ELISA kit for detecting hybrid snakehead aeromonas schubertii antibody and detection method thereof

Abstract

The invention belongs to the technical field of immunodetection, and discloses an ELISA kit for detecting a hybrid snakehead aeromonas schubertii antibody and a detection method thereof. The invention discloses a protein, which is characterized in that immune protein Slr4-like of aeromonas schubertii is analyzed and truncated, and the SLPn protein with a better detection effect on the aeromonas schubertii is obtained through recombinant induced expression. The SLPn protein is used for successfully constructing an ELISA kit for detecting an antibody aiming at aeromonas schubertii of hybrid snakehead and a detection method of the kit, and the kit or the detection method has relatively good sensitivity, specificity and accuracy on the aeromonas schubertii antibody and has very good repeatability and stability; the technical support is provided for the detection of the antibody after the aeromonas schubertii infection or immunization in the cultured hybrid snakehead.

Description

Technical Field

[0001] The invention belongs to the technical field of immune detection, and particularly relates to an ELISA kit for detecting antibodies of hybrid snakehead Aeromonas schubertii and a detection method thereof. Background Art

[0002] Channa maculata (♀) is an important freshwater specialty aquaculture fish species in my country. It is a common name for fish of the Channidae family, including Channa maculata, Channa stigmata, and Channa argus. Hybrid Channa (Channa maculata♀×Channa argus♂) is a hybrid of Channa maculata as the female parent and Channa argus as the male parent. It boasts rapid growth, strong stress tolerance, high survival rate, and transportability, making it a major aquaculture species in the Guangdong and Guangxi regions of my country. In recent years, high-density, intensive aquaculture practices have led to a growing number of aquaculture disease problems. Common pathogens in aquaculture include Nocardia seriolae, Aeromonas schubertii, Flavobacterium cloumnare, hybrid Channa rhabdovirus (HSHRV), and Trichodina. Aeromonas schubertii can invade fish through the gills and intestines, causing septicemia. In snakehead fish, symptoms include punctate or spotty hemorrhages, enlarged and congested livers, numerous slippery, irregularly shaped white spots, and visceral nodules. These infections can cause acute morbidity and mortality, resulting in significant harm. Currently, the disease is primarily treated with antibiotics, while immune control measures are relatively weak.

[0003] Monitoring serum-specific antibody levels is crucial for assessing pathogen infection and vaccine efficacy. Currently, numerous methods exist for detecting antibodies, including traditional agglutination and precipitation tests, as well as immunoassays such as enzyme-linked immunosorbent assays (ELISAs), immunofluorescence techniques, and luminescence immunoassays. However, there is no accurate, simple, and sensitive method for detecting antibodies specific to Aeromonas schubertii. Summary of the Invention

[0004] The first aspect of the present invention aims to provide a protein.

[0005] The second aspect of the present invention aims to provide a nucleic acid molecule encoding the protein of the first aspect of the present invention.

[0006] The third aspect of the present invention aims to provide biological materials related to the nucleic acid molecules of the second aspect of the present invention.

[0007] The fourth aspect of the present invention aims to provide applications of the protein of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, and the biomaterial of the third aspect of the present invention.

[0008] The fifth aspect of the present invention aims to provide a reagent or a kit.

[0009] The sixth aspect of the present invention aims to provide a method.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a protein, wherein the protein is any one of a1) to a3):

[0012] a1) a protein with an amino acid sequence as shown in SEQ ID NO: 7;

[0013] a2) a protein comprising SEQ ID NO: 7 with one or more amino acids substituted and / or deleted and / or added and having the same or similar function as SEQ ID NO: 7;

[0014] a3) A protein having at least 95% homology to SEQ ID NO: 7 and having the same or similar function as SEQ ID NO: 7

[0015] In some embodiments of the present invention, the protein is artificially synthesized using the N-terminal sequence of the Slr4-like gene after codon optimization, ligated into the pET-28a(+) plasmid, transformed into Escherichia coli BL21 competent cells after identification, induced for expression, and then purified by affinity chromatography.

[0016] The second aspect of the present invention provides a nucleic acid molecule encoding the protein of the first aspect of the present invention.

[0017] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:5.

[0018] The third aspect of the present invention provides a biological material related to the nucleic acid molecule of the second aspect of the present invention, wherein the biological material comprises at least one of b1) to b8):

[0019] b1) The nucleic acid molecule according to claim 2;

[0020] b2) an expression cassette containing the nucleic acid molecule described in b1);

[0021] b3) a recombinant vector containing the nucleic acid molecule described in b1);

[0022] b4) a recombinant vector containing the expression cassette described in b2);

[0023] b5) a recombinant cell containing the nucleic acid molecule described in b1);

[0024] b6) a recombinant cell containing the expression cassette described in b2);

[0025] b7) a recombinant cell containing the recombinant vector described in b3);

[0026] b8) A recombinant cell containing the recombinant vector described in b4).

