Antigen detection target of enterocytozoon hepatopenaei, monoclonal antibody for detecting enterocytozoon hepatopenaei and application of monoclonal antibody

Abstract

The invention belongs to the technical field of biological detection and antibody preparation, and particularly relates to an antigen detection target of enterocytozoon hepatopenaei, a monoclonal antibody for detecting enterocytozoon hepatopenaei and application of the monoclonal antibody. A reverse screening technical route is adopted, enriched EHP is adopted to directly immunize mice, then myeloma cells are fused with splenocytes of the immunized mice to prepare hybridoma cells, and monoclonal antibodies are screened; and carrying out immunoblotting detection on EHP total protein by using the screened monoclonal antibody, carrying out mass spectrometry on specific protein bands, and screening to obtain an antigen detection target EHP0019 of EHP. The EHP0019 is a dominant antigen of the EHP, the EHP can be rapidly, accurately and specifically detected by utilizing the monoclonal antibody for specifically detecting the EHP0019 without nucleic acid extraction and amplification, the specificity is high, the sensitivity is high, and a foundation is laid for prevention and control of the hepatopancreatic microsporidiosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection and antibody preparation, and particularly relates to an antigen detection target of shrimp hepatoenteric cytozoon and a monoclonal antibody for detecting shrimp hepatoenteric cytozoon and applications thereof. Background Art

[0002] Penaeus vannamei is one of the most important economic aquatic species in the world. It is highly susceptible to Enterocytozoon hepatopenaei (EHP), a pathogen that infects the hepatopancreas. EHP can cause hepatopancreatic microsporidiosis (HPM), which leads to growth stagnation in shrimp and causes huge economic losses to the shrimp farming industry. There is currently no effective treatment for HPM, so the detection and prevention of EHP are crucial to the control of the disease.

[0003] Nucleic acid is an indispensable component of all living things. By detecting the nucleic acid sequence (including DNA or RNA) of a specific pathogen through nucleic acid amplification technology, it is possible to determine whether the pathogen exists in the sample and thus determine whether it is infected. However, nucleic acid amplification requires the extraction and amplification of nucleic acids, which is time-consuming, prone to aerosol contamination, and requires the use of professional equipment and strict operating environments to carry out testing, which invisibly increases the cost of testing. Therefore, it is necessary to develop reagents for the immediate and rapid detection of EHP for the detection of EHP, thereby effectively preventing and controlling HPM. Summary of the invention

[0004] The invention aims to provide an antigen detection target for shrimp hepatoenteric cysts and a monoclonal antibody for detecting shrimp hepatoenteric cysts and applications thereof, which can rapidly and accurately detect EHP without nucleic acid extraction and amplification, and have strong specificity and high sensitivity.

[0005] The present invention provides application of a reagent for detecting EHP00_19 in preparing a product for detecting shrimp hepatoenterocystis.

[0006] The present invention also provides an antibody for detecting shrimp hepatoenterocystis, wherein the antibody is an antibody against EHP00_19; the antibody comprises a monoclonal antibody and / or a polyclonal antibody.

[0007] Preferably, the monoclonal antibody comprises 1C10 monoclonal antibody;

[0008] The light chain complementary determining region CDR1 of the 1C10 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO.2, the amino acid sequence of the light chain complementary determining region CDR2 comprises LVS, and the light chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.3;

[0009] The heavy chain complementary determining region CDR1 of the 1C10 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO.6, the heavy chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.7, and the heavy chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.8.

[0010] Preferably, the light chain of the 1C10 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO.1;

[0011] The heavy chain of the 1C10 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO.5.

[0012] The present invention also provides a nucleic acid molecule, which encodes the 1C10 monoclonal antibody described in the above technical solution.

[0013] Preferably, the sequence of the nucleic acid molecule encoding the light chain of the 1C10 monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO.4; the sequence of the nucleic acid molecule encoding the heavy chain of the 1C10 monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO.9.

[0014] The present invention also provides a biological material for detecting EHP00_19 and / or shrimp hepatoenterocystis, wherein the biological material is a biological material expressing the 1C10 monoclonal antibody described in the above technical solution; the biological material includes a carrier or a cell.

[0015] The present invention also provides the use of the antibody described in the above technical solution or the 1C10 monoclonal antibody encoded by the nucleic acid molecule or the 1C10 monoclonal antibody expressed by the biological material in one or more of the following:

[0016] (1) Prepare products for detecting EHP00_19;

[0017] (2) preparing products for detecting shrimp hepatocystis;

[0018] (3) Prepare products for the auxiliary prevention of hepatopancreatic microsporidiosis.

[0019] The present invention also provides a kit for detecting EHP00_19 and / or shrimp hepatoenterozoa, the kit comprising a capture antibody and a tracer-labeled detection antibody; the capture antibody is the antibody described in the above technical solution; the tracer-labeled detection antibody comprises a tracer-labeled sheep anti-mouse IgG.

[0020] The present invention also provides a method for detecting EHP00_19 for non-diagnostic purposes, comprising the following steps: mixing a sample to be detected, a capture antibody and a detection antibody labeled with a tracer to form a complex; detecting the tracer signal in the complex to determine the concentration of EHP00_19 in the sample to be detected;

[0021] The capture antibody is the antibody described in the above technical solution;

[0022] The tracer-labeled detection antibody includes tracer-labeled goat anti-mouse IgG.

