1:1000) were used as primary antibodies, with rabbit anti-TgAtg8 polyclonal antibody (previously prepared by our research group) as a positive control. Goat anti-mouse HRP-IgG (1:5000) and goat anti-rabbit HRP-IgG (1:5000) were used as secondary antibodies. Western blotting was used to detect antibody specificity. Recombinant His-TgAtg8 protein was used as a positive control, and total HFF cell protein was used as a negative control.

[0038] (2) IFA detection of monoclonal antibody recognition of endogenous TgAtg8

[0039] One day in advance, HFF cells (3.5 × 10⁻⁶) were inoculated with a spreader. 4 (1) cells were placed in a 24-well plate; the RH TATi1- cells were counted the next day. ΔKu80 The parasite strain was added to HFF cells in 24-well plates at a ratio of 4:1 to the cell strain. After 24 hours, the 24-well plates containing the crawling smears were removed from the incubator. For intracellular parasite IFA, the supernatant culture medium was discarded, and 1 mL of PBS was added to gently shake and wash 3 times, 1 min each time (all shaking was performed on a shaker, the same below). For extracellular parasite IFA, 500 μl of culture medium containing parasites was added to the 24-well plates, and the plates were incubated at 37°C for 1 hour to allow the parasites to precipitate. The supernatant culture medium was then discarded, and 1 mL of PBS was added to gently shake and wash 3 times, 1 min each time.

[0040] Example: Preparation of TgAtg8 mouse monoclonal antibody

[0041] 1. Induction, purification and identification of recombinant protein His-TgAtg8

[0042] To prepare mouse monoclonal antibodies, the antigen is first obtained. The His-TgAtg8 protein is expressed in prokaryotes, such as... Figure 1 As shown in Figure A, both the precipitate and supernatant showed reaction bands at approximately 15 kDa after induction, indicating successful induction of the target protein expression. The supernatant after ultrasonic lysis following induction was purified using a gradient elution with different concentrations of imidazole eluent. The eluted proteins were then analyzed by Western blotting (WB), with anti-His as the primary antibody. Figure 1 As shown in B, the His antibody can specifically recognize the target protein; based on the gradient elution Western blot results, 500mM imidazole elution buffer was selected to elute the purified binding protein in the later stages, as shown in Figure B. Figure 1 As shown in Figure C, the target protein with high purity was obtained. In summary, the His-TgAtg8 protein was successfully expressed and can be used for subsequent steps.

[0043] Antibody titer detection, subtype identification and sequence detection in monoclonal cell supernatant

[0044] ELISA results showed that the serum antibody titer in mice gradually increased with the number of immunizations, reaching 1:128000 after the fourth immunization. Three positive monoclonal hybridoma cell lines were obtained through screening: 15-A12-D2-B1, 16-F9-F5, and 6-B9-E11-C1. Antibody titers were detected using the culture supernatant of these monoclonal cells. The OD value of the highest titer (1:128000) in each group was plotted. The results showed... Figure 2 A), the antibody titers in the supernatants of the three cell cultures all reached 1:128000; combined with Western blotting (WB) detection of endogenous TgAtg8 protein in Toxoplasma gondii using cell supernatants, the supernatant of the 6-B9-E11-C1 cell line failed to recognize endogenous TgAtg8, while the supernatant of the 15-A12-D2-B1 and 16-F9-F5 cell lines could recognize endogenous TgAtg8, but the latter had the best specificity. Therefore, the supernatant of the 16-F9-F5 cell line was selected for antibody subtype identification, subsequent antibody purification, and antibody sequencing; indirect ELISA results showed that the antibody secreted by the cells was IgG1 subtype ( Figure 2 B), the antibody sequence is shown in Table 1.

[0045]

[0046] Effect verification

[0047] To determine whether purified monoclonal TgAtg8 can recognize endogenous TgAtg8 protein from Toxoplasma gondii, whole Toxoplasma gondii protein was extracted and analyzed by Western blotting. The results showed that ( Figure 3 (A, 3B) The monoclonal antibody can specifically recognize endogenous TgAtg8 (pre-immunization mouse serum is a negative control, TgAtg8 rabbit polyclonal antibody is a positive control, and His-TgAtg8 is a positive control); Intracellular and extracellular parasite IFA results show (3C, 3D) that the monoclonal antibody can specifically recognize TgAtg8 and colocalize with the acroplast.

[0048] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A monoclonal antibody against Toxoplasma gondii autophagy-associated protein 8, characterized in that: It includes a first antigen-binding region and a second antigen-binding region. The first antigen-binding region includes a first light chain variable region VL-1 and a first heavy chain variable region VH-1. The second antigen-binding region includes a second light chain variable region VL-1 and a second heavy chain variable region VH-2. The CDR1, CDR2, and CDR3 of the first light chain variable region VL-1 are shown in positions 27-37, 55-57, and 94-102 of SEQ ID NO: 1, respectively. The CDR1, CDR2, and CDR3 of the first heavy chain variable region VH-1 are shown in positions 26-33, 51-58, and 97-106 of SEQ ID NO: 2, respectively. The CDR1, CDR2, and CDR3 of the second light chain variable region VL-2 are shown in positions 27-36, 54-56, and 93-101 of SEQ ID NO: 3, respectively. The CDR1, CDR2, and CDR3 of the second heavy chain variable region VH-2 are shown in positions 26-33, 51-58, and 97-108 of SEQ ID NO: 4, respectively.

2. The Toxoplasma gondii autophagy-associated protein 8 monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the first light chain variable region VL-1 of the first antigen-binding region is shown in SEQ ID NO: 1, and the amino acid sequence of the first heavy chain variable region VH-1 is shown in SEQ ID NO: 2; The amino acid sequence of the second light chain variable region VL-2 of the second antigen-binding region is shown in SEQ ID NO: 3, and the amino acid sequence of the second heavy chain variable region VH-2 is shown in SEQ ID NO:

4.

3. The application of the monoclonal antibody against Toxoplasma gondii autophagy-associated protein 8 as described in claim 1 or 2 in the preparation of a detection kit, wherein the kit is a kit for detecting Toxoplasma gondii autophagy-associated protein 8.

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