Monoclonal antibody targeting EcSSP1 protein and application of monoclonal antibody in detection of microsporidia

Abstract

The invention relates to the technical field of monoclonal antibodies, in particular to a monoclonal antibody targeting EcSSP1 protein and application of the monoclonal antibody in detection of microsporidia. The amino acid sequence of a heavy chain variable region of the monoclonal antibody provided by the invention is as shown in SEQ ID NO.9, and the amino acid sequence of a light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.10. The monoclonal antibody provided by the invention is obtained by screening by taking the recombinant protein EcSSP1 as an immunogen, has relatively high specificity, can be positioned on the surface of microsporidia, has relatively high detection accuracy and sensitivity, can be used for detecting in-vivo microsporidia in the early stage of infection, and can be used for detecting microsporidia in vivo. And if the antibody is positive, the current infection of the microsporidia can be confirmed, the operation is simple and convenient, the subcellular localization research of the microsporidia protein can be carried out by utilizing a multicolor fluorescence labeling method, and the application prospect is relatively good.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection, and particularly to a monoclonal antibody targeting EcSSP1 protein and its application in detecting microsporidia. Background Art

[0002] Microsporidia are a group of obligate intracellular parasitic opportunistic pathogens. Microsporidia exist widely in nature. Their host range is very wide, and they can infect all groups of vertebrates and invertebrates, and are an important pathogen in animal husbandry, agriculture and humans. Encephalitozoon cuniculi is the most intensively studied mammalian microsporidium at present. It was first identified in the brains, spinal cords and kidneys of rabbits with movement disorders. Since then, Encephalitozoon cuniculi has been identified in many mammals including rodents, carnivores, ruminants and other primates. The main mode of infection of Encephalitozoon cuniculi is horizontal transmission through ingestion and inhalation.

[0003] Infection with Encephalitozoon cuniculi usually causes hepatitis, liver failure and peritonitis. Granulomatous hepatitis caused by Encephalitozoon cuniculi is the most common in mammalian infections, and granulomatous hepatitis caused by Encephalitozoon cuniculi has also been reported in AIDS patients. In cases of peritonitis caused by Encephalitozoon cuniculi, most omentums showed focal necrosis and non-granulomatous inflammation. Encephalitozoon cuniculi also exists with disseminated infections caused by fever, renal insufficiency and intractable cough. At present, there is no specific drug for treating microsporidiosis in rabbits. Therefore, it is very important to diagnose Encephalitozoon cuniculi disease in a timely manner.

[0004] The detection methods of microsporidia mainly include etiological detection, molecular biology detection and immunological detection. The detection methods of microsporidia mainly include morphological detection, molecular biology detection and immunological detection. Morphological detection is the earliest detection technology for microsporidia, but it requires specialized technical personnel. Molecular biology detection has high sensitivity and specificity, but its detection cost is relatively high. Immunological detection of microsporidia is a sensitive and specific detection method and is widely used in the diagnosis of microsporidia. For example, Chinese Patent CN116903713A discloses a method for judging whether a person has been infected with microsporidia or has immunity to it by detecting antibodies in serum. However, this detection method requires the body to be infected with the pathogen for a period of time to produce a sufficient amount of antibodies to be detected, there is a window period, and a positive antibody does not necessarily represent a current infection, but may be an immune memory left after a previous infection. Summary of the Invention

[0005] To solve the above problems, the present invention provides a monoclonal antibody targeting the EcSSP1 protein and its application in detecting microsporidia. The monoclonal antibody provided by the present invention can target the surface protein EcSSP1 of microsporidia, has strong specificity and high sensitivity. Using this monoclonal antibody with an indirect immunofluorescence detection method, microsporidia in vivo can be detected at an early stage of infection. Positive antibody indicates a current infection with microsporidia, and the operation is simple, showing good application prospects.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a monoclonal antibody targeting the EcSSP1 protein, including a heavy chain and a light chain. The complementarity-determining regions of the heavy chain include CDR-H1, CDR-H2, and CDR-H3; the complementarity-determining regions of the light chain include CDR-L1, CDR-L2, and CDR-L3.

[0008] The amino acid sequence of CDR-H1 is as shown in SEQ ID NO.12; the amino acid sequence of CDR-H2 is as shown in SEQ ID NO.13; the amino acid sequence of CDR-H3 is as shown in SEQ ID NO.14.

[0009] The amino acid sequence of CDR-L1 is as shown in SEQ ID NO.15; the amino acid sequence of CDR-L2 is as shown in SEQ ID NO.16; the amino acid sequence of CDR-L3 is as shown in SEQ ID NO.17.