[0023] Beneficial effects:

[0024] The present invention adopts a reverse screening technical route, uses the enriched EHP to directly immunize mice, then fuses myeloma cells with spleen cells of the immunized mice to prepare hybridoma cells, and screens monoclonal antibodies; uses the screened monoclonal antibodies to perform Western blot on the total EHP protein, and then performs mass spectrometry analysis on the specific protein bands, and finally screens out the antigen detection target of EHP-EHP00_19. The EHP00_19 described in the present invention is a dominant antigen of EHP, and the monoclonal antibody for specifically detecting EHP00_19 can be used to quickly, accurately and specifically carry out the detection of EHP without extracting nucleic acids or amplifying them, with high specificity and sensitivity, laying a foundation for the prevention and control of hepatopancreatic microsporidiosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0026] Figure 1 For 10 8 Western blot detection results of EHP spore total protein by polyclonal antibody after mice were immunized with EHP spores;

[0027] Figure 2 This is the Western blot test result of negative serum on total protein of EHP spores;

[0028] Figure 3 Western blot results of different monoclonal antibodies screened against EHP spore total protein;

[0029] Figure 4 The results of the specific analysis of EHP using the 1C10 monoclonal antibody indirect ELISA;

[0030] Figure 5 The results are the cut-off value analysis of the indirect ELISA test using the 1C10 monoclonal antibody;

[0031] Figure 6 The results of sensitivity analysis of indirect ELISA for detecting EHP using 1C10 monoclonal antibody;

[0032] Figure 7 Western blot and SDS-PAGE test results of 1C10 monoclonal antibody and EHP spore total protein;

[0033] Figure 8 The results of SDS-PAGE detection of exogenous expression of EHP00_763; among them, lane 1 on the left: uninduced; lanes 2 and 4: induced supernatant; lanes 3 and 5: induced precipitation; lane 6: empty control; lane 1 on the right: bacterial supernatant; 2: flow-through; 3-5: impurity removal; 6-8: target protein elution;

[0034] Fig. 9 The results of SDS-PAGE detection of exogenous expression analysis of EHP00_19; among them, lanes 1 and 5 on the left: uninduced; lanes 2 and 6: induced bacterial solution; lanes 3 and 7: induced supernatant; lanes 4 and 8: induced precipitation; lane 1 on the right: bacterial solution supernatant; 2: flow-through; 3-5: impurity removal; 6-8: target protein elution;

[0035] Fig.10 The Western blot test results of 1C10 monoclonal antibody and recombinant protein; the recombinant protein in the left picture is EHP00_763, and the recombinant protein in the right picture is EHP00_19;

[0036] Fig.11 The results of immunofluorescence localization analysis of 1C10 monoclonal antibody and EHP; from left to right are white light observation, chitin shell localization signal (blue), EHP00_19 protein localization signal (green), EHP nuclear localization signal (red) and fluorescence Merge observation. DETAILED DESCRIPTION

[0037] The present invention provides application of a reagent for detecting EHP00_19 in preparing a product for detecting shrimp hepatoenterocystis.

[0038] In one embodiment, the reagent includes an antibody; in another embodiment, the reagent includes an antibody prepared using the EHP00_19. The accession number of EHP00_19 in NCBI is OQS54511.1, and the specific amino acid sequence is: MFINSLVFSGIPVIECSNNLVLNPAG MDECYPQRKFMTQVDAYAEGFLSNLLRKATIYKYNKQTDQTIKDFVIIKQEAVCVTFPVYKNAVYKAFEEIENKSYDNPEEVDTVLDYIYKSARSNIALFHGRSDLFLEALIQFTATACHNMYGLNMLEKKGENNEDVSISRGLMQWTGQTGYSKLRSCGHYNQRISNLNLYSYETIRSEFKAFVKCYFEGKKRFESPCSMTLQPYLHTISNMAPEETEAFVEDCEGQIFISIKRLNKLNEDGKKIARRLMYYILIKKYTTRFSAVIVASSDN (SEQ ID NO: 10).

[0039] The present invention adopts a reverse screening technical route, uses the enriched EHP to directly immunize mice, then fuses myeloma cells with spleen cells of the immunized mice to prepare hybridoma cells, and screens monoclonal antibodies; uses the screened monoclonal antibodies to perform Western blot on the total EHP protein, and then performs mass spectrometry analysis on the specific protein bands, and finally screens out the antigen detection target of EHP-EHP00_19. The EHP00_19 described in the present invention is the dominant antigen of EHP, and shrimp hepatocystis can be specifically detected by detecting EHP00_19, which lays a foundation for the prevention and control of hepatopancreatic microsporidiosis.

[0040] The present invention also provides an antibody for detecting shrimp hepatoenterocystis, wherein the antibody is an antibody against EHP00_19; the antibody comprises a monoclonal antibody and / or a polyclonal antibody.

[0041] As an embodiment, the method for preparing the polyclonal antibody comprises: using the enriched EHP to directly immunize mice, the number of immunizations is 4 times, and the dose of each immunization is 10 8 As an embodiment, the present invention uses Freund's complete adjuvant and spores mixed in a volume ratio of 1:1 for the first immunization, and injects mice after complete emulsification, and then performs booster immunization every two weeks, and during the booster immunization, a water-soluble adjuvant and an equal volume of EHP are uniformly emulsified for immunization.

[0042] As an embodiment, the monoclonal antibody includes the 1C10 monoclonal antibody; the light chain complementary determining region CDR1 of the 1C10 monoclonal antibody includes the amino acid sequence shown in SEQ ID NO.2, the amino acid sequence of the light chain complementary determining region CDR2 includes LVS, and the light chain complementary determining region CDR3 includes the amino acid sequence shown in SEQ ID NO.3; the heavy chain complementary determining region CDR1 of the 1C10 monoclonal antibody includes the amino acid sequence shown in SEQ ID NO.6, the heavy chain complementary determining region CDR2 includes the amino acid sequence shown in SEQ ID NO.7, and the heavy chain complementary determining region CDR3 includes the amino acid sequence shown in SEQ ID NO.8. As an embodiment, the light chain of the 1C10 monoclonal antibody includes the amino acid sequence shown in SEQ ID NO.1; the heavy chain of the 1C10 monoclonal antibody includes the amino acid sequence shown in SEQ ID NO.5. The subtype of the 1C10 monoclonal antibody of the present invention is IgM, Kappa subtype.

