Monoclonal antibodies and uses thereof, hybridoma cells, cryptosporidium inhibiting drugs, detection reagents, detection kits

Abstract

The application relates to the technical field of biology, and discloses a monoclonal antibody and purposes thereof, a hybridoma cell, a cryptosporidium inhibiting drug, a detection reagent, and a detection kit, wherein the monoclonal antibody comprises a heavy chain region amino acid sequence as shown in SEQ ID NO:1 and a light chain region amino acid sequence as shown in SEQ ID NO:2, the monoclonal antibody can be specifically combined with TRAP-C1 protein with a TSP1 domain in cryptosporidium, and through experiments, it is found that, on the eighth day of infection, the excretion amount of cryptosporidium in the positive control group has reached a peak value and then decreases, it can be seen that, the time of 8 days has enabled the cryptosporidium to be fully reproduced and developed in the mouse, the monoclonal antibody of the application can not only inhibit the reproduction and growth speed of the cryptosporidium in the host in a short time (within 8 days), but also still has a significant inhibiting capacity on the cryptosporidium in the host when the time is prolonged to 10 days.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to monoclonal antibodies and their uses, hybridoma cells, Cryptosporidium inhibitory drugs, detection reagents, and detection kits. Background Technology

[0002] Infection with Cryptosporidium parvum typically begins with the ingestion of Cryptosporidium parvum oocysts. After being ingested into the host's small intestine, the oocysts exocyst, releasing infective sporozoites, which then invade the microvilli of the intestinal epithelium. The intracellular development of Cryptosporidium parvum takes place within the host cell's vacuolars, a unique microenvironment containing cytoplasm. The life cycle of Cryptosporidium parvum includes both asexual and sexual reproduction stages. During asexual reproduction, the parasite releases merozoites into the host's intestinal lumen, allowing them to invade adjacent intestinal epithelial cells. During sexual reproduction, macrogametes and microgametes are formed and combine to form a zygote, which matures within the oocyst. Some of these mature directly exocyst in the host's small intestine, leading to secondary infection, while others are expelled from the host as oocysts into the environment.

[0003] Thrombospondin-related adhesive proteins (TRAPs) are a class of adhesion-related proteins. Only one or two are found in other apocrypha parasites (such as Plasmodium, Toxoplasma gondii, and Eimeria), and functional studies have shown they play important roles. However, the Cryptosporidium genome encodes 12 TRAP proteins (CpTSP1–TSP12), which are also called TSP proteins because they all contain a Thrombospondin (TSP) repeat domain.

[0004] Existing technologies have been extensively studied for the infection problem of Cryptosporidium. For example, Wang Dongqiang of Jilin University pointed out in his master's thesis "Study on the heparin binding characteristics of Cryptosporidium micronematode protein CpTSP4 and its secretion and transport molecular mechanism": "TRAP-C1 is the first TRAP protein recognized and identified in Cryptosporidium. It contains 6 TSP1 domains and 2 apple domains, and plays a key role in the process of host cell invasion. At the same time, it is predicted that it has a cytoplasmic sequence that interacts with actomyosin movement. Subcellular localization studies have shown that it is located at the tip of the sporozoite and belongs to micronematode protein."

[0005] However, the document also points out that: "By comparing with the TSP1 domain, we screened Cryptosporidium microsporidium genome sequences and found 12 genes with TSP1-like domains, including TRAP-C1, which were named TRAP-C1, CpTSP2 to CpTSP12."

[0006] It is not difficult to see from this that existing technology has found through research that the TSP repeating domain may have a relatively important influence on the growth and development of Cryptosporidium.

[0007] Furthermore, Han Fusong of Jilin University, in his dissertation "Localization and Adhesion Function Study of Cryptosporidium TRAP-C2 and TSP9 Proteins," pointed out: "TRAP-C1 and other apical complex TRAP family transmembrane regions all have a short acidic cytoplasmic region and a conserved C-terminal tryptophan residue. Previous in vitro blocking assays using mouse polyclonal antibodies against the Cryptosporidium TRAP-C1 functional domain were conducted to determine their ability to inhibit parasite infection and growth within host cells. However, observations within 48 hours showed only a limited reduction in the number of Cryptosporidium parasites in the developmental stage, indicating that Cryptosporidium parasites possess one or more TSP1 domains at various stages of infection. Antibodies against only one TSP1 domain-containing protein cannot effectively prevent parasite invasion of host cells. In other words, the TSP1 domain may only represent one of the multiple invasion-related functional domains of Cryptosporidium parasites."