[0010] Preferably, the amino acid sequence of the variable region of the heavy chain is as shown in SEQ ID NO.9, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID NO.10.

[0011] Preferably, the nucleotide sequence of the variable region of the heavy chain is as shown in SEQ ID NO.7, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID NO.8.

[0012] The present invention provides the application of the monoclonal antibody described in the above technical solution in one or more of the following:

[0013] 1) Preparation of a product for detecting Microsporidia; 2) Detection of Microsporidia, where the direct purpose of the detection is neither diagnosis nor treatment; 3) Subcellular localization of microsporidia proteins.

[0014] Preferably, the microsporidia include Encephalitozoon.

[0015] Preferably, the microsporidia encephalitidis includes Encephalitozoon cuniculi (E. cuniculi).

[0016] Preferably, the product includes a reagent or a kit.

[0017] The present invention provides a kit for detecting microsporidia, which includes the monoclonal antibody and the immunological detection reagent described in the above technical solution.

[0018] Preferably, the immunological detection reagent includes a fluorescently labeled anti-IgG antibody.

[0019] Preferably, the fluorescent label includes Alexa Fluor 488.

[0020] Advantageous effects:

[0021] The monoclonal antibody provided by the present invention is a monoclonal antibody screened using the recombinant protein EcSSP1 as an immunogen. It has high specificity, can be localized on the surface of microsporidia, has higher detection accuracy and sensitivity, can detect microsporidia in vivo at the early stage of infection, and a positive antibody can confirm the current infection with microsporidia. Moreover, the operation is simple, and the subcellular localization of microsporidia proteins can also be studied using the multi-color fluorescence labeling method, showing good application prospects. Description of the drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0023] Figure 1 It is a result diagram of the expression and purification of the recombinant protein EcSSP1; among them, A: SDS-PAGE analysis result diagram of the purified recombinant protein EcSSP1-His, lanes 1-2: eluted with 20 mM imidazole, lanes 4-5: eluted with 50 mM imidazole, lanes 6-8: eluted with 100 mM imidazole, lane 9: flow-through after loading the sample; B: result diagram of detecting the expressed recombinant protein with His antibody;

[0024] Figure 2 It is a SDS-PAGE analysis result diagram of the monoclonal antibody and different proteins; among them, A: proteins of Cryptococcus neoformans, B: proteins of Escherichia coli, C: proteins of yeast;

[0025] Figure 3 It is a Western blot detection result diagram of the monoclonal antibody; among them, A: Western blot result diagram of the monoclonal antibody reacting with the total protein of E. cuniculi; B: Western blot result diagram of the monoclonal antibody reacting with the recombinant protein EcSSP1;

[0026] Figure 4 The indirect immunofluorescence detection results of the monoclonal antibody; among them, A1 and B1 represent Encephalitozoon cuniculi under white light; A2 and B2 represent the nuclei labeled with DAPI; A3 represents the addition of the monoclonal antibody, and B3 represents the addition of the negative serum; A4 and B4 represent the results of image merging; the scale bar in the lower right corner is 2μm for all. Detailed implementation mode

[0027] The present invention provides a monoclonal antibody targeting the EcSSP1 protein, including a heavy chain and a light chain. The complementary determining regions of the heavy chain include CDR-H1, CDR-H2, and CDR-H3; the complementary determining regions of the light chain include CDR-L1, CDR-L2, and CDR-L3; the amino acid sequence of CDR-H1 is as shown in SEQ ID NO.12; the amino acid sequence of CDR-H2 is as shown in SEQ ID NO.13; the amino acid sequence of CDR-H3 is as shown in SEQ ID NO.14; the amino acid sequence of CDR-L1 is as shown in SEQ ID NO.15; the amino acid sequence of CDR-L2 is as shown in SEQ ID NO.16; the amino acid sequence of CDR-L3 is as shown in SEQ ID NO.17, specifically as follows:

[0028] CDR-H1: DYWIG;

[0029] CDR-H2: NIYPGGFYTHYNEKFKG;

[0030] CDR-H3: RDMIAPFAY;

[0031] CDR-L1: KASQDIKKYMA;

[0032] CDR-L2: YTSTLQP;

[0033] CDR-L3: LQYENLWT.