[0043] The present invention also provides a nucleic acid molecule encoding the 1C10 monoclonal antibody described in the above technical solution. As an embodiment, the sequence of the nucleic acid molecule encoding the light chain of the 1C10 monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO.4; the sequence of the nucleic acid molecule encoding the heavy chain of the 1C10 monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO.9.

[0044] The present invention uses the enriched EHP to directly immunize mice, then fuses myeloma cells with spleen cells of the immunized mice to prepare hybridoma cells, and screens monoclonal antibodies to screen a hybridoma cell line that secretes 1C10 monoclonal antibodies. Since the present invention uses EHP as an immunogen to immunize mice, this immunization method maintains the natural conformation of the pathogen surface protein, enabling the prepared monoclonal antibody (1C10) to specifically recognize the pathogen surface protein.

[0045] The present invention also provides a biological material for detecting EHP00_19 and / or shrimp hepatoenterocystis, wherein the biological material is a biological material expressing the 1C10 monoclonal antibody described in the above technical solution; the biological material includes a carrier or a cell.

[0046] As an embodiment, the vector comprises a basic vector and the nucleic acid molecule described in the above technical solution inserted into the basic vector, or a variant of the nucleic acid molecule described in the above technical solution inserted into the basic vector. The present invention has no strict requirements on the source of the vector, and any vector that can replicate and / or express polynucleotides in eukaryotic or prokaryotic cells including mammalian cells (e.g., human, monkey, rabbit, rat, hamster or mouse cells), plant cells, yeast cells, insect cells and bacteria, such as Escherichia coli (E. coli) can be used.

[0047] The present invention also provides the use of the antibody described in the above technical solution or the 1C10 monoclonal antibody encoded by the nucleic acid molecule or the 1C10 monoclonal antibody expressed by the biological material in one or more of the following: (1) preparing a product for detecting EHP00_19; (2) preparing a product for detecting shrimp hepatoenteroides; (3) preparing a product for assisting in preventing hepatopancreatic microsporidiosis. As an embodiment, the product includes a kit. As an embodiment, the kit is an indirect ELISA detection kit.

[0048] The present invention also provides a kit for detecting EHP00_19 and / or shrimp hepatoenteric cysts, the kit comprising a capture antibody and a tracer-labeled detection antibody; the capture antibody is the antibody described in the above technical solution; the tracer-labeled detection antibody comprises a tracer-labeled goat anti-mouse IgG. As an embodiment, the tracer-labeled detection antibody of the present invention is goat anti-mouse IgG-HRP, purchased from Biosharp. As an embodiment, the kit further comprises a blocking solution and a TMB colorimetric solution, purchased from Beyotime. As an embodiment, the blocking solution is 5% skim milk powder.

[0049] The present invention establishes a kit based on the 1C10 monoclonal antibody described in the technical solution, and can use a 96-well plate to perform high-throughput detection of EHP without extracting nucleic acid. This is the first report of ELISA detection of shrimp hepatocystis, and can specifically detect EHP with high detection sensitivity.

[0050] The present invention also provides a method for detecting EHP00_19 for non-diagnostic purposes, comprising the following steps: mixing a sample to be detected, a capture antibody and a detection antibody labeled with a tracer to form a complex; detecting the tracer signal in the complex to determine the concentration of EHP00_19 in the sample to be detected; the capture antibody is the 1C10 monoclonal antibody described in the above technical scheme; the detection antibody labeled with a tracer includes a tracer-labeled sheep anti-mouse IgG.

[0051] As an embodiment, the present invention uses the sample to be detected to coat the ELISA plate to obtain an antigen-coated ELISA plate. As an embodiment, the coating condition is 37°C for 2 to 3 hours; as another embodiment, the coating condition is 37°C for 3 hours.

[0052] As an embodiment, after obtaining the antigen-coated ELISA plate, the present invention uses a blocking solution to block the antigen-coated ELISA plate to obtain a blocker. As an embodiment, the blocking solution of the present invention is 5% skim milk powder. As an embodiment, the blocking time of the present invention is 1 to 2 hours; as another embodiment, the blocking time of the present invention is 1 hour.

[0053] As an embodiment, after obtaining the blocker, the present invention performs a first incubation on the blocker using a capture antibody to obtain a coated antigen-capture antibody complex. As an embodiment, the incubation concentration of the capture antibody is 7.5-20 μg / well (one well in 96 wells); as another embodiment, the incubation concentration of the capture antibody is 10 μg / well. As an embodiment, the condition of the first incubation is 37°C for 1-2 hours; as another embodiment, the condition of the first incubation is 37°C for 1 hour.

[0054] As an embodiment, after obtaining the coated antigen-capture antibody complex, the present invention uses a tracer-labeled goat anti-mouse IgG to perform a second incubation on the coated antigen-capture antibody complex to obtain a coated antigen-capture antibody-detection antibody complex. As an embodiment, the tracer-labeled goat anti-mouse IgG includes tracer-labeled goat anti-mouse IgG. As an embodiment, the dilution multiple of the tracer-labeled goat anti-mouse IgG is 1:3000 to 5000; as another embodiment, the dilution multiple of the tracer-labeled goat anti-mouse IgG is 1:3000. As an embodiment, the conditions of the second incubation are 37°C for 1 to 1.5 hours; as another embodiment, the conditions of the second incubation are 37°C for 1 hour.

