Application of AP2-F2 protein polyclonal antibody as a positioning tag antibody for Cryptosporidium oocysts

Abstract

The application discloses application of AP2-F2 protein polyclonal antibody as a Cryptosporidium female gamete localization tag antibody. The application carries out truncated prokaryotic expression on AP2-F2 protein of Cryptosporidium parvum, immunizes ICR mice with the purified protein, prepares polyclonal antibody, carries out ELISA and WB detection on the antibody titer, proves that the polyclonal antibody is effective, then carries out indirect immunofluorescence detection on the antibody, determines that the antibody is located on Cryptosporidium female gamete, can be used as a tag to locate Cryptosporidium female gamete, and has an important role for positioning research on unknown proteins of Cryptosporidium.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and specifically relates to the application of AP2-F2 protein polyclonal antibody as a localization tag antibody for female gametes of Cryptosporidium. Background Technology

[0002] Cryptosporidium is an important zoonotic pathogen that parasitizes the gastrointestinal tract. Cryptosporidium has a wide host range; studies have shown that different species can infect a variety of hosts, including mammals, marsupials, birds, amphibians, reptiles, and fish. Currently, nearly 50 species and over 120 genotypes of Cryptosporidium have been identified, with the most prevalent species being *Cryptosporidium hominis*, which infects humans, and *Cryptosporidium parvum*, which infects both humans and livestock. In farmed animals, Cryptosporidium severely impacts cattle farming, being one of the four most important intestinal pathogens causing diarrhea in newborn calves. This pathogen causes watery diarrhea and growth retardation in newborn calves, and severe infections can lead to death, resulting in significant economic losses. Furthermore, Cryptosporidium can also cause diarrhea in sheep and goats, causing substantial economic losses to sheep farming. Cryptosporidiosis is primarily transmitted through the ingestion of food or water contaminated with its oocysts. Cryptosporidium is one of the pathogens that causes severe diarrhea in young children, leading to stunted growth and development, increased burden on the immune system, and susceptibility to secondary infections. In immunocompromised individuals such as HIV patients, organ transplant recipients, and cancer patients, Cryptosporidium infection can cause chronic diarrhea, weight loss, and malnutrition; severe infections can even be life-threatening. Currently, treatment options for Cryptosporidium are extremely limited. Nitazoxanide is the only drug approved by the FDA for clinical treatment. In immunocompetent patients, nitazoxanide can reduce the amount of Cryptosporidium oocysts excreted in feces and alleviate symptoms such as diarrhea, abdominal pain, and vomiting, but it is ineffective in immunocompromised patients. The recently developed gp40 vaccine became the first commercially approved Cryptosporidium vaccine. By immunizing pregnant dairy cows, calves acquire anti-gp40 antibodies through colostrum, effectively preventing severe diarrhea caused by Cryptosporidium parvum infection. Cryptosporidiosis, an infectious disease caused by Cryptosporidium parvum, is an important opportunistic protozoan disease characterized by diarrhea. This disease is caused by the parasite Cryptosporidium infecting the epithelial cells of the gastrointestinal tract in humans and animals. Current understanding of the invasion and development mechanisms of Cryptosporidium parvum is limited, resulting in a lack of effective drugs and vaccines. Therefore, there is an urgent need to develop more key proteins related to Cryptosporidium parvum invasion and development, and to understand their biological functions, thereby laying the foundation for the screening of drug and vaccine targets.

[0003] Cryptosporidium can complete its entire life cycle within a single host, with all stages developing within a single cell type. The parasite undergoes schizogony (asexual reproduction), gametosis, and sporulation. Exocystization is the first step in the infection process; the released sporozoites slide, adhere, and invade the luminal side of the intestinal epithelial cells. After sporozoite invasion, the parasite develops into a trophozoite, which, after an initial growth phase, produces eight merozoites. Cryptosporidium then undergoes gametosis, differentiating into female gametes and male gametophytes. The male gamete undergoes four rounds of nuclear division, producing 16 gametes, while the female gamete cell cycle arrests and remains haploid, simultaneously expanding in size and storing proteins, lipids, and carbohydrates for future oocyst formation. The male gamete is released and fertilizes the female gamete. After fertilization, oocysts are formed through meiosis and sporulation, released from the host cell, and immediately become infectious. Female gamete proteins are crucial for oocyst formation in Cryptosporidium. Therefore, in order to identify more Cryptosporidium-related proteins to further understand growth and development mechanisms and to develop drugs or vaccines against Cryptosporidium, it is necessary to provide more tags that can be used to locate female gametes. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a polyclonal antibody against the AP2-F2 protein.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned AP2-F2 protein polyclonal antibody.

[0006] Another object of the present invention is to provide the application of the above-mentioned AP2-F2 protein polyclonal antibody.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A truncated form of the AP2-F2 protein, the amino acid sequence of which is shown in SEQ ID NO:3.

[0009] The aforementioned biomaterials related to the truncated form of the AP2-F2 protein are any one of the following materials:

[0010] I. A nucleic acid molecule encoding the truncated form of the AP2-F2 protein;

[0011] II. An expression cassette containing the nucleic acid molecules described in I;

[0012] III. A recombinant vector containing the expression cassette described in II;

[0013] IV. Recombinant bacteria containing the recombinant vector described in III;

[0014] V. Transgenic cell lines containing the recombinant bacteria described in IV.

[0015] Furthermore, the nucleotide sequence of the nucleic acid molecule described in I is shown in SEQ ID NO:4.