[0034] As an implementation mode, the amino acid sequence of the variable region of the heavy chain is as shown in SEQ ID NO.9, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID NO.10, specifically as follows:

[0035] SEQ ID NO.9:

[0036] QVQLEQSGAELVRPGTSVKMSCKATGYTFTDYWIGWVKQRPGHGLEWI GNIYPGGFYTHYNEKFKGKATLTVDTSSSTAYMQLSSLTSEDSAIYFCTRRDMI APFAYWGQGTLVTVSASTKGP;

[0037] SEQ ID NO.10:

[0038] DIVLTQTPSSLSPSLGGKVTITCKASQDIKKYMAWYQHKPGKGPRLLINY TSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYENLWTFGGGTKLEI KRADAAPTVS。

[0039] As an embodiment, the nucleotide sequence of the variable region of the heavy chain is as shown in SEQ ID NO.7, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID NO.8.

[0040] The monoclonal antibody provided by the present invention is a monoclonal antibody screened using the recombinant protein EcSSP1 as an immunogen, has high specificity, can be localized on the surface of microsporidia, has higher detection accuracy and sensitivity, can detect microsporidia in vivo through indirect immunofluorescence reaction at the early stage of infection, and a positive antibody can confirm that the test sample is currently infected with microsporidia. Depending on the different infection sites of Encephalitozoon cuniculi and diverse sample types, such as feces, urine, infected tissue samples, etc., and the operation is simple. Moreover, the subcellular localization of microsporidia proteins can also be studied using the multi-color fluorescence labeling method, showing good application prospects.

[0041] Based on the above advantages, the present invention provides the application of the monoclonal antibody described in the above technical solution in one or more of the following:

[0042] 1) Preparing a product for detecting microsporidia; 2) Detecting microsporidia, where the direct purpose of the detection is non-diagnostic and non-therapeutic; 3) Subcellular localization of microsporidia proteins.

[0043] As an embodiment, the microsporidia include Encephalitozoon spp. As an embodiment, the Encephalitozoon spp. include Encephalitozoon cuniculi. As an embodiment, the product for detecting microsporidia can be an immunological detection product. As an embodiment, the immunological detection product can be an indirect immunofluorescence detection product. As an embodiment, the product includes a reagent or a kit. When detecting microsporidia in item 2) of the present invention, the test sample can be an environmental sample (such as soil or water quality) or food, etc., and it can be detected whether the test sample contains microsporidia.

[0044] Based on the above advantages, the present invention provides a kit for detecting microsporidia, which includes the monoclonal antibody described in the above technical solution and an immunological detection reagent.

[0045] As an implementation manner, the immunological detection reagent includes a fluorescently labeled anti-IgG antibody. As an implementation manner, the fluorescent label can be AlexaFluor488.

[0046] To further illustrate the present invention, a monoclonal antibody targeting the EcSSP1 protein provided by the present invention and its application in detecting microsporidia will be described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0047] Example 1 Preparation of antigen

[0048] 1. Construction of recombinant expression plasmid:

[0049] Using the genomic DNA of Encephalitozoon cuniculi as a template, PCR amplification was performed using the upstream primer (SEQ ID NO.1) and the downstream primer (SEQ ID NO.2) to obtain an amplification product; the reaction system for the PCR amplification was: 2×Primer STARMax Premix 25 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, template 1 μL, supplemented with ddH2O to 50 μL; the reaction program for the PCR amplification was: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 10 s, 35 cycles; final extension at 72°C for 7 min; the Encephalitozoon cuniculi was preserved by the National Key Laboratory of Efficient Breeding and Utilization of Resource Insects, Southwest University, Chongqing, and was disclosed in Chinese Patent CN116903713A;

[0050] SEQ ID NO.1: 5'-acaaggccatggctgatatcatgtgcgactatgagctgaag-3';

[0051] SEQ ID NO.2: 5'-tggtgctcgagtgcggccgcaaagattctccctgtaagtgct-3'.

[0052] The amplification product was ligated between the BamHI and HindIII sites of the pET32a(+) prokaryotic expression vector by homologous recombination to obtain a recombinant expression plasmid.

[0053] 2. The recombinant expression plasmid constructed in step 1 was transformed into Escherichia coli (E. coli Rosetta, purchased from Beijing TransGen Biotech Co., Ltd.) to obtain a bacterial solution containing the recombinant expression plasmid.