[0008] It is evident that the current understanding of the TRAP-C1 protein is that it can affect the development and reproduction of Cryptosporidium in host cells (in vitro infection model) to a certain extent. However, when the treatment time exceeds 48 hours, only the number of Cryptosporidium microsporidium in the developmental stage is reduced to a limited extent. It only inhibits one of the 12 genes in Cryptosporidium that have TSP1-like domains, and cannot effectively prevent the parasite from invading the host cell.

[0009] The problem this solution aims to address is: how to provide a TRAP-C1 monoclonal antibody that inhibits Cryptosporidium infection and development. Summary of the Invention

[0010] The purpose of this application is to provide a TRAP-C1 monoclonal antibody that can specifically bind to the TRAP-C1 protein with the TSP1 domain in Cryptosporidium. Experiments have shown that this monoclonal antibody can not only inhibit the proliferation and development of Cryptosporidium in host cells in a short period of time, but also has a significant inhibitory effect on Cryptosporidium in the host when the time is extended to 10 days.

[0011] Unless otherwise specified in this application: nM represents nanomoles per liter, μM represents micromoles per liter, mM represents millimoles per liter, and M represents moles per liter;

[0012] To achieve the above objectives, this application discloses a TRAP-C1 monoclonal antibody, which includes a heavy chain region amino acid sequence as shown in SEQ ID NO: 1 and a light chain region amino acid sequence as shown in SEQ ID NO: 2.

[0013] > TRAP-C1_Heavy antibody amino acid sequence (SEQ ID NO: 1)

[0014] QVQLQQPGSELVRPGASVKLSCKASGYTFTSYLMHWVKQRPGQGLEWIGSIYPGSGTTNYDEKFKSKATLTVDTSSTTAYMQLSSLTSEDSAVYYCSVYSYRFAYWGQGTLVTVSAAKTT

[0015] > TRAP-C1_Light antibody amino acid sequence (SEQ ID NO: 2)

[0016] DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCIQYANFPRTFGGGTKLEIKRADAAPTV

[0017] Preferably, the nucleotide sequence corresponding to the amino acid sequence of the heavy chain region of the monoclonal antibody is as shown in SEQ ID NO: 3;

[0018] The nucleotide sequence corresponding to the amino acid sequence of the light chain region of the monoclonal antibody is shown in SEQ ID NO: 4.

[0019] TRAP-C1_Heavy antibody nucleotide sequence (SEQ ID NO: 3)

[0020] GCAGCAGCCTGGGTCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACATTCACCAGCTACTTGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAGTATTTATCCTGGTAGTGGTACTACTAACTACGATGAG AAGTTCAAGAGCAAGGCCACACTGACTGTAGACACATCCTCCACCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTTCAGTCTACTCGTACAGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCGGCCAAAACGACAC

[0021] TRAP-C1_Light antibody nucleotide sequence (SEQ ID NO: 4)

[0022] ATTCTGATGACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAGTCAGTCTCACTTGTCGGGCAAGTCAGGAAATTAGTGGTTACTTAAGCTGGCTTCAGCAGAAACCAGATGGAACTATTAAACGCCTGATCTACGCCGCATCCACTTTAGATTCTGGTGTC CCAAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCAGCCTTGAGTCTGAAGATTTTGCAGACTATTACTGTATACAATATGCTAATTTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTA

[0023] In addition, this application also discloses a hybridoma cell expressing the above-mentioned monoclonal antibody, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 20, 2025, with accession number GDMCC No:67322, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and taxonomically named Mus musculus.

[0024] In addition, this application also discloses the use of the monoclonal antibody specifically binding to the TRAP-C1 protein as described above.

[0025] In addition, this application also discloses the use of the monoclonal antibody as described above to prepare a drug for inhibiting the proliferation and / or development of Cryptosporidium.

[0026] In addition, this application also discloses a drug containing the aforementioned monoclonal antibody.

[0027] In addition, this application also discloses a TRAP-C1 detection reagent containing the above-mentioned monoclonal antibody.

[0028] In addition, this application also discloses a Cryptosporidium detection kit containing the above-mentioned detection reagents.