[0016] A method for preparing polyclonal antibodies against Cryptosporidium microsporidium AP2-F2 protein using the above-mentioned truncated AP2-F2 protein or AP2-F2 protein as an antigen.

[0017] Furthermore, the amino acid sequence of the AP2-F2 protein is shown in SEQ ID NO:3.

[0018] Further, the method involves obtaining polyclonal antibodies by immunizing animals with the truncated form of the AP2-F2 protein or the AP2-F2 protein.

[0019] Furthermore, the animal includes a mouse, preferably an ICR mouse.

[0020] Furthermore, the method includes the following steps:

[0021] S1. Clone the nucleotide fragment shown in SEQ ID NO:4 and construct a recombinant plasmid;

[0022] S2. Transform the recombinant plasmid obtained in step S1 into competent cells, induce expression by IPTG, and obtain the target protein after purification.

[0023] S3. Using the protein obtained in step S2 as an antigen, immunize animals to prepare antiserum, and then purify it to obtain the polyclonal antibody.

[0024] Furthermore, the recombinant plasmid described in step S1 uses pESUMO as its base plasmid.

[0025] Furthermore, the competent cells mentioned in step S2 are BL21(DE3) and TSsetta(DE3).

[0026] Furthermore, the conditions for IPTG-induced expression are: temperature 37±2℃, rotation speed 180±50rpm / min, IPTG concentration 1±0.5mM, and time 18±2 hours.

[0027] A polyclonal antibody against Cryptosporidium microsporidium AP2-F2 protein was obtained by the above method.

[0028] The application of the aforementioned Cryptosporidium microsporidium AP2-F2 protein polyclonal antibody is any one of the following applications:

[0029] A. Application as a localization tag antibody for female gametes of Cryptosporidium microsporidium;

[0030] B. Application in the preparation of products for screening and / or identifying female gamete proteins of Cryptosporidium microsporidium;

[0031] C. Application in the preparation of products specifically for detecting Cryptosporidium microsporidium AP2-F2 protein;

[0032] D. Application in the preparation of products for detecting female gametes of Cryptosporidium microsporidium.

[0033] A Cryptosporidium microsporidium ELISA kit containing a polyclonal antibody against the aforementioned Cryptosporidium microsporidium AP2-F2 protein.

[0034] Furthermore, the Cryptosporidium microsporidium detection kit comprises at least one of the following components: washing solution, blocking solution, substrate chromogenic solution, stop solution, ELISA plate coated with the above-mentioned Cryptosporidium microsporidium AP2-F2 protein truncated body or AP2-F2 protein, detection antibody, and enzyme-labeled antibody; wherein the detection antibody is the above-mentioned Cryptosporidium microsporidium AP2-F2 protein polyclonal antibody, and the enzyme-labeled antibody is HRP-labeled goat anti-rabbit IgG.

[0035] The above-mentioned Cryptosporidium ELISA kit is used for the detection of Cryptosporidium female gametes or Cryptosporidium female gamete proteins for non-diagnostic purposes.

[0036] This invention, through prokaryotic expression of the AP2-F2 protein of Cryptosporidium microsporum and the preparation of polyclonal antibodies, determined that the AP2-F2 protein is located on the female gamete. The prepared antibodies have good specificity and sensitivity and can be used for the localization of novel proteins. As a localization tag for Cryptosporidium female gametes, it helps to identify Cryptosporidium female gamete proteins, lays the foundation for the study of Cryptosporidium female gamete proteins, and thus provides a new direction for the development of drugs and vaccines against Cryptosporidium diseases.

[0037] The present invention has the following advantages and effects compared with the prior art:

[0038] This invention involves truncating the AP2-F2 protein of Cryptosporidium parvum for prokaryotic expression, purifying the protein, and immunizing ICR mice to prepare a polyclonal antibody. This antibody can recognize the AP2-F2 protein in Cryptosporidium parvum. ELISA detection showed that the anti-AP2-F2 polyclonal antibody and the immunized serum remained positive even after a dilution of 1 / 25600, indicating good antibody titer.

[0039] By using HA antibody and AP2-F2 polyclonal antibody on HA-tagged Cryptosporidium strains with uncharacterized proteins for indirect immunofluorescence experiments, it can be determined that they are commonly located as female gamete proteins. This can be used for the localization study of unknown Cryptosporidium proteins. Immunofluorescence localization can also be used to localize Cryptosporidium female gametes, which can be used to develop more protein-based drugs or vaccines against Cryptosporidium diseases. It plays an important role in the characterization and localization of unknown proteins. Attached Figure Description

[0040] Figure 1 The image shows the amplification results of the AP2-F2 truncated-1 and AP2-F2 truncated-2 fragments of the Cryptosporidium microsporum gene.

[0041] Figure 2 The image shows the PCR amplification results of the pESUMO vector for the recombinant expression plasmid pESUMO-AP2-F2.

[0042] Figure 3 Figures showing the expression of AP2-F2 by SDS-PAGE gel electrophoresis (a, b, and d) and Western blot (c) (a, b: SDS-PAGE results of AP2-F2 expressing bacteria and expression time exploration; c: Western blot verification results of AP2-F2 induced band His tag; d: SDS-PAGE identification results of AP2-F2 recombinant protein expression form).

[0043] Figure 4 Figure 1 shows the purification results of AP2-F2 recombinant protein and the protein concentration determination results by BCA method (a: SDS-PAGE results of AP2-F2 recombinant protein purification; b: protein concentration determination of AP2-F2 recombinant protein by BCA method).