[0027] In some embodiments of the present invention, the expression cassette comprises a 5' transcription control region, an open reading frame encoding the fusion protein of the first aspect of the present invention, a translation control signal, a 3' untranslated region (3'UTR) and a transcription termination signal.

[0028] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0029] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a viral vector.

[0030] In some embodiments of the present invention, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxvirus vector, a Sendai virus vector, and a herpes simplex virus vector.

[0031] In some embodiments of the present invention, the non-viral vector comprises at least one of a plasmid vector, a cationic polymer vector, chitosan, polyethyleneimine, a nanoparticle vector, and a liposome.

[0032] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a phage, a cosmid, an F cosmid, or an artificial chromosome.

[0033] In some embodiments of the present invention, the recombinant expression vector uses pET-28a(+) as the original expression vector.

[0034] In some embodiments of the present invention, the plasmid vector may be any plasmid, and the viral vector may be any virus.

[0035] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new species of plants or animals.

[0036] In some embodiments of the present invention, the cell lines include cell lines for expressing antigen epitope peptides, such as Expi293F; cell lines for preparing hybridomas, such as Sp-2 / 0. Those skilled in the art can expect that other common engineered cell lines in the art can also achieve the same or similar technical effects of the present invention.

[0037] The fourth aspect of the present invention provides the use of the protein of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, or the biomaterial of the third aspect of the present invention in c1) to c6):

[0038] c1) Detection of Aeromonas Schubert;

[0039] c2) preparing a product for detecting Aeromonas schubertii;

[0040] c3) detecting whether the sample to be tested is infected with Aeromonas schubertii;

[0041] c4) preparing and testing whether the sample to be tested is infected with Aeromonas schubertii;

[0042] c5) evaluating the immune efficacy of vaccines against Aeromonas schubertii;

[0043] c6) preparing a product for evaluating the immune effect of a vaccine against Aeromonas schubertii;

[0044] The above applications are for non-disease diagnosis.

[0045] In some embodiments of the present invention, the product includes a kit, a reagent strip, a reagent, or a chip.

[0046] The fifth aspect of the present invention provides a reagent or kit comprising the protein of the first aspect of the present invention.

[0047] In some embodiments of the present invention, the reagent or kit further comprises at least one of a capture antibody, an enzyme-labeled secondary antibody detection antibody, a solid phase carrier, a coating solution, a washing solution, a substrate color development solution, a stop solution, a positive standard, and a negative standard.

[0048] In some embodiments of the present invention, the solid phase carrier is an ELISA plate.

[0049] In some embodiments of the present invention, the protein according to the first aspect of the present invention is coated on the surface of a solid support.

[0050] In some embodiments of the present invention, the capture antibody is a mouse anti-chickenhead IgM monoclonal antibody.

[0051] In some embodiments of the present invention, the enzyme-labeled secondary antibody is goat immune mouse IgG labeled with horseradish peroxidase (HRP).

[0052] In some embodiments of the present invention, the color developing solution comprises TMB color developing solution.

[0053] In some embodiments of the present invention, the stop solution is H2SO4 solution.

[0054] In some embodiments of the present invention, the coating solution is phosphate buffered saline (PBS, pH 7.4).

[0055] The sixth aspect of the present invention provides a method for detecting whether a sample to be tested is infected with Aeromonas schubertii for non-diagnostic purposes or a method for evaluating the immune effect of a vaccine against Aeromonas schubertii, comprising the step of using the protein of the first aspect of the present invention or the reagent or kit of the fifth aspect of the present invention to detect the sample to be tested.

[0056] In some embodiments of the present invention, ELISA is used to detect the sample.

[0057] In some preferred embodiments of the present invention, the ELISA includes direct ELISA, indirect ELISA, competitive ELISA, and double antibody ELISA sandwich method.

[0058] In some more preferred embodiments of the present invention, the method specifically comprises the following steps:

[0059] Coating: coating the protein of the first aspect of the present invention onto a solid phase carrier;

[0060] Closed;

[0061] Sample addition: Add the sample to be tested, incubate, and set up negative and positive controls at the same time;

[0062] Primary antibody reaction: adding the capture antibody of the fifth aspect of the present invention and incubating;

[0063] Secondary antibody reaction: adding the enzyme-labeled secondary detection antibody of the fifth aspect of the present invention and incubating;

[0064] Color development;

[0065] Stop: Add stop solution and measure OD 450 nm value;

[0066] Result determination: OD of the sample to be tested 450 nm value / negative serum OD 450 nm≥2.1 is positive, otherwise it is negative. In some embodiments of the present invention, the concentration of the protein coating is 0.5-20 μg / mL.

[0067] In some preferred embodiments of the present invention, the concentration of the protein coating is 2-16 μg / mL.

[0068] In some more preferred embodiments of the present invention, the concentration of the protein coating is 4-8 μg / mL.

[0069] In some embodiments of the present invention, the capture antibody has an effective concentration of 1:(1000-8000).

[0070] In some preferred embodiments of the present invention, the effective concentration of the capture antibody is 1:(1000-6000).