[0029] Compared with the prior art, this application has the following advantages:

[0030] This application provides a monoclonal antibody that specifically binds to the TRAP-C1 protein in Cryptosporidium containing the TSP1 domain. Experiments have shown that, on the 8th day after infection, the amount of Cryptosporidium excreted in the positive control group of mice has reached its peak and then decreased. Therefore, it is not difficult to infer that the number of Cryptosporidium reaches the K value and no longer increases after 8 days. It is further evident that 8 days is sufficient for Cryptosporidium to proliferate and develop in mice. However, the monoclonal antibody of this application not only inhibits the proliferation and development of Cryptosporidium in the host in a short period of time (within 8 days), but also has a significant inhibitory effect on Cryptosporidium in the host when the time is extended to 10 days. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the immunization results in mice;

[0032] Figure 2 This is a schematic diagram of WB-specific screening for hybridoma cell lines 28, 29, 31, and 32.

[0033] Figure 3 A schematic diagram comparing the in vitro insect resistance of hybridoma cell lines No. 28, 29, 31, and 32;

[0034] Figure 4 This is a schematic diagram of the amino acid sequences of the heavy and light chains of a monoclonal antibody.

[0035] Figure 5 This is a schematic diagram of the heavy and light chains of a monoclonal antibody.

[0036] Figure 6 A comparative graph of the experimental groups during the mouse weight gain detection process;

[0037] Figure 7A comparative graph of different experimental groups during the detection of fecal oocyst excretion in mice;

[0038] Figure 8 This is a schematic diagram of ileum sections from mice in each experimental group after HE staining.

[0039] Figure 9 A comparison chart of villus length, villus diameter, crypt depth, and the ratio of villus height to crypt depth for each experimental group. Detailed Implementation

[0040] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0041] Example 1

[0042] 1.1 TRAP-C1 antigen design

[0043] Based on existing Cryptosporidium proteome sequence information, bioinformatics techniques and literature search analysis were used to screen TRAP antigen targets. Then, an integrated antigen epitope screening system was established in our laboratory to calculate interaction regions and affinities with antigen-presenting and effector-related proteins such as Toll-like receptors, B-cell antigen receptors, and T-cell antigen receptors, thereby screening for optimal peptide epitopes. These epitopes were synthesized and modified by Genscript Biotech Inc. (Nanjing Genscript Biotech Co., Ltd.)

[0044] 1.2 Preparation of hybridoma cells

[0045] 1.2.1 Myeloma cell resuscitation and expansion

[0046] Revive frozen myeloma cells 1-2 weeks in advance, culture them in basal medium to the logarithmic growth phase, and ensure cell viability ≥95%.

[0047] 1.2.1.1 Antigen Pretreatment

[0048] Adjust the concentration of TRAP-C1 to 10 μg / time, emulsify it evenly with the adjuvant, and set aside.

[0049] 1.2.2 Mouse Immunization

[0050] First immunization: Mice were immunized by intraperitoneal injection of the emulsified TRAP-C1-Freud's complete adjuvant mixture, with each mouse receiving 0.2 mL.

[0051] Second and third immunizations: Two weeks after the first immunization, the second and third booster immunizations are given with TRAP-C1-French incomplete adjuvant mixture, with the same injection method as the first immunization.

[0052] Shock immunization: Three days before cell fusion, administer an adjuvant-free antigen solution via intraperitoneal injection at the same dosage as before to enhance the antibody secretion capacity of spleen B cells.

[0053] Immunization results in mice as follows Figure 1 As shown.

[0054] 1.2.3 Cell Fusion

[0055] Preparation of feeder cells: Mice from the same batch were euthanized by cervical dislocation, and spleens were aseptically removed. Cells were dispersed by grinding with PBS, filtered, and centrifuged to collect the cell suspension. The cell concentration was adjusted to 1.11 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 cell / mL, with a plate density of 90 μL.

[0056] Cell suspension preparation: Mice were euthanized by cervical dislocation, spleens were aseptically removed, cells were dispersed by grinding with PBS, filtered, centrifuged to collect immunized mouse cells, and the cells were resuspended.

[0057] Myeloma cell collection: Collect myeloma cells in the logarithmic growth phase, centrifuge and wash 2-3 times, and collect the precipitate.

[0058] Cell fusion: Mix immune cells and myeloma cells at a ratio of 10:1, centrifuge and discard the supernatant; slowly add PEG solution, incubate for 1 min, and then add culture medium to terminate the fusion.

[0059] Centrifugation and washing: Centrifuge at 1000 rpm / min for 5 min, discard the supernatant, and resuspend the cells in culture medium containing HAT.

[0060] 1.2.4 Screening and Culture

[0061] Inoculation and culture: The fused cell suspension was inoculated into 10 96-well plates, totaling 960 wells, with 90 μL per well, and cultured in a 37°C, 5% CO2 incubator.