[0044] Figure 5 Figure 1 shows the results of Western blot detection for AP2-F2 polyclonal antibody specificity (a: Western blot results of recombinant protein detection in AP2-F2 polyclonal antibody negative serum; b: Western blot results of recombinant protein detection in AP2-F2 polyclonal antibody positive serum; c: Western blot results of whole-worm protein detection in AP2-F2 polyclonal antibody positive serum).

[0045] Figure 6 The image shows the IFA localization results of AP2-F2 protein in the oocysts and sporozoites of Cryptosporidium microphyllum.

[0046] Figure 7Image showing the IFA localization of AP2-F2 protein during intracellular development in Cryptosporidium microsporidium culture (Hoechst-stained somatic cell nucleus (blue) and HA-tagged antibody-bound AP2-F2 protein (red), lectin (VVL)-stained somatic cells (green), scale bar = 2 μm).

[0047] Figure 8 The graph shows the results of AP2-F2 polyclonal antibody titer testing (a) shows the serum titer measurement; b) shows the serum positive result.

[0048] Figure 9 This is a diagram showing the results of the localization analysis of uncharacterized proteins. Detailed Implementation

[0049] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0050] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0051] The gene cgd2_3490 encoding the Cryptosporidium microsporidium AP2-F2 protein is located on chromosome 3 of Cryptosporidium microsporidium and encodes the Cryptosporidium microsporidium mucin AP2-F2. Its predicted molecular weight is 129.12 kDa, and analysis revealed that it lacks a signal peptide. The nucleotide sequence of the gene encoding this protein is shown in SEQ ID NO:2. Due to the large size of the AP2-F2 protein, this invention subsequently selected to truncate it for prokaryotic expression. Ultimately, two peptide segments, 328aa to 409aa and 709aa to 1074aa, were selected for prokaryotic expression, resulting in the truncated AP2-F2 prokaryotic protein. The amino acid sequence is shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO:4. A polyclonal antibody with good specificity was prepared by immunizing ICR mice. This polyclonal antibody specifically binds to the Cryptosporidium microsporidium AP2-F2 protein and specifically locates in the female gametes of Cryptosporidium, which can be used for the localization study of unknown Cryptosporidium proteins. The polyclonal antibody showing that the Cryptosporidium microsporidium AP2-F2 protein can serve as a localization tag for female gametes can be used to co-localize with uncharacterized proteins to determine whether they are female gamete proteins or are located in female gametes. Details are as follows:

[0052] Example 1: Construction of AP2-F2 recombinant protein expression bacteria

[0053] 1. Cloning of the Cryptosporidium microsporidium gene AP2-F2

[0054] (1) Cryptosporidium microsporidium gDNA extraction: Cryptosporidium microsporidium oocysts (isolated and subcultured from dairy cow feces samples from Heilongjiang Province, China) were first subjected to repeated freeze-thaw cycles to break the cell walls, and then the genomic gDNA was extracted according to the Qiagen DNeasy Blood & Tissue Kit instructions for later use.

[0055] (2) Cloning of the target gene: Two peptide segments (328aa to 409aa and 709aa to 1074aa) of the *Cryptospora microsporum* cgd2_3490 gene were truncated and expressed in prokaryotes. The gene sequence was amplified using PCR. Among them:

[0056] Upstream primer for fragment 1 pAP2-F2-1-F: 5'-ACCGCGAACAGATTGGAGGTGGAGGATTAGGAGTGACCC-3', downstream primer for fragment 1 pAP2-F2-1-R: 5'-CCAGTGCCTGTGCTTGGCCCATATTGCACTGATTTCTTACTTG-3'.

[0057] upstream primer for fragment 2: pAP2-F2-2-F: 5'-GGGCCAAGCACAGGCAC-3',

[0058] Downstream primer for fragment 2: pAP2-F2-2-R: 5'-TCGAATTCGGATCCTCTAGTAGATGATAATGAAGCAATCC-3'.

[0059] Using the extracted Cryptosporidium microsporidium gDNA as a template, PCR amplification was performed according to the system and procedure shown in Tables 1 and 2 below to obtain the truncated expression fragment of the Cryptosporidium microsporidium AP2-F2 gene. The PCR product was recovered using a PCR product purification kit. The concentration and purity of the purified product were determined, and the product was stored at -20℃ for later use. Figure 1 The images show the amplification results of the AP2-F2 truncated-1 and AP2-F2 truncated-2 fragments of the Cryptosporidium microsporidium gene. As can be seen, the PCR amplification products are the same size as expected, indicating successful amplification.

[0060] 2. Cloning of the pESUMO expression vector

[0061] In this experiment, the pESUMO vector was used for prokaryotic expression of the protein. The pESUMO empty vector plasmid was obtained by PCR, and the primer sequences are as follows:

[0062] pSUMO-F:5'-TAAACTAGAGGATCCGAATT-3'

[0063] pSUMO-R: 5'-ACCTCCAATCTGTTCGCGGT-3'

[0064] The reaction system and procedure for PCR amplification are shown in Tables 1 and 2 below. PCR products were recovered using a PCR product purification kit.

[0065] Figure 2 The results show the PCR amplification of the recombinant expression plasmid pESUMO-AP2-F2 and the pESUMO vector. As can be seen, the PCR amplification product is the same size as expected, indicating successful amplification.