[0055] After obtaining the coated antigen-capture antibody-detection antibody complex, the present invention detects the tracer signal in the coated antigen-capture antibody-detection antibody complex to determine the concentration of EHP00_19 in the sample to be detected, thereby detecting EHP. The method established by the present invention can perform high-throughput detection of EHP without extracting nucleic acid, with high specificity and high sensitivity. The results of the embodiment show that the sensitivity of detecting EHP is 5×10 4 Spores.

[0056] To further illustrate the present invention, the antigen detection target for shrimp hepatoenteric cytosis and the monoclonal antibody for detecting shrimp hepatoenteric cytosis provided by the present invention and their applications are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0057] Example 1

[0058] 1. Enrichment of EHP

[0059] (1) 100 whiteleg shrimps were collected from shrimp ponds infected with EHP. The hepatopancreas was put into a grinder and ground for 3 min. Sterile ddH2O was added for dilution. The homogenate filtrate was then filtered through a gauze and collected.

[0060] (2) The homogenate filtrate obtained in step (1) was divided into 50 mL centrifuge tubes, centrifuged at 750 rpm for 5 min at room temperature, the supernatant was collected, and the precipitate was discarded;

[0061] (3) centrifuging the supernatant collected in step (2) at 12,000 rpm for 5 min, discarding the supernatant, and collecting the precipitate, wherein the precipitate contains the initially enriched EHP;

[0062] (4) resuspend the precipitate collected in step (3) with 40 mL of sterile ddH2O, centrifuge at 16°C, 12000 rpm for 10 min, and collect the precipitate;

[0063] (5) Repeat step (4) once;

[0064] (6) The collected precipitate was resuspended in 2.0 mL of sterile ddH2O, transferred to a 10 mL centrifuge tube, and Percoll medium was added for density gradient centrifugation. The Percoll concentrations from bottom to top were 90% (2 mL), 60% (3 mL), 45% (2 mL), and 30% (2 mL). Centrifuged at 12000 rpm for 45 min.

[0065] (7) After centrifugation, the spores in the 90% percoll and 60% percoll layers were collected, washed five times with sterile 1× PBS (pH 7.4), and centrifuged at 12,000 rpm for 10 min. The precipitate after washing was the enriched EHP;

[0066] (8) Finally, add 1× PBS (pH 7.4) buffer to resuspend the spores, count them using a hemocytometer, and store them at -20°C until use.

[0067] 2. Immunization of mice with EHP

[0068] The enriched EHP was used as an immunogen to immunize Balb / c mice using subcutaneous multi-point immunization. 8EHP was used for 4 immunizations. For the first immunization, Freund's complete adjuvant was mixed with spores in a volume ratio of 1:1, and injected into mice after complete emulsification. Then, booster immunization was performed every two weeks. During the booster immunization, water-soluble adjuvant (purchased from NEST) was used to emulsify the same volume of EHP and then immunize.

[0069] 3. Determination of screening antigens

[0070] (1) ELISA screening was performed using pure EHP spores as coating antigens (untreated EHP spores were directly coated by gradient dilution). The specific detection steps are as follows:

[0071] Use pure EHP spores to coat a 96-well ELISA plate and place it at 4°C overnight; discard the liquid in the wells, add 200 μL of 0.01M PBST to each well, wash three times, each time for 5 minutes; add 200 μL of PBST containing 5% (W / V) skimmed milk powder to each well, block at 37°C for 2 hours or at 4°C overnight; discard the liquid in the wells, add 200 μL of 0.01M PBST to each well, wash three times, each time for 5 minutes; use mouse negative serum as a negative control, and dilute the serum after four immunizations in step 2 and the mouse negative serum continuously starting from 1:200 with PBST. After the dilution is completed, draw 100 μL and add it to the coated plate; incubate at 37°C for 1.5 hours or at 4°C overnight; discard the liquid in the wells, add 200 μL of 0.01M PBST, wash three times, each wash for 5 minutes; HRP-labeled goat anti-mouse antibody was diluted and added to each well 100 μL, incubated at 37°C for 50 minutes; the liquid in the well was discarded, 200 μL of 0.01M PBST was added to each well, and washed three times, each wash for 5 minutes; 200 μL of TMB colorimetric solution was added to each well, and incubated at 37°C for 10-15 minutes; 50 μL of stop solution was added to each well, and the absorbance value of each well at a wavelength of 450 nm was measured with an enzyme-labeled instrument. The test results are shown in Table 1.

[0072] Table 1 ELISA test results of EHP pure spores (OD 450 )

[0073]

[0074] Note: Serum OD to be tested 450 >Negative serum OD 450 ×2.1 is considered positive, otherwise it is negative, the same below.

[0075] According to Table 1, the optimum coating amount of pure EHP spores is 4×10 6 When pure spores were used for coating, the coating values ​​were unstable due to the large size of the particles. The coating effect was poor when untreated EHP spores were used directly. This may be due to the presence of impurities or the physical properties of the spores that hindered the presentation of effective antigens.

[0076] (2) Based on the conclusion of step (1), in order to improve the coating efficiency and the reliability of ELISA results, the spores were pretreated (mechanically disrupted), resuspended in RIPA lysis buffer, and mechanically disrupted for two rounds, each with 6 cycles, to prepare EHP total protein. The EHP total protein obtained by mechanical disruption was used as the coating antigen, and ELISA screening was performed in the same manner as step (1). The results are shown in Table 2.

[0077] Table 2 ELISA test results of EHP total protein (OD 450 )

[0078]

[0079] According to Table 2, when the coating amount of EHP total protein per well was 8 μg, the positive signal intensity was not as strong as that when EHP spores were used for coating. This indicates that although RIPA lysis buffer can effectively lyse spores and extract total protein, it may not be the best choice to directly use the lysed total protein for coating in ELISA.