[0062] Medium replacement maintenance: Replace half of the HAT medium on the 3rd day of culture, and replace the entire HAT medium on the 7th day, and observe cell growth.

[0063] Hybridoma cell screening: After one week of culture, and after observing clone formation under a microscope, the culture supernatant of all 960 strains was collected for antibody detection.

[0064] 1.2.5 Cloning Culture and Antibody Detection

[0065] Antibody detection: The specificity of the target antibody in the supernatant was detected by ELISA, and cell lines with OD values ​​greater than 1.5 were screened.

[0066] Limiting dilution cloning: Digest and disperse positive well cells, serially dilute with HT medium, and seed into new 96-well plates, ensuring that each well contains only 1 cell.

[0067] Repeated cloning: After culturing for 7 days, test for antibodies again, pick positive single clone wells, and repeat cloning 2-3 times to ensure cell line purity.

[0068] 1.2.6 Cell Expansion and Preservation

[0069] Expanded culture: The cloned cells that stably secrete specific antibodies are transferred from 96-well plates to 24-well plates, and then expanded to culture flasks for large-scale culture.

[0070] Cryopreservation: Collect cells in the logarithmic growth phase, add cryopreservation solution containing 10% dimethyl sulfoxide (DMSO), aliquot into cryovials, and store in liquid nitrogen after gradient cooling for later use.

[0071] 1.3 TRAP-C1 antibody specificity detection

[0072] First, the OD value of the hybridoma cells obtained above was tested by ELISA, and four candidate cell lines (No. 28, No. 29, No. 31, and No. 32) with the highest OD value, i.e. the strongest antibody affinity, were screened out.

[0073] Subsequently, the antibody specificity of the four candidate cell lines TRAP-C1 monoclonal antibodies was detected by Western blotting. The main methods are as follows: First, the recombinant TRAP-C1 protein was prepared, and the pET-30a-TRAP-C1 recombinant plasmid was transfected into Escherichia coli BL21(DE3) expression bacteria to induce expression. The bacterial lysate was used as the detection antigen for SDS-PAGE electrophoresis, transfer, and blocking.

[0074] The culture supernatants (cells 28, 29, 31, and 32) of the four candidate TRAP-C1 monoclonal antibody cell lines selected from the above-mentioned hybridoma cells were diluted 1000-fold using antibody dilution buffer. The blocked NC membranes were washed three times with PBST and then incubated with the above-mentioned antibodies overnight at 4°C. IgG was used as a control.

[0075] The following day, after washing the NC membrane three times with PBST, the NC membrane was incubated with HRP-labeled goat anti-mouse IgG antibody diluted 1:5000 at room temperature for 1 hour. Finally, ECL developing solution was added to the NC membrane, and the protein bands were detected using a multifunction imaging system.

[0076] refer to Figure 2 Western blot (WB) screening revealed that the culture supernatant of monoclonal antibody cell line No. 29 exhibited superior specific binding to the TRAP-C1 antigen.

[0077] 1.4 Inhibitory effect of TRAP-C1 monoclonal antibody on in vitro infection of Cryptosporidium pravum

[0078] Furthermore, the culture supernatants of monoclonal antibody cell lines (cells 28, 29, 31, and 32) were subjected to in vitro infection inhibition tests. The specific test method was as follows:

[0079] HCT-8 cells were seeded into 24-well plates at a density of 500,000 cells per well. When the cell density reached 70%, 125,000 *C. parvum* oocysts were infected. Before *C. parvum* infection, sporozoites were incubated with different concentrations of TRAP-C1 antibody at 37°C for 2 hours to ensure complete blocking of the TRAP-C1 protein on the sporozoite surface. After 24 hours of *C. parvum* infection, RNA was extracted and reverse transcribed into cDNA. The expression level of the cDNA gene was detected by qPCR to assess the changes in *C. parvum* load in the cells.

[0080] The primer sequences for detecting C gene expression are CF: TGGTTCCGATTTTACGCGAA and CR: AACGTCAATTAGGTCTCAAATGACC;

[0081] The primer sequences for the GAPDH internal reference gene are GF: GAAGGTGAAGGTCGGAGTC and GR: GAAGATGGTGATGGGATTTC.

[0082] The reaction system for reverse transcription is shown in Table 1, and the reaction conditions are shown in Table 2. The reaction system for qPCR is shown in Table 3, and the reaction conditions are shown in Table 4. After the reaction, the Ct values ​​were used to calculate the relative gene expression level using the 2-ΔΔCt method, with GAPDH used as an internal reference gene.