[0066] Table 1. PCR amplification reaction system

[0067]

[0068] Table 2. PCR Amplification Reaction Procedure

[0069]

[0070] 3. Construction of recombinant expression plasmids

[0071] The purified PCR product and pESUMO expression vector were ligated for homologous recombination according to the system in Table 3 below. The recombination reaction system is shown in Table 3, and the reaction conditions were 37℃ for ligation for 45 min.

[0072] Table 3. Multi-fragment homologous recombination system

[0073]

[0074] Note: a / b / c = (fragment length × 0.02) ÷ concentration of purified PCR product. Recommended fragment usage is [0.02 × number of cloning vector base pairs] ng. Use at least 10 ng for each fragment.

[0075] 4. Transformation of recombinant plasmids

[0076] Take 10 μL of the ligation product and place it in DH5α competent cells. Gently tap to mix and incubate on ice for 30 min. Heat shock in a 42°C water bath for 60 s, then immediately incubate on ice for 3 min. Add 800 μL of antibiotic-free LB liquid culture medium to the cells and incubate at 37°C on a shaker at 200 rpm for 1–2 h. Centrifuge at 5000 rpm for 5 min at room temperature, discard 700 μL of supernatant in a clean bench, resuspend the remaining cells with a pipette tip, mix well, and transfer the bacterial culture to an LB agar plate containing ampicillin. Spread evenly using a spreader. Mark the plate and incubate at room temperature for 30 min. Invert the plate in a 37°C incubator and incubate for 14–24 h until single colonies grow. Observe the density and size of the colonies during growth to facilitate subsequent experiments.

[0077] One of the recombinant plasmids with correct sequencing was selected and transformed into strains BL21(DE3) and TSsetta(DE3). Single clones with correct sequencing were selected for subsequent experiments.

[0078] Example 2: Expression and detection of AP2-F2 recombinant protein

[0079] 1. Induced expression of recombinant proteins

[0080] The correctly identified single-clone colonies were inoculated into LB medium for protein expression induction, and the expression conditions were explored. The specific steps are as follows:

[0081] Fresh bacterial culture was inoculated into 10 mL of ampicillin LB medium at a 1:100 ratio and cultured at 37°C on a shaker until the absorbance (OD600) reached approximately 0.6-0.8. 5 mL of the culture was taken as the uninduced control group, and IPTG was added to the remaining 3 mL of culture to a final concentration of 1 M to create the induction group. The culture was then continued at 37°C on a shaker. 1 mL of the culture was collected at four time points: 3 hours, 6 hours, 12 hours, and 18 hours.

[0082] 2. SDS-PAGE gel electrophoresis detection

[0083] (1) Sample preparation: OD of all the samples to be tested as described above. 600 Set the temperature to 1, centrifuge at 12000 rpm for 1 min at room temperature to collect bacterial cells, discard the supernatant, and resuspend in PBS. Take 40 μL of bacterial culture for each sample into a 1.5 mL centrifuge tube, add 10 μL of 5×SDS protein loading buffer, mix well, boil in a water bath for 10 min, and finally centrifuge at 12000 rpm for 1 min at 4 °C. Store the samples at -20 °C for later use.

[0084] (2) Preparation of 10% SDS-PAGE gel: Take 5 mL of separating gel premix, 50 μL of 10% AP and 2.5 μL of TEMED, mix well and pour into a 1 mm glass gel plate, seal with ddH2O, and let stand at room temperature for about 25 min. After the separating gel solidifies, discard the ddH2O. Take 1 mL of stacking gel premix, 10 μL of 10% AP and 1 μL of TEMED, mix well and pour into the upper layer of the separating gel, insert a 1 mm comb, and let stand until the stacking gel solidifies.

[0085] (3) SDS-PAGE gel electrophoresis: Add 8 μL of protein standard marker to the first well, then add the sample, and finally add an appropriate amount of electrophoresis buffer. Electrophoresis at a constant voltage of 120V for about 60 minutes.

[0086] After electrophoresis, the gel was stained with Coomassie Brilliant Blue on a shaker at room temperature for 30 minutes. After staining, the gel was transferred to destaining solution until the background was colorless. Finally, the gel was photographed using a gel imaging system to observe protein expression.

[0087] The results are as follows Figure 3 As shown in Figures a and b, a specific band of approximately 70 kDa was induced in both expression bacteria, slightly larger than the predicted molecular weight of 6His-SUMO-AP2-F2-6His (60.8 kDa). The optimal induction conditions were 37°C, 180 rpm / min, and 1 mM IPTG induction for 18 hours.

[0088] 3. Validation by Western blot analysis of proteins.

[0089] (1) SDS-PAGE gel electrophoresis: Follow the steps above. After electrophoresis, cut off the gel containing the sample lane and soak it in the transfer solution for 1-2 minutes.

[0090] (2) Prepare for membrane transfer. Cut a PVDF membrane and 6 layers of filter paper to a size similar to the SDS-PAGE gel. Soak the PVDF membrane in methanol for 3-5 minutes. Stack the components in the transfer solution in the following order: from negative electrode (black plate), sponge pad, 3 layers of filter paper, protein gel, PVDF membrane, 3 layers of filter paper, sponge pad to positive electrode (white plate).

[0091] (3) Transfer: Place the assembled transfer assembly into the tank, add an appropriate amount of pre-cooled transfer solution, and transfer at a constant current of 250mA for 48 minutes.

[0092] (4) Closure: After the transfer is completed, rinse the PVDF membrane with TBST, then add 5% (v / v) skim milk powder and incubate overnight at 4°C.