[0071] In some more preferred embodiments of the present invention, the effective concentration of the capture antibody is 1:(1000-4000).

[0072] In some embodiments of the present invention, the working concentration of the enzyme-labeled secondary detection antibody is 1:(10,000-30,000).

[0073] In some preferred embodiments of the present invention, the working concentration of the enzyme-labeled secondary detection antibody is 1:(10000-25000).

[0074] In some more preferred embodiments of the present invention, the working concentration of the enzyme-labeled secondary detection antibody is 1:(10,000-20,000).

[0075] In some embodiments of the present invention, the incubation condition of the sample to be tested is 20-40° C. for 30-50 min.

[0076] In some preferred embodiments of the present invention, the incubation condition of the sample to be tested is 20-37° C. for 30-50 min.

[0077] In some more preferred embodiments of the present invention, the incubation condition of the sample to be tested is 20-37° C. for 30-40 min.

[0078] In some embodiments of the present invention, the incubation conditions in the primary antibody reaction are 20-40° C. for 30-70 min.

[0079] In some preferred embodiments of the present invention, the incubation conditions in the primary antibody reaction are 35-40° C. for 30-60 min.

[0080] In some more preferred embodiments of the present invention, the incubation conditions in the primary antibody reaction are 35-37° C. for 40-50 minn.

[0081] In some embodiments of the present invention, the incubation conditions in the secondary antibody reaction are 20-40° C. for 30-50 min.

[0082] In some preferred embodiments of the present invention, the incubation conditions in the secondary antibody reaction are 20-37° C. for 30-45 min.

[0083] In some more preferred embodiments of the present invention, the incubation conditions in the secondary antibody reaction are 25-37° C. for 30-45 min.

[0084] When performing quantitative detection, a standard substance is used in the sample addition step to construct a standard curve, and the specific content is calculated based on the standard curve.

[0085] The beneficial effects of the present invention are:

[0086] The present invention analyzes and truncates the immune protein Slr4-like of Aeromonas Schubert, and then obtains the SLPn protein (the N-terminal half domain sequence of Slr4-like) through recombinant induced expression, which has a good detection effect on Aeromonas Schubert. Using this SLPn protein, an ELISA kit and detection method for detecting antibodies against Aeromonas Schubert in hybrid snakehead snakeheads were successfully constructed. The kit or detection method has good sensitivity (sensitivity of 1:1280 for hybrid snakehead serum detection), specificity, and accuracy for Aeromonas Schubert antibodies, and has excellent repeatability and stability. Furthermore, the method does not require the use of complex detection equipment, has low technical requirements for operation, and is low-cost, providing technical support for the detection of Aeromonas Schubert infection or antibodies after immunization in cultured hybrid snakehead snakeheads. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0088] Figure 1 This is the SDS-PAGE picture of inactivated Aeromonas schubertii.

[0089] Figure 2 Western blotting images for the identification of immunogenic proteins of Aeromonas schubertii.

[0090] Figure 3 The results are for immunogenic protein spectrum identification.

[0091] Figure 4 Figure 4 is the PCR amplification image of Slr4-like gene; M, DL2000; 1-4, Slr4-like gene fragments.

[0092] Figure 5 Schematic diagram of the predicted three-dimensional structure of SLP protein.

[0093] Figure 6 SDS-PAGE images of SLP, SLPn and SLPc recombinant proteins; M, protein marker; 1, purified SLP recombinant protein; 2, purified SLPn recombinant protein; 3, purified SLPc recombinant protein.

[0094] Figure 7The indirect ELISA method for detecting the specific antibody to Aeromonas schubertii constructed in Example 5 was used to detect the positive and negative serum samples of Aeromonas schubertii. DETAILED DESCRIPTION

[0095] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0096] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0097] In the examples, mouse anti-hybrid snakehead IgM monoclonal antibodies, Aeromonas schubertii, Nocardia sphenodon, Flavobacterium columnaris, hybrid snakehead rhabdovirus, and neuronecrosis virus were deposited at the Animal Husbandry and Aquatic Products Research Center of Guangdong Haid Group Co., Ltd. Bacteria were identified by morphology, physiology, biochemistry, and molecular biology, while viruses were confirmed by molecular biology and viral pathological morphology. Mouse anti-hybrid snakehead IgM monoclonal antibodies were kindly provided by Professor Kong Weiguang of the Institute of Hydrobiology, Chinese Academy of Sciences.

[0098] The primary antibody diluent, secondary antibody diluent, washing solution, color development solution, and stop solution in the examples were all purchased from Huzhou Yingchuang Biotechnology Co., Ltd. Goat anti-mouse IgG-HRP was purchased from Abcam.