[0083] Table 1 Reverse transcription reaction system

[0084] reagents volume 5×ABScript III RT Mix 4 μL 20×gDNA Remover Mix 1 μL Total RNA 1000 ng <![CDATA[Nuclease-free H2O]]> Add 20 μL

[0085] Table 2 Reverse transcription reaction conditions

[0086] temperature time 37℃ 2 min 55℃ 12 min 85℃ 5 min 4℃ Hold

[0087] Table 3 qPCR reaction system

[0088] reagents volume 2×Universal SYBR Green Fast qPCR Mix 10 μL <![CDATA[Nuclease-free H2O]]> 6 μL Upstream primer (10 μM) 1 μL Downstream primer (10 μM) 1 μL cDNA 2 μL

[0089] Table 4 qPCR reaction conditions

[0090]

[0091] Results analysis:

[0092] refer to Figure 3 Cell line 29 had the lowest insect load, indicating the best in vitro insect resistance. Further analysis of its specificity test results confirmed that cell line 29 performed best and was selected for subsequent tests and experiments. It should be noted that hybridoma cell line 29 was deposited on November 20, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No: 67322), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and taxonomically named *Mus musculus*.

[0093] 1.5 Sequence analysis of TRAP-C1 monoclonal antibody

[0094] The antibody secreted by cell line No. 29 was sequenced by Guangdong Botang Stem Cell Technology Co., Ltd., and its amino acid sequence and structure are as follows: Figure 4-5 As shown;

[0095] The amino acid sequence of the TRAP-C1 heavy chain region is shown in SEQ ID NO: 1;

[0096] The amino acid sequence of the TRAP-C1 light chain region is shown in SEQ ID NO: 2;

[0097] The nucleotide sequence of the TRAP-C1 heavy chain region is shown in SEQ ID NO: 3;

[0098] The nucleotide sequence of the TRAP-C1 light chain region is shown in SEQ ID NO: 4;

[0099] Among them, the complementarity-determining regions (CDRs) of the TRAP-C1 heavy chain are: CDR1 (amino acid sequence GYTFTSYL).

[0100] CDR2 (amino acid sequence is IYPGSGTT);

[0101] CDR3 (amino acid sequence is SVYSYRFAY);

[0102] The complementarity-determining regions (CDRs) of the TRAP-C1 light chain are: CDR1 (amino acid sequence QEISGY);

[0103] CDR2 (amino acid sequence is AAS);

[0104] CDR3 (amino acid sequence is IQYANFPRT).

[0105] Example 2

[0106] TRAP-C1 monoclonal antibody (No. 29) inhibits C. parvum infection in vivo.

[0107] 2.1 Experimental animals and grouping

[0108] IFN-γ- / - mice were fed for one week before the experiment to acclimatize to the new environment, with free access to food and water. Three days before infection, the mice were tested by microscopic examination and PCR to ensure they were not infected with Cryptosporidium.

[0109] Referring to Table 5, the mice were randomly divided into 4 groups, regardless of sex, with 4 mice in each group.

[0110] Ctrl 1: Do not infect sporozoites.

[0111] Ctrl 2: 4×10 5 Mice were infected by gavage with freshly decapitated sporozoites.

[0112] Ctrl 3: 4×10 5 Freshly decapitated sporozoites were incubated with 10 μg / mL mouse IgG antibody at 37°C for 2 h, and then mice were infected by gavage.

[0113] TG: 4×10 5 Freshly decapitated sporozoites were incubated with 10 μg / mL TRAP-C1 antibody at 37°C for 2 h, and then mice were infected by gavage.

[0114] Table 5 Grouping Information of Experimental Animals

[0115] Experimental Groups deal with quantity Age (W) route of infection Infection time (D) Ctrl 1 (Ctrl) PBS 4 5 Gavage 10 Ctrl 2 (C. parvum) <![CDATA[4×10 5 individual spores]]> 4 5 Gavage 10 Ctrl 3 (IgG+C. parvum) <![CDATA[4×10 sporozoites after IgG treatment]]> 5 ​ 4 5 Gavage 10 TG (TRAP-C1+C. parvum) <![CDATA[4 x 10 sporozoites after TRAP-C1 Ab treatment 5 > 4 5 Gavage 10

[0116] 2.2 Mouse weight gain detection

[0117] Starting from day 1 of infection with oocysts, each mouse was weighed daily, and the weight gain rate of mice in different groups was statistically analyzed and calculated.