[0093] (5) Primary antibody incubation: Discard the blocking solution, dilute the anti-His-tagged rabbit monoclonal antibody with 5% skim milk powder at 1 / 1,000, and incubate the PVDF membrane at 37°C for 1 hour.

[0094] (6) Secondary antibody incubation: After primary antibody incubation, rinse 3 times with TBST for 5 minutes each time, dilute HRP-labeled goat anti-rabbit IgG monoclonal antibody with 5% skim milk powder at 1 / 10000, and incubate at 37°C for 1 hour.

[0095] (7) Observation: After incubation with the secondary antibody, wash with TBST 3 times for 5 minutes each time, develop color with ECL chemiluminescence reagent, and observe and take pictures in the imaging system.

[0096] The results are as follows Figure 3 As shown in Figure c, the results of Western blot analysis using an anti-His tag monoclonal antibody as the primary antibody showed that the expressed bacteria after induction exhibited a specific band of approximately 70 kDa compared to before induction.

[0097] 4. Validation of recombinant protein expression form

[0098] (1) Expand the culture of bacterial solution: Take positive expression bacteria, revive the strain, and add an appropriate amount of bacterial solution to ampicillin-resistant LB medium for culture.

[0099] (2) Induction culture: When the OD of the above bacterial solution is 100%, the induction culture is 100 600 When the pH is 0.6–0.8, add 1 mmol / L IPTG and incubate at 37°C in a shaker for 18 h.

[0100] (3) Collect bacterial cells: Centrifuge the induced bacterial solution multiple times and collect it in 50 mL centrifuge tubes. Centrifuge at 8,000 r / min for 20 min at 4℃ each time. Discard the supernatant. Add 30 mL of PBS to each tube of bacterial cells for resuspending. Centrifuge under the same conditions twice. Finally, resuspend with 10 mL of PBS and store at -80℃ for later use.

[0101] (4) Ultrasonic disruption: Repeat the freeze-thaw cycle at -80℃ and 4℃ 3 times, add protease inhibitor at 1 / 100, power 50%, work for 2 seconds, pause for 3 seconds, and sonicate on ice until the bacterial solution is clear. The time is about 40 minutes.

[0102] (5) Take 80 μL of the ultrasonically disrupted bacterial culture. Centrifuge the remaining bacterial cells at 10,000 r / min, 4℃ for 30 min, and take 80 μL of the supernatant. Resuspend the precipitate in 10 mL of dissolving buffer, mix well, and incubate overnight in a shaker at 4℃. Take 80 μL of the precipitate. Prepare the sample and perform SDS-PAGE gel electrophoresis for detection.

[0103] Figure 3The SDS-PAGE gel electrophoresis results in the middle d showed that lane 3 and lane 4 were the supernatant and inclusion bodies separated from the bacterial cell lysate after induction, respectively. Both showed bands of about 70 kDa, and the band in the inclusion body was significantly stronger than the band in the supernatant, indicating that the recombinant protein rAP2-F2 was mainly expressed in large quantities in the form of inclusion bodies.

[0104] Finally, we determined the optimal induction conditions to be 37℃, 180 rpm / min, and 1 mM IPTG induction for 18 hours, and cultured the expression bacteria under these conditions for purification of the rAP2-F2 protein. These results demonstrate that the recombinant protein rAP2-F2 was successfully expressed in *E. coli*.

[0105] 5. Purification and validation of recombinant proteins

[0106] Supernatant protein purification

[0107] (1) Sample preparation: Inclusion bodies were dissolved overnight at 4°C using 8 mol / L urea. The supernatant was centrifuged at 12000 r / min for 10 min at 4°C, and the resulting supernatant was used for protein purification.

[0108] (2) Equilibration: Add 2 mL of Ni-NTA His Bind Resin nickel column to the chromatography column, and equilibrate the packing material with 5-10 column volumes of Hisbinding Buffer to place the packing material in the same buffer system as the target protein, thereby protecting the protein.

[0109] (3) Sample loading: After equilibration, add the broken protein sample and incubate on a horizontal shaker at 4°C for 3 hours or overnight to ensure full contact with the packing material and improve the recovery rate of the target protein. Collect the effluent. Record it as "sample loading flow-through".

[0110] (4) Washing: Wash with 10-20 column volumes of Hisbinding Buffer to remove non-specifically adsorbed proteins and collect the effluent. Record the effluent as "washing buffer".

[0111] (5) Elution: Elute the target protein with imidazole at different concentrations. The imidazole concentrations were 40mM, 80mM, 120mM, 160mM, 200mM, 240mM, 280mM, 320mM, 360mM, and 400mM. The elution order was from low to high concentration, with 1-2 column volumes eluted for each concentration. Collect the eluent. Record it as "elution buffer".

[0112] (6) Cleaning the resin: Use 3 column volumes of Hisbinding Buffer and 5 column volumes of deionized water balance packing in sequence.

[0113] (7) Resin preservation: Finally, equilibrate with 5 column volumes of 20% ethanol, and then store in an equal volume of 20% ethanol at 4°C to prevent the packing material from being contaminated by bacteria.

[0114] 6. BCA method for protein concentration determination

[0115] The purified protein was analyzed using the Beyotime BCA protein concentration assay kit. The specific steps are as follows:

[0116] (1) Preparation of protein standards:

[0117] (a) Add 1.2 mL of protein standard preparation solution to one tube of protein standard (30 mg BSA), dissolve thoroughly, and prepare a 25 mg / mL protein standard solution. It can be used immediately after preparation or stored at -20°C for long-term storage.