[0054] 3. Induced expression and purification of recombinant protein EcSSP1 (antigen):

[0055] Inoculate the bacterial solution in step 2 into 300 mL of LB medium containing ampicillin, and culture it in a shaker at 37 °C and 180 rpm. When it grows to the logarithmic phase (OD 600 is 0.6), add 300 μL of 100 mM IPTG and induce at 37 °C for 4 h. Collect the induced bacterial solution, resuspend the cell pellet with Buffer A solution, and ultrasonically disrupt it on ice for 30 min; the formula of the Buffer A solution is: 100 mM NaCl, 10 mM Tris-HCl, and then adjust the pH to 8.0. After disruption, centrifuge at 12,000 rpm and 4 °C for 30 min, and transfer the protein supernatant to a 50 mL centrifuge tube.

[0056] Mix 50% Ni-NTA, and pipette 1 mL of it into the column. Add 20 mL of sterile water for rinsing, and then add 20 mL of Buffer A (100 mM NaCl, 10 mM Tris-HCl, pH = 8.0) for equilibration. Subsequently, bind the protein supernatant to the nickel column, and control the flow rate to be 1 drop every 3 seconds. First, use 20 mM imidazole to remove the impurity proteins, and then use 50 mM and 100 mM imidazole to elute the target protein. All the liquids added to the column need to be filtered through a 0.22 μm filter membrane, and the results are as Figure 1 shown in A. The results show that: after purification, the expression product obtains a recombinant protein EcSSP1 with a relatively high purity at 40 kDa, and the amino acid sequence is as shown in SEQ ID NO.11, specifically as follows:

[0057] MCDYELKTPIINMGERIFEFLKNYEDQYKKAVVLFLTRILSQIDGFAPSYPSADYEPLIEQLETLGVTVPSNMAADLAALDAAEATSLAGTIRANAQKVIGDLLARVNTMCYLDLMSLVTSGLFASQVTSAFSNTQPIITIAGNDLFTKQMAVFQRLPGTLPSAAITAITNALQANKNNFVTFFTTQTTNLQTDVQNALTALITALTTLTSTTSTEFTQFANSEIGALTGRIF.

[0058] 4. Detection of recombinant protein EcSSP1 with His antibody

[0059] (1) Electrophoresis: Mix 40 μL of the purified recombinant protein EcSSP1 in step 3 with 10 μL of 5× SDS-PAGE loading buffer, boil for 10 min, and take 10 μL of it for SDS-PAGE;

[0060] (2) Transfer membrane: Place the filter paper soaked in transfer buffer on the semi-dry transfer apparatus, then place the PVDF membrane and PAGE protein gel, and finally place the filter paper soaked in transfer buffer again. The parameters of the transfer apparatus are set as voltage 25V, current 1.5A, and transfer time 15 min;

[0061] (3) Blocking: After the electrotransfer is completed, place the PVDF membrane into 20 mL of 5% skim milk blocking solution and block it at 37 °C for 2 h;

[0062] (4) Dilute the mouse anti-His antibody (His mouse monoclonal antibody, purchased from abcam company) with the primary antibody diluent at a working concentration of 1:1000 (v / v). Incubate the antibody with the PVDF membrane at room temperature for 2 h or overnight at 4 °C, and wash it 3 times with 1×TBST, 10 min each time;

[0063] (5) Dilute the goat anti-mouse IgG-HRP (goat anti-mouse IgG HRP, purchased from biosharp company) with PBS at 1:8000 (v / v). Then co-incubate the secondary antibody with the PVDF membrane at room temperature for 40 min, and wash it 3 times with 1×TBST, 10 min each time;

[0064] (6) After the PVDF membrane reacts with the ECL chemiluminescent substrate in the dark, observe the color development result of the PVDF membrane using an imager.

[0065] The results are as Figure 1 shown in B below. The His antibody reacts with the recombinant target protein at 40 kDa, which is consistent with the expected result.

[0066] Example 2 Preparation of Monoclonal Antibody

[0067] 1. Animal Immunization

[0068] For the first immunization, mix the antigen prepared in Example 1 and complete Freund's adjuvant evenly at a volume ratio of 1:1, and immunize 4 BALB / c mice by subcutaneous multi-site injection. The protein injection dose for each mouse is 100 μg each time. Then perform 3 subsequent immunizations. For the latter 3 immunizations, mix the antigen and incomplete Freund's adjuvant evenly at a volume ratio of 1:1 and immunize the mice by subcutaneous multi-site injection. The protein injection dose for each mouse is 100 μg each time. Before fusion, mix the antigen and PBS evenly at a volume ratio of 1:1, and immunize by intraperitoneal injection. The dose of the immunized protein is 50 μg each time, and a total of 3 immunizations are required, with an interval of 24 h between each immunization.