[0099] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0100] Example 1 Identification of immunogenic proteins of Aeromonas schubertii

[0101] 1. Preparation of serum positive for Aeromonas Schubert antibodies

[0102] The laboratory-preserved Aeromonas Schubert was revived and expanded using LB liquid culture medium, and 50 mL of the culture medium with a density of 10 9 cfu / mL of bacterial culture solution was washed twice with phosphate buffer and inactivated with 0.1% formaldehyde solution at 4°C for 48 h. After neutralizing the formaldehyde with sodium thiosulfate, the bacteria were collected and prepared into 1×10 9cfu / mL of inactivated bacteria, stored at 4°C after quality inspection. Use SEPPIC's ISA 201 adjuvant, stir and mix with the inactivated Schubert Aeromonas to prepare an emulsified vaccine for use. Select 50 healthy hybrid snakeheads weighing 80-100 grams, and immunize them with the emulsified vaccine by intraperitoneal injection, 100 μL per tail, immunize twice, and each immunization is 15 days apart. 15 days after the booster immunization, collect blood from the tail vein of all fish, let it stand for 24 hours, and then centrifuge to separate the serum. Combined with the ELISA test to evaluate the antibody titer, select the one with a relatively high antibody titer (serum diluted 40 times, OD 450nm Serum samples with a value between 1.0 and 1.5) were considered as strongly positive serum for the Schubert Aeromonas antibody. The serum was diluted 40 times and the OD 450nm Serum samples with values ​​between 0.3 and 0.6 were regarded as weakly positive sera for Aeromonas Schubert antibodies and were combined and stored at -80°C for future use.

[0103] 2. Identification of immunogenic proteins

[0104] Resuscitated Aeromonas schuberti were disrupted and analyzed by SDS-PAGE. The membranes were then transferred to PVDF membranes for Western blotting analysis as follows: First, the membranes were blocked in PBS containing 5% BSA at 37°C for 1 hour. The membranes were washed three times with PBST (PBS containing 0.05% Tween-20) for 5 minutes each, then incubated with serum positive for Aeromonas schuberti (1:40) for 1 hour at 37°C. After three washes with PBST, the membranes were incubated with mouse anti-hybrid snakehead IgM monoclonal antibody (1:3000) for 1 hour at 37°C. After three washes with PBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG monoclonal antibody (1:8000) for 1 hour at 37°C. Finally, the membranes were developed in DAB colorimetric solution and the reaction was terminated by washing with deionized water. The immunogenic proteins corresponding to the obvious band regions were cut out from the SDS-PAGE gel and sent to Zhongke Xinbiology for mass spectrometry analysis.

[0105] SDS-PAGE and Western blotting results are as follows Figure 1 、 Figure 2 As shown in Figure 2, there is a clear color band in the area around 45kDa, which corresponds to a high-abundance protein band on the SDS-PAGE gel. Figure 3), the protein with the highest match was identified as hypothetical protein WL1483_4046 [Aeromonas schubertii]. NCBI alignment analysis revealed it to be a bacterial S-layer protein and a Slr4-like protein. The Slr4-like protein consists of 459 amino acids, including a 19-amino acid signal peptide (GenBank: ALP43465.1), with an estimated molecular weight of 47.1 kDa.

[0106] Example 2 Preparation of recombinant expressed protein

[0107] Using the genomic DNA of laboratory-maintained Aeromonas Schubertii as a template, the full-length Slr4-like gene (signal peptide removed) was amplified by PCR, and the nucleotide sequence was obtained after sequencing. After codon optimization of the sequence, three genes, including the full-length Slr4-like sequence without the signal peptide, the N-terminal half domain sequence of Slr4-like, and the C-terminal half domain sequence of Slr4-like, were artificially synthesized. After digestion with BamHI and HindIII, the gene fragments were ligated into the pET-28a(+) plasmid and, after identification, transformed into Escherichia coli BL21 competent cells. Expression was induced by IPTG, and the recombinant protein was isolated and purified by affinity chromatography. The details are as follows:

[0108] 1. Strains and reagents

[0109] E. coli BL21 (DE3) was purchased from TakaRa; the kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; and the protein purification nickel column was purchased from Merck.

[0110] 2. Expression vector construction

[0111] 2.1 PCR amplification

[0112] Based on the predicted sequence of the S-layer protein Slr4-like gene of Aeromonas Schubert published in NCBI, upstream and downstream amplification primers were designed to remove the signal peptide sequence. The primer sequences are as follows:

[0113] Slr4-F: GTTGTTGATTTTGATGCGGCC (SEQ ID NO: 1);

[0114] Slr4-R:GAATGCGTTCATGGTGTTCAG (SEQ ID NO: 2);

[0115] The genomic DNA of Aeromonas Schubert was extracted from the laboratory and used as a template. The gene sequence was obtained by PCR amplification using the bacterial genome extraction kit. PCR amplification was performed using the Ex Taq polymerase system from Sangon Biotechnology. The PCR reaction procedure was as follows: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 58°C annealing for 30 seconds, and 72°C extension for 60 seconds for 35 cycles; a final extension at 72°C for 10 minutes, and storage at 4°C. After the reaction, agarose gel electrophoresis was performed. The electrophoresis results are shown in the figure. Figure 4 The PCR product was recovered from the agarose gel using a standard agarose gel DNA recovery kit. The recovered product was ligated into a T vector and sent to a biotechnology company for sequencing, obtaining the Slr4-like gene sequence of the laboratory-maintained Aeromonas schuberti.