[0118] Results Analysis: Reference Figure 6 The control group (Ctrl 1) mice gained an average weight of about 45%, while the Cryptosporidium microsporidium infection group (Ctrl 2) gained only about 13%, indicating that their weight gain was significantly limited.

[0119] Mice in the TRAP-C1 antibody-treated sporozoite-infected group (TG) gained 35% body weight, while mice in the mouse IgG antibody-treated sporozoite-infected group (Ctrl 3) gained only 15%. Notably, the weight gain rate of the TRAP-C1 antibody-treated group was 2.33 times that of the mouse IgG control group.

[0120] 2.3 Detection of fecal oocyst content

[0121] Starting from day 4 after infection with oocysts, we collected fresh feces from each mouse daily, weighed them, and stored them at 4°C. DNA was then extracted using the Tool DNA Kit. A standard curve for fecal oocyst content was established, and the expression level of Cp18s-LC2 was detected by qPCR using gene-specific primers to determine the C. parvum oocyst content per gram of feces.

[0122] Results Analysis: Reference Figure 7 Cryptosporidium microsporidium oocysts were detectable in mouse fecal samples as early as day 4, with peak excretion intensity on day 8. At this point, the number of oocysts per gram of feces (OPG) in the sporozoite-infected group treated with mouse IgG antibodies was 5.11 × 10⁻⁶. 6 There was no significant difference compared to the group infected with sporozoites alone; however, the OPG in the TRAP-C1 antibody-treated group was only 1.89 × 10⁻⁶. 6 The OPG in the TRAP-C1 antibody-treated group was equivalent to 36.98% of that in the mouse IgG-treated group (p<0.01). Furthermore, on day 4, the OPG in the TRAP-C1 antibody-treated group was only 16.69% of that in the mouse IgG group. These results indicate that the infectivity of Cryptosporidium microsporidia sporozoites treated with TRAP-C1 antibody was significantly reduced in the mouse model.

[0123] 2.4 Histopathological observation

[0124] Mice were sacrificed 10 days after infection with *C. parvum*. A 5 cm section of the distal small intestine from the anterior end of the cecum was harvested, immersed in 10% formalin buffer, then embedded in paraffin and sectioned transversely to a thickness of 5 µm. The sections were flattened and mounted on poly-L-lysine-treated slides to prevent detachment, dried, and then subjected to routine hematoxylin and eosin (HE) staining. Intestinal pathology was analyzed in different groups of mice. Five sections were selected from each mouse, and five intact intestinal villi were selected from each section. Villus length, villus diameter, crypt depth, and intestinal mucosal thickness were measured.

[0125] Results Analysis: Reference Figure 8-9 The results showed that the ileal villi of the infected mice were disordered and the intestinal mucosa was atrophied. Measurement of the intestinal villi coefficient revealed that *C. parvum* sporozoite infection caused villi atrophy, shortening, thickening, and crypt hyperplasia, accompanied by a decrease in the villi length-to-crypt depth ratio. Sporozoites treated with TRAP-C1 antibody significantly reduced the intestinal villi coefficient, indicating that TRAP-C1 antibody can effectively alleviate intestinal damage in mice caused by *Cryptosporidium* infection.

[0126] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A TRAP-C1 monoclonal antibody, characterized in that, The monoclonal antibody comprises the heavy chain region amino acid sequence as shown in SEQ ID NO: 1 and the light chain region amino acid sequence as shown in SEQ ID NO:

2.

2. The TRAP-C1 monoclonal antibody according to claim 1, characterized in that, The nucleotide sequence corresponding to the amino acid sequence of the heavy chain region of the monoclonal antibody is shown in SEQ ID NO: 3; The nucleotide sequence corresponding to the amino acid sequence of the light chain region of the monoclonal antibody is shown in SEQ ID NO:

4.

3. A hybridoma cell expressing the monoclonal antibody according to any one of claims 1-2, characterized in that, The hybridoma cells were deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 20, 2025, with accession number GDMCC 67322, and taxonomically named... Mus musculus .

4. A Cryptosporidium inhibitory agent, characterized in that, Contains the monoclonal antibody as described in any one of claims 1-2.

5. A reagent for detecting TRAP-C1, characterized in that, Contains the monoclonal antibody as described in any one of claims 1-2.

6. A Cryptosporidium detection kit, characterized in that, It contains the detection reagent as described in claim 5.

Images