[0069] 2. Preparation of Feeder Cells

[0070] Take a Kunming (KM) mouse in good growth condition. After sacrificing it by cervical dislocation (avoid squeezing its internal organs), immerse it in 75% ethanol for 5 minutes. Then take out the mouse and place it in a laminar flow hood. Use sterile forceps to lift the epidermis of the mouse's abdominal cavity and cut the epidermis with scissors to fully expose the peritoneum of the mouse. Use a 10 mL syringe to aspirate 10 mL of HT medium (or HAT medium), then inject it into the mouse's abdominal cavity. Gently massage the mouse's abdomen with an alcohol cotton ball and then aspirate the liquid. Transfer the liquid into a trough tube, dilute it with 50 - 80 mL of HT medium (or HAT medium), and then dispense 100 μL per well into a 96-well cell culture plate. Place it in an incubator at 37°C with 5% CO2 for culture. Observe whether there is contamination of the feeder cells the next day. If there is contamination, it needs to be prepared again.

[0071] 3. Cell fusion

[0072] Sacrifice the mouse by cervical dislocation and take out its swollen spleen. Place it in a sterile petri dish with 3 mL of serum-free and antibiotic-free medium. Then use two syringe tips to gently scrape the spleen in one direction to disperse the splenocytes.

[0073] Wash the SP2 / 0 cells (8 - 10 flasks) in a T25 cell flask once with serum-free and antibiotic-free RPMI-1640 medium (Hyclone), then blow them down from the cell flask and transfer them into a 50 mL centrifuge tube.

[0074] Mix the SP2 / 0 cells and the splenocytes passed through a cell sieve at a cell number ratio of 1:5 - 1:10 in a 50 mL centrifuge tube. Add serum-free and antibiotic-free RPMI-1640 medium to 30 mL. After mixing evenly, centrifuge at 1000 rpm for 10 minutes and discard all the supernatant.

[0075] Gently tap the bottom of the centrifuge tube to make the cells at the bottom loose and uniform. Aspirate 1 mL of PEG-1500 preheated to 37°C and add it to the centrifuge tube within 45 s - 60 s, gently stirring while adding.

[0076] Subsequently, add a total of 30 mL of serum-free and antibiotic-free RPMI-1640 medium preheated to 37°C to the centrifuge tube to terminate the action of PEG-1500. The specific operation is as follows: add 2 mL in the first 30 s, add 8 mL in the second 30 s, and add 20 mL in the last 30 s. Place it at 37°C for 10 minutes, then centrifuge at 1000 rpm for 10 minutes and discard the supernatant.

[0077] Resuspend the cells with HAT medium and transfer them to a trough tube. Add 50 - 80 mL of HAT medium for dilution, and then dispense 100 μL per well into a 96-well cell culture plate paved with feeder cells. Place it in an incubator at 37°C with 5% CO2 for culture.

[0078] 4. Subcloning and Screening

[0079] One day before subcloning, feeder cells were seeded into 96-well plates. Then, the cells in the wells identified as positive by ELISA were resuspended and diluted 10-fold to 10 6 . Subsequently, the diluted cells were added to the 96-well plates containing feeder cells, 100 μL per well, and cultured in an incubator at 37 °C with 5% CO2 for a total of 4 rounds of subcloning.

[0080] The cell supernatant after the 4th round of subcloning was collected. After expanding the cell clusters in the wells identified as positive by ELISA, the RNA of the cells was extracted, and then cDNA was synthesized. The heavy-chain variable region (VH) and light-chain variable region (VL) of the antibody were amplified by PCR using degenerate primers for the antibody, and then sequenced to identify the heavy-chain variable region and light-chain variable region of the monoclonal antibody. The nucleotide sequences of the degenerate primers for the antibody are as follows:

[0081] Heavy-chain forward primer: 5'-CAGGTACAGCTCGAGCAGTCAGG-3' (SEQ ID NO.3);

[0082] Heavy-chain reverse primer: 5'-TGGGCCCTTGGTGCTAGCTGAGACGGTGACC-3' (SEQ ID NO.4);

[0083] Light-chain forward primer: 5'-GAYATTGTGMTSACMCARWCTMCA-3' (SEQ ID NO.5);

[0084] Light-chain reverse primer: 5'-GGATACAGTTGGTGCAGCATC-3' (SEQ ID NO.6);

[0085] The sequencing results of the monoclonal antibody are as follows:

[0086] Heavy-chain variable region sequence (SEQ ID NO.7):