[0080] (3) Based on the conclusions of steps (1) and (2), the spores were resuspended in RIPA lysis buffer and incubated on ice for 30 min to obtain EHP after incubation. The EHP after RIPA incubation and pure EHP spores were used as coating antigens, respectively, at a rate of 4 × 10 6 The spores were coated and screened by ELISA according to step (1). The results are shown in Table 3.

[0081] Table 3 ELISA test results of EHP after lysate incubation (OD 450 )

[0082] Dilution multiple 1：400 1：800 1：1600 1：3200 1：6400 1：12800 1：25600 1：51200 RIPA incubation 2.389 2.514 1.831 1.492 0.974 0.462 0.232 0.151 Not incubated 0.858 0.406 0.378 0.285 0.221 0.148 0.100 0.148 Negative control 0.318 0.137 0.093 0.071 0.06 0.089 0.062 0.057

[0083] According to Table 3, after RIPA incubation treatment, the performance in ELISA coating was better, and it was speculated that RIPA incubation promoted the exposure of EHP spore surface proteins.

[0084] Example 2

[0085] Preparation of monoclonal antibodies

[0086] 1. Detection of polyclonal antibody titer

[0087] (1) The RIPA-incubated EHP obtained in step 3 of Example 2 was used as the coating antigen, and ELISA detection was performed in the same manner as in step 3; wherein the coating concentration was 4×10 6 spores, and normal mouse serum was diluted in the same proportion as a negative control. The test results are shown in Table 4.

[0088] Table 4 Test results of polyclonal antibody titer (OD 450 )

[0089] Potency 1：3200 1：6400 1：12800 1：25600 1：51200 1：102400 1：204800 1：409600 Rat No.1 1.75648 1.38562 0.91812 0.66148 0.47728 0.2807 0.20802 0.17276 Mouse No.2 1.08456 0.69432 0.39252 0.20638 0.19266 0.1401 0.08588 0.07066 Mouse No. 3 1.57998 1.03348 0.57146 0.3839 0.24208 0.16546 0.12434 0.1367 Mouse No. 4 0.98238 0.69226 0.3766 0.2743 0.19256 0.13364 0.10936 0.12394 Number 5 Mouse 0.77618 0.5622 0.36842 0.23828 0.17014 0.13444 0.11092 0.13326 Negative control 0.09848 0.10008 0.0919 0.11136 0.12402 0.11386 0.10962 0.13376

[0090] Note: Serum OD to be tested 450 >Negative serum OD 450 ×2.1 is the maximum dilution ratio of serum titer, and cell fusion can be performed when the titer is >12800.

[0091] According to Table 4, the titer of the mouse polyclonal antibody No. 1 immunized with EHP spores can reach 1:102400, which meets the fusion requirements.

[0092] (2) Western blot analysis of polyclonal antibodies and native proteins

[0093] After four immunizations, blood was collected from the eyeballs of mice No. 1 to obtain polyclonal antibodies, and Western blot was performed. The results showed that the EHP polyclonal antiserum could specifically recognize the total protein of EHP spores ( Figure 1 ), and negative serum cannot recognize EHP spore total protein ( Figure 2 ), indicating that the polyclonal antibody of EHP natural protein prepared by the present invention has good specificity, which lays a foundation for subsequent experiments.

[0094] 2. Cell Fusion

[0095] (1) Three days before cell fusion, booster immunization was performed with 10 8 The spores were mixed with saline at a ratio of 1:1 (V / V) and injected into the peritoneal cavity of mice.

[0096] (2) Preparation of feeder layer cells: 7-8 week old female Balb / c mice sacrificed by cervical dislocation were soaked in 75% alcohol for 5 min. Preheated incomplete culture medium was injected into the mouse peritoneal cavity in a clean bench. The peritoneal wall was blown and washed several times. The cells in the peritoneal cavity were fully suspended and transferred to a sterile centrifuge tube. The process was repeated 2-3 times. Centrifugation was performed at 1000 rpm for 5 min. The supernatant was discarded and the cells were resuspended. The cells were washed three times. 1640 complete culture medium supplemented with HAT was added to resuspend the cells. The cells were diluted to the required volume. 100 μL was added to each well of a 96-well plate. The plate was cultured in a cell culture incubator at 37°C and 5% CO2 saturated humidity for later use.

[0097] (3) Preparation of spleen cells: The mice were killed by cervical dislocation and immersed in 75% alcohol for 5 min for disinfection. The spleen was removed with sterile forceps and the connective tissue was removed. The spleen was ground on a mesh and an appropriate amount of incomplete culture medium was added to prepare a cell suspension. The suspension was centrifuged at 1000 rpm for 5 min and the cells were washed three times. 10 8The spleen cells were 2×10 7 ~5×10 7 SP2 / 0 cells were mixed in a 50 mL sterile centrifuge tube, and incomplete culture medium was added to 30 mL. The tube was mixed thoroughly, and centrifuged at 1000 rpm for 10 min. The supernatant was aspirated as much as possible, and the bottom of the tube was tapped to make the precipitated cells loose and uniform. The tube was preheated in a 40°C water bath, and 1 mL of PEG-1500 preheated to 40°C was added. Then, 30 mL of incomplete culture medium preheated to 37°C was added within 90 s to terminate the fusion. The tube was allowed to stand at 37°C for 10 min, and centrifuged at 1000 rpm for 10 min. The supernatant was discarded, and 5 mL of HAT culture medium was added. The precipitated cells were gently pipetted to suspend and mix. Then, HAT culture medium was added to 80-100 mL, and the tube was dispensed into a 96-well cell culture plate containing feeder layer cells, with 100-150 μL per well, and the growth of hybridoma cells was observed.