[0118] (b) Take an appropriate amount of 25 mg / mL protein standard and dilute it to a final concentration of 0.5 mg / mL. For example, take 20 μl of 25 mg / mL protein standard and add 980 μl of diluent to prepare a 0.5 mg / mL protein standard. The standard should be diluted with the same solution as the protein sample. However, for simplicity, the standard can also be diluted with 0.9% NaCl or PBS. The diluted 0.5 mg / mL protein standard can be stored at -20°C for a long time.

[0119] (2) Preparation of BCA working solution: Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1) and mix thoroughly. For example, add 100 μL of BCA reagent B to 5 mL of BCA reagent A, mix well, and prepare 5.1 mL of BCA working solution. The BCA working solution is stable at room temperature for 24 hours.

[0120] (3) Protein concentration determination:

[0121] (a) Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to the wells of a 96-well plate, and add standard diluent to bring the total to 20 μL. This is equivalent to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively.

[0122] (b) Add an appropriate volume of sample to the wells of the 96-well plate. If the sample volume is less than 20 μL, add standard diluent to bring it to 20 μL. Please record the sample volume.

[0123] (c) Add 200 μL of BCA working solution to each well and incubate at 37°C for 20-30 minutes.

[0124] (d) Measure the absorbance at A562, or other wavelengths between 540-595 nm, using an ELISA reader.

[0125] (e) Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0126] Figure 4 The figures show the purification results of the AP2-F2 recombinant protein and the protein concentration determination results using the BCA method. It can be seen that the purified AP2-F2 protein band is relatively uniform. The BCA method was used to determine the recombinant protein concentration, and the results showed that the concentrated protein concentration was 1.22 mg / mL.

[0127] Example 3: Preparation and titer detection of polyclonal antibodies

[0128] 1. The prepared recombinant protein was used to immunize ICR mice to obtain polyclonal antibodies. The specific steps are as follows:

[0129] (1) The initial immunization protein dose was 200 μg / mouse. The protein solution was diluted with PBS to 100 μL and mixed with an equal volume of Freund's complete adjuvant and emulsified.

[0130] (2) Subcutaneous injection: The total injection volume for each mouse is 200 μL. The emulsifying agent is injected subcutaneously at multiple points on the back of the mouse using a 1 mL syringe. After the injection, the injection site can be massaged appropriately to increase the absorption of immunogen.

[0131] (3) A second immunization was administered 14 days after the first immunization, followed by immunizations every 7 days for a total of 4 immunizations. The protein dose for the second, third, and fourth immunizations was 100 μg / mouse. The protein solution was diluted to 100 μL with PBS and emulsified with an equal volume of Freund's incomplete adjuvant. Subcutaneous injection was administered as above.

[0132] (4) After the fourth immunization, a small amount of blood was collected using the orbital prick method to determine the serum antibody titer. A serum titer exceeding 10... 5 Then, all the blood was collected using the ocular blood sampling method.

[0133] 2. ELISA detection of polyclonal antibody titer

[0134] (1) Coating: Use recombinant protein with a final concentration of 5 μg / mL as the coating solution, add 100 μL to each well of the microplate, and incubate overnight at 4°C.

[0135] (2) Blocking: Discard the coating solution, add PBST to wash 3 times, 5 min each time, add 100 μL of 1% BSA to each well, and block in a humidified box at 37°C for 1 h.

[0136] (3) Primary antibody incubation: Discard the blocking solution. Add 200 μL of PBST to each well, except for the second column. Then add 0.5 μL and 1 μL of purified anti-AP2-F2 polyclonal antibody or antiserum to the first and second columns, respectively. A negative serum control is also included. Serial dilutions are performed starting from the second column. The liquid is discarded after dilution, and the solution is incubated in a humidified incubator at 37°C for 1 hour.

[0137] (4) Secondary antibody incubation: Discard the primary antibody, wash as before, add 100 μL of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG diluted 1 / 100,000 times to each well, and incubate in a humidified chamber at 37°C for 1 h.

[0138] (5) Color development: Discard the secondary antibody, wash as before, add 100 μL of TMB substrate solution to each well, and incubate in a humidified chamber at 37°C for 10 min. If there is a reaction, different shades of blue will be observed.

[0139] (6) Termination: Add 50 μL of termination solution (2M H2SO4) to each well to terminate the reaction, which will result in different shades of yellow.

[0140] (7) Detection reading: OD was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 450 The values ​​are plotted on a line graph, and the P / N ratio (sample absorbance / negative sample value) is calculated.

[0141] (8) Detection: Place the sample in an ELISA reader to detect OD450. A P / N ratio greater than 2.1 indicates a positive result.

[0142] The results are as follows Figure 8 As shown, after four immunizations, the antibody level reached a high level, with the P / N ratio of absorbance (OD) of the post-immunization serum to the pre-immunization serum being much greater than 2.1. This indicates the antibody is effective, and the result is positive. Analysis of the test results revealed that the AP2-F2 antiserum remained positive even after a 1 / 25600 dilution, indicating that the prepared AP2-F2 antiserum has good titer.