[0087] 5'-CAGGTACAGCTCGAGCAGTCAGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGATGTCCTGCAAGGCTACTGGATACACCTTCACTGACTACTGGATAGGTTGGGTTAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAAATATTTACCCTGGAGGTTTTTATACTCACTACAATGAGAAGTTCAAGGGCAAGGCCACTCTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCCATCTATTTCTGTACAAGAAGAGACATGATCGCCCCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCAGCTAGCACCAAGGGCCCA-3';

[0088] Light chain variable region sequence (SEQ ID NO.8):

[0089] 5'-GATATTGTGCTGACCCAGACTCCATCCTCACTGTCTCCATCTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCAAGCCAAGACATTAAGAAGTATATGGCTTGGTACCAACACAAGCCTGGAAAAGGTCCTAGGCTGCTCATAAATTACACATCTACATTACAGCCAGGCATCCCATCAAGGTTCAGTGGAAGTGGGTCTGGGAGAGATTATTCCTTCAGCATCAGCAACCTGGAGCCTGAAGATATTGCAACTTATTATTGTCTACAGTATGAGAATCTGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAGCGGGCTGATGCTGCACCAACTGTATCC-3'.

[0090] 5. Monoclonal antibody titer detection:

[0091] (1) Mix the antigen prepared in Example 1 (0.5 μg / well) thoroughly with the ELISA coating solution and add 100 μL per well to the ELISA plate, and incubate overnight at 4°C;

[0092] (2) The ELISA coating solution in the spin-drying plate was washed three times with PBST for 5 minutes each time. Then, 200 μL of ELISA blocking buffer was added to each well and incubated at 37 °C for 2 hours. After blocking, it was washed three times with PBST;

[0093] (3) The monoclonal antibody and negative serum obtained in step 4 were serially diluted with PBS at volume ratios of 1:51200, 1:102400, 1:204800, 1:409600, 1:819200, and 1:1638400. Then, 100 μL was added to each well, with three replicates per well, and incubated at 37 °C for 2 hours. After incubation, it was washed three times with PBST;

[0094] (4) Goat anti-mouse IgG-HRP was diluted with PBS at a dilution ratio of 1:5000 (v / v). 100 μL was added to each well and incubated at 37 °C for 1 - 2 hours. After incubation, it was washed three times with PBST;

[0095] (5) TMB chromogenic solution was added, 200 μL per well, and incubated at 37 °C in the dark for 30 minutes. Then, 2 M H2SO4 was added to terminate the reaction, 50 μL per well;

[0096] (6) The microtiter plate was placed in an ELISA reader and detected at an absorption wavelength of 450 nm. When the absorbance value of the test well was more than 2.1 times that of the negative well (P / N > 2.1) at the same dilution factor, it was considered positive;

[0097] The results are shown in Table 1. Even when the monoclonal antibody was diluted to 1:1638400, the antigen (recombinant protein EcSSP1) could still react with the monoclonal antibody but not with the negative serum, indicating that the monoclonal antibody screened in the present invention has good sensitivity and a titer of 10 6 levels.

[0098] Table 1 Detection results of monoclonal antibody titer

[0099] Dilution ratio 1:51200 1:102400 1:204800 1:409600 1:819200 1:1638400 Monoclonal antibody 3.25 3.16 2.99 2.92 2.53 2.17 Negative serum 1.09 0.37 0.37 0.41 0.38 0.37 P / N 2.98 8.54 8.08 7.12 6.66 5.86

[0100] 6. Monoclonal antibody specificity experiment:

[0101] (1) Electrophoresis: Cryptococcus neoformans, Escherichia coli, and Saccharomyces cerevisiae were ultrasonically disrupted respectively to obtain three kinds of protein solutions. 40 μL of each protein solution was mixed with 10 μL of 5×SDS-PAGE loading buffer, boiled for 10 minutes, and 10 μL of it was taken for SDS-PAGE;

[0102] (2) Membrane transfer: Place the filter paper soaked in transfer buffer on the semi-dry membrane transfer instrument, then place the PVDF membrane and PAGE protein gel, and finally place the filter paper soaked in transfer buffer again. The parameters of the membrane transfer instrument are set as voltage 25V, current 1.5A, and transfer time 15 min;

[0103] (3) Blocking: After the electrotransfer is completed, place the PVDF membrane into 20 mL of 5% non-fat milk blocking solution and block it at 37 °C for 2 h;