[0098] 3. Preparation of Hybridoma Cell Lines

[0099] (1) Remove the feeder layer cells in advance and plate them in a 96-well plate;

[0100] (2) After the positive hybridoma cells have grown into cell clusters in the 96-well plate, they are resuspended in 200 μL of HT medium, 100 μL is retained for expansion and culture, and the remaining 100 μL is diluted in a 10-fold gradient ratio. The diluted cells are plated at 100 μl / well in a 96-well plate from which the feeder layer cells have been removed and cultured. Positive hybridoma cells were screened by indirect ELISA, and a total of 4 monoclonal cell lines were screened, namely hybridoma cells 6D9-e4, 2D10, 2E11 and 1C10. Western blot confirmed that the antibodies secreted by the above 4 cell lines can recognize EHP natural protein ( Figure 3 ).

[0101] 4. Purification of Monoclonal Antibodies

[0102] (1) Preparation of ascites: 12-week-old Balb / c mice were selected and 400 μL of Freund's incomplete adjuvant was injected into their peritoneal cavity one week in advance. The abdomen of the mouse was gently stroked to make it evenly dispersed. The positive hybridoma cells cultured were blown up with 1640 medium and then incubated at 10 6 10 cells / mouse were injected into the abdominal cavity of mice, and the abdomen of the mice was gently stroked to make them spread evenly; about 10 days after injection, the abdomen of the mice showed obvious swelling, and the status of the mice was observed every day. When the abdomen swelled to the maximum, the ascites was collected with a 50mL syringe needle; the ascites was centrifuged at 5000rpm for 5min to remove fat and blood cells, and the supernatant was collected and stored at -80℃;

[0103] (2) Antibody purification: Protein L antibody purification column was used to affinity purify the collected ascites to obtain four EHP monoclonal antibodies with high concentration and purity, namely 6D9-e4, 2D10, 2E11 and 1C10. The subtype identification is shown in Table 5.

[0104] Table 5 Results of subtype identification of monoclonal antibodies

[0105] Monoclonal antibodies 6D9-e4 1C10 2E11 2D10 IgG1 0.1060 0.0810 0.1050 0.0830 IgG2a 0.1060 0.0810 0.1170 0.0840 IgG2b 0.1100 0.0810 0.1000 0.0730 IgG3 0.0990 0.0920 0.1100 0.0780 IgA 0.0810 0.0720 0.0920 0.0700 IgM 0.4040 0.4530 0.4700 0.4800 Kappa 0.4290 0.4700 0.5070 0.4920 Lambda 0.0750 0.0740 0.0870 0.0720

[0106] Note: OD was read by microplate reader 450 The well with the highest OD value corresponds to the corresponding subtype.

[0107] It can be seen from Table 5 that the subtypes of the above four antibodies are all IgM, Kappa subtype.

[0108] Example 3

[0109] Establishment of indirect ELISA method for detecting EHP

[0110] (1) Determination of the optimal monoclonal antibody

[0111] The affinity of the antibodies purified above was compared at the same concentration to determine the optimal monoclonal antibody. The results are shown in Table 6.

[0112] Table 6 Comparison of antibody affinity at the same concentration (OD 450 )

[0113] Antibody amount (μg) 20.0000 10.0000 5.0000 2.5000 1.2500 0.625 0.3125 0.1563 6D9-e4 0.5790 0.3740 0.2430 0.1830 0.1690 0.1350 0.1280 0.1200 1C10 3.0190 1.9150 1.2020 0.9400 0.6250 0.3530 0.2120 0.2050 2D10 0.3730 0.3660 0.2170 0.1900 0.1620 0.1420 0.1080 0.1130 2E11 0.4790 0.1920 0.1680 0.1380 0.1300 0.1100 0.1140 0.1240 Mouse negative serum 0.2600 0.1610 0.1340 0.1190 0.1140 0.1110 0.0925 0.0800

[0114] It can be seen from Table 6 that the 1C10 monoclonal antibody has the best effect among the four antibodies. Therefore, 1C10 was subsequently selected for the establishment of the indirect ELISA detection method. The sequence information of the 1C10 monoclonal antibody is shown in Table 7.

[0115] Table 7 1C10 monoclonal antibody sequence information

[0116]

[0117]

[0118] (2) Determination of optimal antigen coating concentration and optimal antibody incubation concentration

[0119] Indirect ELISA was performed using the chessboard titration method. The EHP treated with the lysate in step 3 of Example 2 was used as the antigen. 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6, 5×10 6 Each well was coated and incubated. The primary antibody (1C10 antibody) was incubated with 20μg, 10μg, 5μg, 2.5μg, 1.25μg, 0.625μg, 0.3125μg, 0.156μg and 0.078μg in columns 1 to 10, and negative serum was set as a control. The experimental data was measured by the P / N value. The larger the P / N value, the better the effect. The results showed that the optimal coating antigen was 5×10 6 / well, the optimal antibody incubation concentration was 10 μg / well (Table 8).

[0120] Table 8 Detection results of different antigen coating amounts and antibody incubation amounts (OD 450 )

[0121]

[0122]

[0123] (3) Determination of optimal coating conditions

[0124] After determining the optimal monoclonal antibody, optimal antigen coating concentration, and optimal antibody incubation concentration, the OD values ​​of different coating conditions (coating at 37°C for 1 h, 37°C for 2 h, 37°C for 3 h, and 4°C for 12 h) were measured. 450 The P / N value was analyzed and the results showed that the P / N value was the largest at 37°C for 3 h, which was 6.069869 (Table 9).