[0143] Example 4: Specificity detection of polyclonal antibodies

[0144] 1. Western blot detection

[0145] To verify the expression of the target protein in Cryptosporidium sporozoites, this invention reacts sporozoite protein extracted from oocysts with an anti-AP2-F2 polyclonal antibody and detects the prokaryotic expression of the recombinant protein AP2-F2. The specific steps are as follows:

[0146] Obtaining sporozoites: Take 1×10 8One oocyst was placed in a low-absorption centrifuge tube, and the oocysts were treated by centrifugation at 13200 rpm for 5 min at 4 °C, and the supernatant was discarded. The oocysts were then resuspended in 1 mL of 0.5% sodium hypochlorite solution and treated on ice for 10 min. The oocysts were washed three times with PBS, centrifuged at 13200 rpm for 3 min at 4 °C each time, and the supernatant was discarded. Finally, the oocysts were washed three times with PBS under the same centrifugation conditions; the supernatant was discarded, and the oocysts were resuspended in a mixture of 40 μL of cell lysis buffer (RIPA) and protease inhibitor (1 / 100) and incubated at 4 °C on a shaker for 4 h.

[0147] Recombinant protein and sporozoite protein samples were processed and subjected to SDS-PAGE gel electrophoresis. Western blot analysis was then performed. Recombinant protein and sporozoite-extracted protein were used as antigens and incubated with anti-AP2-F2 polyclonal antibody (primary antibody) and pre-immune negative rabbit serum, respectively, both diluted 1 / 2000. Secondary antibody incubation was performed with HRP-labeled goat anti-rabbit IgG (1 / 4000 dilution). Finally, the samples were developed using ECL chemiluminescence immunoassay and observed and photographed using an imaging system.

[0148] The results are as follows Figure 5 As shown, the sporozoite protein incubated with pre-immunization serum did not react with the purified recombinant protein, nor did any bands appear; however, the purified recombinant protein incubated with post-immunization serum showed a specific band of approximately 70 kDa; similarly, the sporozoite protein incubated with post-immunization serum also showed a band of approximately 70 kDa. This indicates that the antibody can specifically recognize the native protein AP2-F2 in Cryptosporidium microsporum, and that the polyclonal antibody exhibits good specificity.

[0149] 2. Indirect immunofluorescence localization (IFA)

[0150] IFA of sporozoites and oocysts: Decapitated oocysts and decapitated sporozoites were directly resuspended and spot-coated onto 6-well glass plates and allowed to air dry.

[0151] Intracellular IFA: HCT-8 cells were infected via sporozoites. After 2 hours, the culture medium in the culture plate was discarded, and the cells were washed three times with PBS to remove any incompletely invading sporozoites and oocysts. Fresh 1640 cell culture medium (2% FBS) was added, and the cells were cultured for 3, 4, 6, 12, 24, and 48 hours. After the culture was completed, the culture medium in the culture plate was discarded, and the cells were washed three times with PBS.

[0152] The specific steps of IFA in sporozoites and oocysts, and IFA during the intracellular stage are as follows:

[0153] Fixation: Add 100 μL of polyethanol to the sample and fix for 15 min. Then rinse three times with 150 μL of PBS buffer for 5 min each time.

[0154] Permeation: Add 0.5% (v / v) of Triton-100 to the sample, treat for 15 min and then rinse, using the same rinsing method as above.

[0155] Blocking: Add 100 μL of 5% BSA solution to the sample and incubate at room temperature for 30 min.

[0156] Primary antibody incubation: For intracellular IFA, dilute the anti-AP2-F2 polyclonal antibody 1 / 800 (working concentration 0.7 μg / mL) with 5% BSA and add 100 μL of antibody dilution to the sample; for sporozoite IFA, dilute the anti-AP2-F2 polyclonal antibody with 5% BSA and the anti-EF1a rabbit polyclonal antibody 1 / 800, add 20 μL of antibody dilution to the sample, incubate at 37°C for 1 h, and then rinse as above.

[0157] Secondary antibody incubation: Sporozoite IFA diluted 1 / 800 with 5% BSA. 594-labeled goat anti-mouse IgG and Alexa 488-labeled goat anti-rabbit IgG was used as the secondary antibody, and 20 μL of antibody dilution buffer was added to the sample; intracellular IFA was diluted 1 / 800 with 5% BSA. 594-labeled goat anti-mouse IgG and 1 / 500 diluted VVL were added to the sample, and 100 μL of antibody dilution solution was added. The sample was incubated at 37°C in the dark for 1 hour, and then rinsed using the same method as above.

[0158] DAPI staining: Dilute the DAPI stock solution 500 times with PBS, add 50 μL of DAPI staining solution (working concentration of 5 μg / mL) to the sample, let stand at room temperature for 5 min, then rinse as above, and finally rinse once with ddH2O.

[0159] Mounting: Add one drop of anti-fluorescence quenching agent (No-Fade Mounting Medium) to the sample, cover with a coverslip, and apply nail polish around the edges to seal the slide. After the nail polish is completely dry, the sample can be observed and photographed under a fluorescence microscope, or stored at 4°C in the dark for later use.

[0160] IFA assay was performed using AP2-F2 murine polyclonal antibody to observe oocysts and sporozoites. Results are as follows: Figure 6 As shown, AP2-F2 is not expressed in the oocyst and sporozoite stages of Cryptosporidium microlucum.

[0161] This invention involves infecting HCT-8 cells with oocysts and performing IFA assays at 3h, 12h, 36h, and 48h. AP2-F2 protein (red) was labeled with a murine polyclonal antibody, and Cryptosporidium was stained with volatile lectin (VVL). Results are as follows... Figure 7 As shown, after sporozoites invade and form trophozoites, the trophozoites develop into tetranuclear schizonts, which further divide into mature octanuclear schizonts. No AP2-F2 fluorescence signal was observed at any of these stages. Upon entering the male and female gamete stages, no fluorescence was observed in the male gametes, while a strong fluorescence signal was observed in the female gametes. This indicates that the AP2-F2 protein is not expressed in the trophozoite, schizont, and male gamete stages, and is expressed only in the female gamete stage.