[0116] 2.2 Optimizing gene fragment synthesis

[0117] The obtained Slr4-like gene sequence of Aeromonas schubertii (signal peptide removed) was codon-optimized, and the optimized sequence is shown in SEQ ID NO: 3. The optimized Slr4-like gene full-length sequence, N-terminal half sequence (SEQ ID NO: 4), and C-terminal half sequence (SEQ ID NO: 5) were sent to a biological company for artificial synthesis. Two restriction enzyme sites, BamHI and HindIII, were added at the upstream and downstream positions and inserted into the pET-28a(+) multiple cloning site to obtain three recombinant plasmids. Each was transformed into E. coli Top10 and, after correct sequence identification, stored in a -80°C freezer.

[0118] 2.3 Construction of expression strain

[0119] Three strains of E. coli containing the recombinant plasmid were shake-cultured, and the plasmids were extracted. The recombinant plasmids were then transformed into expression-competent E. coli DH3 (BL21) cells using the heat shock method. The transformed bacterial suspension was spread onto LB solid medium containing kanamycin (Kan+) and incubated in an inverted position at 37°C for 12–18 hours. Six to eight positive clones were selected, and the insert fragments were identified by colony PCR and sequenced. Strains with the correct sequence were then selected for expression analysis and shake-cultured to obtain recombinant strains.

[0120] 3. Induced expression and purification of recombinant protein

[0121] Take the recombinant strain expressing three proteins and streak culture, pick out single clones, inoculate into LB broth medium (Kan+), shake and culture at 37℃ with 220rpm overnight, and inoculate into 100mL culture medium at a ratio of 1%. Culture until OD 600When the pH is around 0.6, add IPTG to a final concentration of 1 mM. Induce expression at 37°C for 6 hours and harvest the cells. Centrifuge the collected bacterial solution, remove the pellet, and resuspend it in sterile PBS (1:10 ratio). Add 20 μL of the bacterial solution to 1 / 4 of the volume in 5× loading buffer (final concentration of 1×), boil for 15 minutes, and analyze on SDS-PAGE gel.

[0122] The remaining resuspended bacterial solution was centrifuged, washed twice with PBS solution, and resuspended. The suspension was broken into clarification using an ultrasonic disruptor in an ice bath. After centrifugation, the supernatant was collected and filtered with a 0.45 μm filter membrane for later use. The recombinant protein was purified by affinity chromatography. After determining the purification conditions by exploring the imidazole concentration, impurities were eluted with a buffer containing 40 mM imidazole, and the target protein was eluted with a buffer containing 400 mM imidazole to obtain high-quality purified protein. The recombinant protein was lyophilized after gradient dialysis, and the lyophilized protein was resuspended in inactivated PBS to obtain the recombinant proteins SLP (i.e., the protein encoded by the full-length sequence of Slr4-like), SLPn (i.e., the protein encoded by the N-terminal half domain sequence of Slr4-like), and SLPc (i.e., the protein encoded by the C-terminal half domain sequence of Slr4-like).

[0123] The amino acid sequences of SLP, SLPn, and SLPc are SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. The molecular weights of the three recombinant proteins are predicted to be 48.7 kDa, 26.2 kDa, and 26.0 kDa, respectively. The predicted structure of the SLP protein is as follows: Figure 5 The concentration of recombinant protein was determined by commercial BCA protein quantification kit and analyzed by SDS-PAGE (see Figure 6 ) The protein size and purity were analyzed. The size of the recombinant protein was consistent with the prediction and the purity was greater than 90%. The protein concentration was adjusted to 1 mg / mL and stored at -80°C after aliquoting.

[0124] VVDFDAASPAVPKFASEIKFDATNGTTLTNAGNILDVKSKVGFSISQNNQRYVRFDITGAEFGANGAITLAGASGNFVSTVSSSGKTFVIFEIKDPNQATLADAVLTYPLNIKVKAGSTVNLTYKLFETAVAAVNNDPAQALYTKSGAVATFTPALLAKKVDTTPTKIDVTLESKKFVGGGVTNTVGKVAIGAATDVLWTTGVQTVMGDLVAAGSKVEVTADLSAGKKQAADATKVDVTALKLNGVDATAIDSTKATFDLVAALGDVNTDPATTVDLVFEADGKTAMNETTFAAKYLVTASATATTGDVAIGDLSTVTKNGSTKEANLVLAPDTSYTNMVRISNTSTIAGKFFITVIADGKSVTFALSDVAGQPASLDAGASTKQMKVADIYAAAAKGLALTGDKKLRLKVEGEVGSLSLQNYTISKDGNALNTMNAF(SEQ ID NO:6).