[0125] Table 9 Detection results of different coating conditions (OD 450 )

[0126] Coating conditions 37℃ coating for 1h 37℃ coating for 2h 37℃ coating for 3h 4℃ coating for 12h P 0.3830 0.5283 0.9267 0.6782 N 0.1284 0.1180 0.1527 0.1425 P / N 2.983641 4.477401 6.069869 4.7592

[0127] (4) Determination of the optimal blocking solution

[0128] According to the optimized conditions, the selection of blocking solution was optimized, and the commonly used blocking solutions 5% skim milk powder, 1% BSA and 3% BSA were selected for blocking, and the OD was measured by an enzyme marker. 450 , the P / N value was analyzed, and the results showed that the BSA background value (N value) was high, and skimmed milk powder was better as the blocking solution (Table 10).

[0129] Table 10 Detection results of different blocking solutions (OD 450 )

[0130] Blocking solution 1% BSA 3% BSA 5% skim milk powder P 3.2323 3.1213 1.762 N 0.7233 0.6873 0.169 P / N 4.468 4.541 10.426

[0131] (5) Determination of the optimal closure time

[0132] According to the selected blocking solution, set different blocking time for comparison and measure OD by microplate reader. 450 , the P / N value was analyzed and the results showed that the optimal sealing time was 1h (Table 11).

[0133] Table 11 Test results of different closure times (OD 450 )

[0134] Closure time 1h 1.5h 2h P 1.442 1.826 1.101 N 0.123 0.18 0.104 P / N 11.787 10.144 10.587

[0135] (6) Determination of the optimal incubation time for primary antibody

[0136] According to the determined conditions, the primary antibody incubation time was optimized, three gradients were set from 1h to 2h, and the OD was measured by an enzyme-labeled instrument. 450 , the P / N value was analyzed, and the results showed that the optimal incubation time for the primary antibody was 1 h (Table 12).

[0137] Table 12 Detection results of different primary antibody incubation times (OD 450 )

[0138] Primary antibody incubation time 1h 1.5h 2h P 0.964 0.972 0.8910 N 0.1830 0.1910 0.2060 P / N 5.268 5.089 4.325

[0139] (7) Determination of the optimal dilution ratio of the secondary antibody

[0140] Sheep anti-mouse IgG-HRP (Biosharp) was used as the secondary antibody. The dilution ratio of the secondary antibody was optimized according to the determined conditions. The initial concentration of the secondary antibody was 2 mg / mL. 1:3000, 1:5000, 1:8000 and 1:10000 were selected for comparison. The OD was measured by an enzyme-labeled instrument. 450 , the P / N value was analyzed and the results showed that the optimal dilution ratio was 1:3000 (Table 13).

[0141] Table 13 Detection results of different secondary antibody dilution ratios (OD 450 )

[0142] Secondary antibody dilution ratio 1:3000 1:5000 1:8000 1:10000 P 0.7460 0.4220 0.3080 0.2860 N 0.2130 0.1612 0.1200 0.1370 P / N 3.502 2.618 2.567 2.088

[0143] (8) Determination of the optimal incubation time for secondary antibodies

[0144] Sheep anti-mouse IgG-HRP (Biosharp) was used as the secondary antibody. The incubation time of the secondary antibody was optimized according to the determined conditions. The OD was measured by an enzyme-labeled instrument at 45 min, 1 h, 1.5 h, and 2 h. 450 , the P / N value was analyzed, and the results showed that the optimal incubation time for the secondary antibody was 1 h (Table 14).

[0145] Table 14 Detection results of different secondary antibody incubation times (OD 450 )

[0146] Secondary antibody incubation time 45min 1h 1.5h 2h P 1.097 1.673 1.326 1.2265 N 0.111 0.089 0.098 0.123 P / N 9.916 18.658 13.439 9.945

[0147] (9) Determination of the optimal color development time

[0148] Based on the above optimized conditions, the color development time was optimized, and three time gradients of 10min, 15min, and 20min were set. The OD was measured by an enzyme marker. 450 , the P / N value was analyzed, and the results showed that the optimal color development time was 20 min (Table 15).

[0149] Table 15 Detection results of different color development time (OD 450 )

[0150] Color development time 10min 15min 20min P 0.668 1.329 1.593 N 0.088 0.104 0.109 P / N 7.562 12.738 14.659

[0151] Example 4

[0152] Specificity test

[0153] According to the indirect ELISA detection method established in Example 3, six pathogens under the same quantity, including Encephalitozoon hellem (Eh), Encephalitozooncuniculi (Ec), Nosema bombycis (Nb), Vairimorphanecatrix (Vn), Bombyx mori Nuclear Polyhedrosis Virus (NPV) and Enterocytozoon hepatopenaei (EHP), were detected. The results showed that only EHP was detected as positive, and the detection of other pathogens was negative, indicating that the indirect ELISA has a strong specificity for detecting EHP ( Figure 4 ).

[0154] Example 5

[0155] Threshold detection

[0156] Normal white shrimp hepatopancreas samples and pathogens were selected, including Encephalitozoon hellem (Eh), Encephalitozoon cuniculi (Ec), Nosemabombycis (Nb), Vairimorpha necatrix (Vn) and Bombyx mori Nuclear Polyhedrosis Virus (NPV), 5 samples of each, for a total of 30 samples. The indirect ELISA system established in Example 3 was used for detection, and the OD was measured by an enzyme marker. 450 The sample was statistically analyzed based on the readings. The results showed that the sample mean (X) = 0.247, standard deviation (SD) = 0.041, and critical value = 3 × standard deviation (SD) + mean (X), that is, the critical value is 0.37. 450 >0.37 is positive, when OD 450 <0.37 is negative ( Figure 5 ).