[0162] Example 5: Localization analysis of uncharacterized proteins

[0163] A gene-editing marker strain of an uncharacterized protein was constructed, and a 3×HA tag was inserted into the 3' end of a gene editing an unknown localized protein (obtained from Cryptosporidium microsporum transcriptome data, its amino acid sequence is shown in SEQ ID NO:5) using CRISPR / Cas9 technology. HCT-8 cells were then infected using this marker strain as described above. HA antibody (rat-derived) and a prepared AP2-F2 polyclonal antibody (mouse-derived) were then used as primary antibodies; Alexa 488-labeled goat anti-rat IgG and Alexa 594-labeled goat anti-mouse IgG was used as a secondary antibody, and indirect immunofluorescence (IFA) experiments were performed in the same manner as described above.

[0164] The IFA results of uncharacterized proteins and AP2-F2 proteins during intracellular development in Cryptosporidium microsporidium culture are as follows: Figure 9 As shown, female gametes of Cryptosporidium (red) were labeled with AP2-F2 polyclonal antibody, and uncharacterized proteins (green) were labeled with commercial HA antibody. The uncharacterized proteins were found to be dotted and located close to the nucleus of female gamete cells, but no expression of the uncharacterized proteins was detected in male gametophytes, indicating that the uncharacterized proteins are located in female gametes and are not expressed in male gametophytes.

[0165] In summary, this invention prepares a polyclonal antibody by prokaryotic expression of the AP2-F2 protein of Cryptosporidium, followed by protein purification and immunization of ICR mice. Indirect immunofluorescence detection confirms the antibody's localization on the female gametes of Cryptosporidium. This antibody specifically recognizes Cryptosporidium female gametes and can be used to study the localization of other uncharacterized Cryptosporidium proteins, serving as a localization tag protein for Cryptosporidium female gametes.

[0166] 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 truncated form of the AP2-F2 protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

3.

2. The biomaterial containing the AP2-F2 protein truncated form as described in claim 1, characterized in that: The biomaterial is selected from any one of the following: I. A nucleic acid molecule encoding the truncated form of the AP2-F2 protein, the nucleotide sequence of which is shown in SEQ ID NO:4; II. An expression cassette containing the nucleic acid molecules described in I; III. A recombinant vector containing the expression cassette described in II; IV. Recombinant bacteria containing the recombinant vector described in III; V. Transgenic cell lines containing the recombinant bacteria described in IV.

3. A method for preparing polyclonal antibodies against Cryptosporidium microsporidium AP2-F2 protein using the truncated form of AP2-F2 protein as an antigen, characterized in that: Polyclonal antibodies were obtained by immunizing animals with the truncated AP2-F2 protein.

4. The method according to claim 3, characterized in that: The animal in question is an ICR mouse; The method includes the following steps: S1. Clone the nucleotide fragment shown in SEQ ID NO:4 and construct a recombinant plasmid; S2. Transform the recombinant plasmid obtained in step S1 into competent cells, induce expression by IPTG, and obtain the target protein after purification. S3. Using the protein obtained in step S2 as an antigen, immunize animals to prepare antiserum, and then purify it to obtain the polyclonal antibody.

5. The method according to claim 4, characterized in that: The recombinant plasmid mentioned in step S1 uses pESUMO as its base plasmid; The competent cells mentioned in step S2 are BL21 (DE3) or TSsetta (DE3). The conditions for IPTG-induced expression were: temperature 37±2℃, rotation speed 180±50 rpm / min, IPTG concentration 1±0.5mM, and time 18±2 hours.

6. A polyclonal antibody against Cryptosporidium microsporidium AP2-F2 protein, characterized in that: Obtained by the method described in any one of claims 3-5.

7. The application of the polyclonal antibody against Cryptosporidium microsporidium AP2-F2 protein as described in claim 6, characterized in that: The application mentioned is any one of the following: A. Application in the preparation of antibodies targeting female gametes of Cryptosporidium microsporidium; B. Application in the preparation of products for screening and / or identifying female gamete proteins of Cryptosporidium microsporidium; C. Application in the preparation of products specifically for detecting Cryptosporidium microsporidium AP2-F2 protein; D. Application in the preparation of products for detecting female gametes of Cryptosporidium microsporidium.

8. A Cryptosporidium microsporidium ELISA kit, characterized in that: A polyclonal antibody containing the Cryptosporidium microsporidium AP2-F2 protein as described in claim 6.

9. The Cryptosporidium microsporidium ELISA kit according to claim 8, characterized in that: The Cryptosporidium microsporidium detection kit comprises at least one of the following components: washing buffer, blocking buffer, substrate chromogenic solution, stop solution, ELISA plate coated with the truncated form of Cryptosporidium AP2-F2 protein, detection antibody, and enzyme-labeled antibody; The detection antibody is the Cryptosporidium microsporidium AP2-F2 protein polyclonal antibody as described in claim 6, and the enzyme-labeled antibody is HRP-labeled goat anti-rabbit IgG.

10. The use of the Cryptosporidium microsporidium ELISA kit of claim 8 or 9 in the detection of Cryptosporidium microsporidium female gametes or Cryptosporidium microsporidium female gamete proteins for non-diagnostic purposes.

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