[0125] VVDFDAASPAVPKFASEIKFDATNGTTLTNAGNILDVKSKVGFSISQNNQRYVRFDITGAEFGANGAITLAGASGNFVSTVSSSGKTFVIFEIKDPNQATLADAVLTYPLNIKVKAGSTVNLTYKLFETAVAAVNNDPAQALYTKSGAVATFTPALLAKKVDTTPTKIDVTLESKKFVGGGVTNTVGKVAIGAATDVLWTTGVQTVMGDLVAAGSKVEVT(SEQ ID NO:7).

[0126] ADLSAGKKQAADATKVDVTALKLNGVDATAIDSTKATFDLVAALGDVNTDPATTVDLVFEADGKTAMNETTFAAKYLVTASATATTGDVAIGDLSTVTKNGSTKEANLVLAPDTSYTNMVRISNTSTIAGKFFITVIADDGKSVTFALSDVAGQPASLDAGASTKQMKVADIYAAAEAGGLALTGDKKLRLKVEGEVGSLSLQNYTISKDGNALNTMNAF(SEQ ID NO:8).

[0127] Example 3 Aeromonas Schubert Antibody Negative Serum and Quality Control

[0128] 1. Preparation of negative serum for Aeromonas Schubert antibodies

[0129] Fifty healthy hybrid snakehead fish weighing 80-100 grams were selected. The fish were required to be pathogen-free throughout the entire culture process, with no detection of susceptible pathogens such as Aeromonas schuberti, Nocardia sphenobarbus, or rhabdovirus. Blood was collected from the tail vein of each fish, allowed to rest for 24 hours, and then centrifuged to separate the serum. Antibody titers were assessed using an ELISA test. Serum samples without an immune response were designated as negative for Aeromonas schuberti antibodies. The samples were then pooled, aliquoted, and stored at -80°C until further use.

[0130] 2. Aeromonas Schubert antibody test quality control product

[0131] The specificity of the ELISA detection method for Aeromonas schubertii antibodies of the present invention was evaluated using sera positive for antibodies to four common pathogens in hybrid snakehead culture as quality control products.

[0132] Quality control preparation and testing methods: Cultures of Nocardia scabra, Flavobacterium columnaris, hybrid snakehead rhabdovirus, and neural necrosis virus were separately prepared. After inactivation and quality control, the bacteria or viruses were emulsified with ISA201 adjuvant to prepare the respective vaccines. Fifty healthy hybrid snakehead fish weighing 80-100 g were selected and immunized intraperitoneally with the prepared inactivated vaccine, 100 μL per fish, three times with 15-day intervals between immunizations. Seven days after the last immunization, blood was collected from the caudal vein of all immunized fish, and antibody titers were assessed using a neutralization test or latex agglutination test. Serum samples with high titers of antibacterial and antiviral antibodies (antibody titers greater than 8 in virus neutralization or bacterial agglutination tests) were selected as quality controls and stored at -80°C until needed.

[0133] Example 4 ELISA reactivity of three recombinant proteins

[0134] An indirect ELISA method was used to detect the immunological reaction between the serum antibodies of hybrid snakehead fish immunized with Aeromonas Schubert and the three recombinant proteins (prepared in Example 2). The ELISA assay was performed on the solid-phase coated substrates SLP, SLPn, and SLPc. The specific operation was carried out as follows:

[0135] (1) Coating protein: dilute the three recombinant proteins to 8, 4, and 2 μg / mL (in 0.5 M carbonate buffer) respectively, coat the polystyrene reaction plate at 100 μL / well, incubate at 37°C for 1 h and 4°C overnight. The next day, add washing solution at 200 μL / well, wash three times, and pat dry.

[0136] (2) Blocking: Add 200 μL / well of PBS solution containing 5% BSA and block at 37°C for 2 h. Discard the blocking solution. Add 200 μL / well of washing solution and wash three times in succession. Pat dry.

[0137] (3) Add serum samples: Dilute the positive and negative serum for Aeromonas Schubert antibodies prepared in Example 3 at a 1:40 ratio, add 100 μL / well, and incubate at 37°C for 30 min. Add 200 μL / well of washing solution, wash three times, and pat dry.

[0138] (4) Incubation with primary antibody: Add mouse anti-hybrid snakehead IgM monoclonal antibody diluted 1:4000 in primary antibody diluent to each well as the primary antibody, 100 μL / well, and incubate at 37°C for 50 min. Add washing solution at 200 μL / well, wash three times in succession, and pat dry;

[0139] (5) Incubation with secondary antibody: HRP-labeled goat anti-mouse IgG was added to each well, diluted with secondary antibody diluent to 1:10,000, 100 μL / well, and incubated at 37°C for 45 min; washing solution was added at 200 μL / well, washed 4 times continuously, and patted dry;

[0140] (6) Color development: Add 100 μL of TMB color development solution to each well and develop the color for 15 min at room temperature (20-25°C, the same below) in the dark. After color development, add 50 μL of stop solution to each well to terminate the reaction.