[0157] Example 6

[0158] Sensitivity test

[0159] According to the indirect ELISA detection system established in Example 3 and the critical value determined in Example 3, the purified EHP spores were gradiently diluted and protein samples were extracted for detection. The results showed that the indirect ELISA detection sensitivity was 5×10 4 Spores ( Figure 6 ).

[0160] Example 7

[0161] Reverse screening of EHP antigen detection targets

[0162] In order to explore the target recognized by the 1C10 monoclonal antibody obtained in Example 2, the purified 1C10 monoclonal antibody was used to perform Western blot with EHP total protein; at the same time, SDS-PAGE and Coomassie Brilliant Blue staining were performed with the same protein, and the positive bands of Western blot ( Figure 7 The proteins at the position of 25 kDa to 35 kDa in the middle of the peptide were cut and analyzed by LC-MS / MS mass spectrometry. The LC-MS / MS mass spectrometry results were analyzed to screen possible candidate target proteins, and two candidate target proteins, EHP00_763 and EHP00_19, were initially screened out (Table 16). Among them, the accession number of EHP00_763 protein in NCBI is OQS53743.1; the accession number of EHP00_19 protein in NCBI is OQS54511.1.

[0163] Table 16 LC-MS / MS mass spectrometry data analysis results

[0164]

[0165] Example 8

[0166] To further verify whether the candidate protein identified by LC-MS / MS mass spectrometry in Example 7 is the target protein of 1C10, the coding genes of EHP00_763 protein and EHP00_19 protein were inserted into pET-28a plasmid, respectively, to construct exogenous expression vectors pET-28a-EHP00_19 and pET-28a-EHP00_763; the constructed exogenous expression vectors were transformed into expression strain BL21, and pET-28a-EHP00_763 was detected to express in the supernatant of 16°C, 0.4 mM IPTG induction for 22 h ( Figure 8 ), pET-28a-EHP00_19 was expressed in the pellet induced by 0.1 mM IPTG at 16°C for 22 h ( Fig. 9 ). Western blot detection was performed using the 1C10 monoclonal antibody obtained in Example 2. The results showed that the monoclonal antibody 1C10 could specifically recognize EHP00_19 ( Fig.10 , right), EHP00_763 ( Fig.10 , left), indicating that the target protein of EHP detected by 1C10 monoclonal antibody is EHP00_19.

[0167] Example 9

[0168] Referring to the literature (ImK, Mareninov S, DiazM FP, et al. An introduction to forming immunofluorescence staining [M]. Springer New York, 2019.), the 1C10 monoclonal antibody obtained in Example 2 was used to carry out immunolocalization analysis (immunofluorescence assay, IFA) to explore the protein localization of EHP00_19. The IFA results showed that EHP00_19 was localized on the spore wall ( Fig.11 ), indicating that EHP00_19 is the spore wall protein of EHP.

[0169] It can be seen from the above content that the technical solution provided by the present invention can quickly, accurately and specifically carry out the detection of EHP without the need for nucleic acid extraction and amplification, and has strong specificity and high sensitivity.

[0170] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of the reagent for detecting EHP00_19 in the preparation of products for detecting hepatoenterozoa in shrimp.

2. Detection of antibodies against shrimp hepatocellular carcinoma, characterized in that: The antibody is an antibody against EHP00_19; the antibody includes a monoclonal antibody and / or a polyclonal antibody.

3. The antibody according to claim 2, characterized in that The monoclonal antibody comprises 1C10 monoclonal antibody; The light chain complementary determining region CDR1 of the 1C10 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO.2, the amino acid sequence of the light chain complementary determining region CDR2 comprises LVS, and the light chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.3; The heavy chain complementary determining region CDR1 of the 1C10 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO.6, the heavy chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.7, and the heavy chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.

8.

4. The antibody according to claim 3, characterized in that The light chain of the 1C10 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO.1; The heavy chain of the 1C10 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO.

5.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the 1C10 monoclonal antibody according to claim 3 or 4.

6. The nucleic acid molecule according to claim 5, characterized in that The sequence of the nucleic acid molecule encoding the light chain of the 1C10 monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO.4; the sequence of the nucleic acid molecule encoding the heavy chain of the 1C10 monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO.

9.

7. A biological material for detecting EHP00_19 and / or shrimp hepatoenterocystis, characterized in that: The biological material is a biological material that expresses the 1C10 monoclonal antibody according to claim 3 or 4; the biological material includes a vector or a cell.

8. Use of the antibody according to any one of claims 2 to 4, the 1C10 monoclonal antibody encoded by the nucleic acid molecule according to claim 5 or 6, or the 1C10 monoclonal antibody expressed by the biomaterial according to claim 7 in one or more of the following: (1) Prepare products for detecting EHP00_19; (2) preparing products for detecting shrimp hepatocystis; (3) Prepare products for the auxiliary prevention of hepatopancreatic microsporidiosis.

9. A kit for detecting EHP00_19 and / or shrimp hepatoenterocystis, characterized in that: The kit comprises a capture antibody and a tracer-labeled detection antibody; the capture antibody is the antibody according to any one of claims 2 to 4; the tracer-labeled detection antibody comprises a tracer-labeled goat anti-mouse IgG.

10. A method for detecting EHP00_19 for non-diagnostic purposes, characterized in that: The method comprises the following steps: mixing a sample to be detected, a capture antibody and a detection antibody labeled with a tracer to form a complex; detecting a tracer signal in the complex to determine the concentration of EHP00_19 in the sample to be detected; The capture antibody is the antibody according to any one of claims 2 to 4; The tracer-labeled detection antibody includes tracer-labeled goat anti-mouse IgG.

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