[0104] (4) Dilute the monoclonal antibody obtained in step 4 with the primary antibody diluent at a working concentration of 1:1000 (v / v). Incubate the antibody with the PVDF membrane at room temperature for 2 h or overnight at 4 °C, and wash it 3 times with 1×TBST, 10 min each time;

[0105] (5) Dilute goat anti-mouse IgG-HRP (goat anti-mouse IgG HRP, purchased from biosharp company) with PBS at 1:8000 (v / v). Then co-incubate the secondary antibody with the PVDF membrane at room temperature for 40 min, and wash it 3 times with 1×TBST, 10 min each time;

[0106] (6) After the PVDF membrane reacts with the ECL chemiluminescent substrate in the dark, observe the color development result of the PVDF membrane using an imager.

[0107] Results Figure 2 As shown, the monoclonal antibody does not react with the proteins of Cryptococcus neoformans, Escherichia coli, and yeast, indicating good specificity.

[0108] Example 3

[0109] 1. Detection of recombinant protein EcSSP1 by monoclonal antibody

[0110] (1) Electrophoresis: Mix 40 μL of the recombinant protein EcSSP1 prepared in Example 1 with 10 μL of 5×SDS-PAGE loading buffer, boil for 10 min, and take 10 μL of it for SDS-PAGE;

[0111] (2) Membrane transfer: Place the filter paper soaked in transfer buffer on the semi-dry membrane transfer instrument, then place the PVDF membrane and PAGE protein gel, and finally place the filter paper soaked in transfer buffer again. The parameters of the membrane transfer instrument are set as voltage 25V, current 1.5A, and transfer time 15 min;

[0112] (3) Blocking: After the electrotransfer is completed, place the PVDF membrane into 20 mL of 5% non-fat milk blocking solution and block it at 37 °C for 2 h;

[0113] (4) Dilute the EcSSP1 monoclonal antibody screened in Example 2 with a primary antibody diluent at a working concentration of 1:1000 (v / v). Incubate the antibody with the PVDF membrane at room temperature for 2 h or overnight at 4 °C. Wash 3 times with 1×TBST, 10 min each time;

[0114] (5) Dilute goat anti-mouse IgG-HRP (goat anti-mouse IgG HRP, purchased from Biosharp) with PBS at 1:8000 (v / v). Then co-incubate the secondary antibody with the PVDF membrane at room temperature for 40 min, and then wash 3 times with 1×TBST, 10 min each time;

[0115] (6) After the PVDF membrane reacts with the ECL chemiluminescent substrate in the dark, observe the color development result of the PVDF membrane using an imager.

[0116] The results are as Figure 2 shown in A below. The monoclonal antibody can react with the recombinant protein EcSSP1 at 40 kDa, which is consistent with the expected result.

[0117] 2. Detection of total protein of E. cuniculi by monoclonal antibody

[0118] (1) Electrophoresis: Mix 40 μL of the total protein extracted from Encephalitozoon cuniculi with 10 μL of 5×SDS-PAGE loading buffer, boil for 10 min, and take 10 μL of it for SDS-PAGE;

[0119] (2) Transfer: Place the filter paper soaked in the transfer buffer on the semi-dry transfer apparatus, then place the PVDF membrane and the PAGE protein gel, and finally place the filter paper soaked in the transfer buffer again. The parameters of the transfer apparatus are set as voltage 25 V, current 1.5 A, and transfer time 15 min;

[0120] (3) Blocking: After electrotransfer is completed, place the PVDF membrane into 20 mL of 5% skim milk blocking solution and block at 37 °C for 2 h;

[0121] (4) Dilute the EcSSP1 monoclonal antibody screened in Example 2 with a primary antibody diluent at a working concentration of 1:1000 (v / v). Incubate the antibody with the PVDF membrane at room temperature for 2 h or overnight at 4 °C. Wash 3 times with 1×TBST, 10 min each time;

[0122] (5) Dilute goat anti-mouse IgG-HRP (goat anti-mouse IgG HRP, purchased from Biosharp) with PBS at 1:8000 (v / v). Then co-incubate the secondary antibody with the PVDF membrane at room temperature for 40 min, and then wash 3 times with 1×TBST, 10 min each time;

[0123] (6) After the PVDF membrane reacts with the ECL chemiluminescent substrate in the dark, an imager is used to observe the color development result of the PVDF membrane.

[0124] The results are as Figure 2 shown in B below. The monoclonal antibody can recognize a single band of the total protein of E. cuniculi at 55 kDa, which is consistent with the expected result.