[0141] (7) Reading: Read OD in a microplate reader 450nm The absorbance value of

[0142] (8) Calculate the positive serum OD 450nm Value / negative serum OD 450nm , that is, the P / N value.

[0143] According to the above ELISA method, the three recombinant proteins were evaluated for strong positive results with the antibodies of Aeromonas Schubert (serum diluted 40 times, OD 450nm value around 1.0) and weakly positive (serum diluted 40 times, OD 450nm The reactivity of the two serum types (values ​​between 0.3 and 0.6) was evaluated to screen for the optimal solid-phase coating. During the experiment, blank control wells without serum were set up. The results are shown in Table 1.

[0144] Table 1 ELISA test results of different antigens and different sera

[0145]

[0146] The results showed that among the three recombinant proteins, SLPn showed the best ELISA reactivity with both strong and weakly positive sera for Aeromonas schuberti antibodies, with a higher P / N ratio and good specificity. Therefore, SLPn was selected as the solid-phase coating for the detection of Aeromonas schuberti antibodies.

[0147] Example 5 ELISA kit for detecting antibodies to Aeromonas Schubert

[0148] An ELISA kit for detecting Aeromonas schubertii antibodies comprises a coated plate (i.e., an ELISA plate coated with SLPn prepared in Example 2), a capture antibody (mouse anti-hybrid snakehead IgM monoclonal antibody), an enzyme-labeled secondary detection antibody (HRP-labeled goat anti-mouse IgG), a washing solution, a substrate developer, a stop solution, a positive standard (i.e., the Aeromonas schubertii antibody detection quality control product prepared in Example 2), and a negative standard (i.e., the Aeromonas schubertii antibody-negative serum prepared in Example 2).

[0149] The above-mentioned coated plate was prepared as follows: SLPn protein was diluted to 8 μg / mL with 0.5 M carbonate buffer, 0.1 mL was added to each well of the ELISA plate, and the plate was coated at 4°C for 15 h; 200 μL of blocking solution was added to each well, the plate was blocked at room temperature for 2 h, the blocking solution was discarded, and the plate was dried at room temperature for 2 h; the coated plate was obtained, and the coated plate and desiccant were placed in an aluminum foil bag, vacuum-sealed, and stored for later use.

[0150] Example 6 Construction of indirect ELISA detection method for specific antibodies to Aeromonas Schubert

[0151] 1. Optimal concentrations of antigens and serum antibodies

[0152] The ELISA solid phase is coated with different concentrations of protein antigen, and a gradient dilution of positive serum is used as the sample. Mouse anti-hybrid snakehead IgM monoclonal antibody and HRP-labeled goat anti-mouse IgG dilution are fixed. The optimal antigen concentration and serum sample dilution are determined by the checkerboard titration method. The specific steps are as follows:

[0153] (1) Coating protein: SLPn was diluted to 16, 8, 4, 2, 1, and 0.5 μg / mL, respectively, and coated on polystyrene reaction plates at 100 μL / well. The plates were incubated at 37°C for 1 h and at 4°C overnight. The next day, washing solution was added at 200 μL / well, and the plates were washed three times in succession and patted dry.

[0154] (2) Blocking: Add 200 μL / well of PBS solution containing 5% BSA and block at 37°C for 2 h. Discard the blocking solution. Add 200 μL / well of washing solution and wash three times in succession. Pat dry.

[0155] (3) Add serum samples: dilute positive and negative serum at 1:20, 1:40, 1:80, and 1:160, add 100 μL / well, and incubate at 37°C for 30 min. Add 200 μL / well of wash buffer, wash three times, and pat dry.

[0156] (4) Incubation with primary antibody: Add mouse anti-hybrid snakehead IgM monoclonal antibody diluted 1:4000 in primary antibody diluent to each well as the primary antibody, 100 μL / well, and incubate at 37°C for 50 min. Add washing solution at 200 μL / well, wash three times in succession, and pat dry;

[0157] (5) Incubation with secondary antibody: HRP-labeled goat anti-mouse IgG was added to each well, diluted with secondary antibody diluent at 1:10,000, 100 μL / well, and incubated at 37°C for 45 min; washing solution was added at 200 μL / well, washed 4 times continuously, and patted dry;

[0158] (6) Color development: Add 100 μL of TMB color development solution to each well and develop the color for 15 min at room temperature in the dark. After color development, add 50 μL of stop solution to each well to terminate the reaction.

[0159] (7) Reading: Read OD in a microplate reader 450nm The absorbance value of

[0160] (8) Calculate the positive serum OD 450nm Value / negative serum OD 450nm , also known as the P / N ratio, was used to determine the optimal dilution of the antigen and serum samples. During the experiment, a blank control without sample was set up. The results are shown in Tables 2 and 3.

[0161] Table 2 ELISA test results of serum with different concentrations of antigen and different dilution ratios

[0162]

[0163]

[0164] Table 3 P / N values ​​of ELISA reactions

[0165]

[0166] The results showed that according to the P / N value analysis of the test results, the maximum P / N value was when the recombinant protein SLPn coating concentration was 8μg / mL and the serum dilution multiple was 80 times, which were the optimal conditions for the two.