[0125] Example 4 Indirect Immunofluorescence Detection of Monoclonal Antibody

[0126] (1) Fixation of E. cuniculi: At the central position of a glass slide, 50 μL of a 0.1 g / L polylysine solution (Sigma, P3150) is added and then air-dried to obtain a polylysine-treated glass slide for later use; 10 μL of E. cuniculi at a concentration of 10 6 cells / mL is pipetted onto the polylysine-treated glass slide and allowed to stand for 5 min; 50 μL of a 40 g / L paraformaldehyde solution is added to the glass slide and fixed for 25 min; the excess liquid is aspirated, and 100 μL of a 0.01 mol / L PBS solution is added to the culture well to immerse the sample, and the sample is gently shaken and washed 3 times, 5 min each time;

[0127] (2) Blocking: After aspirating the liquid, 50 μL of the blocking solution is added to cover the spores and treated for 1 h; the blocking solution is a PBS solution containing 0.05% (v / v) Tween 20, 2% (v / v) Triton X-100, 0.5 g / L BSA, and 10% (v / v) goat serum;

[0128] (3) Labeling E. cuniculi by indirect immunofluorescence: After aspirating the blocking solution, the sample is washed 3 times with the washing solution, 5 min each time, and the washing solution is a PBS solution containing 0.05% (v / v) Tween 20; the monoclonal antibody and negative serum screened in Example 1 are diluted with the blocking solution at a ratio of 1:200 (v / v) to prepare the primary antibody working solution, and then 50 μL of the primary antibody working solution is added to immerse the spores and incubated for 1 h; the primary antibody working solution is aspirated, and the sample is washed 3 times with the washing solution, 5 min each time; Alexa Fluor 488-labeled goat anti-mouse IgG is diluted with the blocking solution at a ratio of 1:1000 (v / v) to prepare the secondary antibody working solution, and then 50 μL of the secondary antibody working solution is added to immerse the spores and incubated at room temperature for 1 h; the secondary antibody working solution is aspirated, and the sample is washed 3 times with the washing solution, 5 min each time;

[0129] (4) DAPI staining of E. cuniculi nuclei: Add 50 μL of 1 μg / mL DAPI staining solution to cover the spores, and incubate at room temperature for 30 min; aspirate the staining solution, add washing solution and wash 3 times, 5 min each time; aspirate the liquid, take 5 μL of anti-fluorescence quenching mounting medium and drop it onto the glass slide, cover with a coverslip, and seal the coverslip with nail polish;

[0130] (5) Observe and take pictures with a laser confocal microscope.

[0131] The results are as Figure 3 shown that the monoclonal antibody can be localized on the surface of Encephalitozoon cuniculi.

[0132] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody targeting EcSSP1 protein, comprising a heavy chain and a light chain, characterized in that: The complementary determining region of the heavy chain includes CDR-H1, CDR-H2 and CDR-H3; the complementary determining region of the light chain includes CDR-L1, CDR-L2 and CDR-L3; The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.12; the amino acid sequence of the CDR-H2 is shown in SEQ ID NO.13; the amino acid sequence of the CDR-H3 is shown in SEQ ID NO.14; The amino acid sequence of the CDR-L1 is shown in SEQ ID NO.15; the amino acid sequence of the CDR-L2 is shown in SEQ ID NO.16; and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO.

17.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.9, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.

10.

3. The monoclonal antibody according to claim 1 or 2, characterized in that The nucleotide sequence of the variable region of the heavy chain is shown in SEQ ID NO.7, and the nucleotide sequence of the variable region of the light chain is shown in SEQ ID NO.

8.

4. Use of the monoclonal antibody according to any one of claims 1 to 3 in one or more of the following: 1) Preparation of products for detecting microsporidia; 2) Detection of microsporidia, wherein the direct purpose of the detection is non-diagnostic and non-therapeutic; 3) Subcellular localization of microsporidia proteins.

5. The use according to claim 4, characterized in that: The microsporidia include Encephalitozoon.

6. The use according to claim 5, characterized in that: The encephalitogenic microsporidia include E. cuniculi.

7. The use according to any one of claims 4 to 6, characterized in that: The product comprises a reagent or a kit.

8. A kit for detecting microsporidia, characterized in that: The invention comprises the monoclonal antibody and immunological detection reagent according to any one of claims 1 to 3.

9. The kit according to claim 8, characterized in that The immunological detection reagent includes fluorescently labeled anti-IgG antibodies.

10. The kit according to claim 9, characterized in that The fluorescent label includes Alexa Fluor488.

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