[0167] 2. Optimal concentrations of primary and secondary antibodies

[0168] The optimal dilutions of the primary and secondary antibodies were determined using a checkerboard titration method. An ELISA plate was coated with 8 μg / mL of SLPn, and 80-fold diluted positive or negative serum was added. Different concentration gradients of mouse anti-hybrid snakehead IgM monoclonal antibody (primary antibody) and HRP-labeled goat anti-mouse IgG (secondary antibody) were set up. The optimal concentration of the antibodies was determined based on the P / N value. The specific operation was carried out as follows:

[0169] (1) Coating protein: Dilute SLPn to 8 μg / mL and coat the polystyrene reaction plate at 100 μL / well. Incubate at 37°C for 1 h or 4°C overnight. The next day, add washing solution at 200 μL / well, wash three times, and pat dry.

[0170] (2) Blocking: Add 200 μL / well of PBS solution containing 5% BSA and block at 37°C for 2 h. Discard the blocking solution; add 200 μL / well of washing solution, wash three times in a row, and pat dry.

[0171] (3) Add serum samples: dilute positive and negative serum at 1:80, add 100 μl / well, and incubate at 37°C for 30 min. Add 200 μl / well of wash buffer, wash three times, and pat dry.

[0172] (4) Incubation with primary antibody: Add mouse anti-hybrid snakehead IgM monoclonal antibody at dilutions of 1:1000, 1:2000, 1:4000, and 1:8000, respectively, 100 μL / well, and incubate at 37°C for 50 min. Add washing solution at 200 μL / well, wash three times, and pat dry.

[0173] (5) Incubation with secondary antibody: add HRP-labeled goat anti-mouse IgG at dilutions of 1:10,000, 1:15,000, 1:20,000, and 1:25,000, respectively, 100 μL / well, and incubate at 37°C for 45 min; add washing solution at 200 μL / well, wash four times continuously, and pat dry;

[0174] (6) Color development: Add 100 μL of TMB color development solution to each well and develop the color for 15 min at room temperature in the dark. After color development, add 50 μL of stop solution to each well to terminate the reaction.

[0175] (7) Reading: Read OD in a microplate reader 450nm The absorbance value of

[0176] (8) Calculate the positive serum OD 450nm Value / negative serum OD 450nm , i.e., the P / N value, was used to determine the optimal dilution of the primary and secondary antibodies. The results are shown in Tables 4 and 5.

[0177] Table 4 ELISA test results of the first and second antibodies at different dilutions

[0178]

[0179] Table 5 P / N values ​​of ELISA reactions

[0180]

[0181] The results showed that according to the P / N value analysis of the test results, the optimal concentration of mouse anti-hybrid snakehead IgM monoclonal antibody (first antibody) was 1:4000, and the optimal concentration of HRP-labeled goat anti-mouse IgG (secondary antibody) was 1:15000.

[0182] 3. Optimal incubation conditions

[0183] Based on the optimization results, in the Aeromonas Schubert antibody test, an ELISA plate was coated with 8 μg / mL of SLPn, and 80-fold dilutions of positive and negative sera were added. Mouse anti-hybrid snakehead IgM monoclonal antibody (primary antibody) was diluted 1:4000, and commercial HRP-labeled goat anti-mouse IgG (secondary antibody) was diluted 1:8000. This experiment will further optimize the optimal incubation conditions (temperature and time) for the test sera, primary antibody, and secondary antibody.

[0184] 3.1 Optimization of serum incubation conditions

[0185] The specific operation is as follows:

[0186] (1) Coating protein: Dilute SLPn to 8 μg / mL and coat the polystyrene reaction plate at 100 μL / well. Incubate at 37°C for 1 h or 4°C overnight. The next day, add washing solution at 200 μL / well, wash three times, and pat dry.

[0187] (2) Blocking: Add 200 μL / well of PBS solution containing 5% BSA and block at 37°C for 2 h. Discard the blocking solution; add 200 μL / well of washing solution, wash three times in a row, and pat dry.

[0188] (3) Add serum samples: dilute positive and negative serum at 1:80 and add 100 μL / well. Set the incubation conditions as follows: ① 37°C, 50 min; ② 37°C, 30 min; ③ room temperature (20-25°C), 50 min; ④ room temperature, 30 min. Add washing solution at 200 μL / well, wash three times in a row, and pat dry.

[0189] (4) Incubation with primary antibody: Add 1:4000 diluted mouse anti-hybrid snakehead IgM monoclonal antibody, 100 μL / well, and incubate at 37°C for 50 min. Add 200 μL / well of washing solution, wash three times in a row, and pat dry.

[0190] (5) Incubation with secondary antibody: Add 1:15000 diluted HRP-labeled goat anti-mouse IgG, 100 μL / well, incubate at 37°C for 45 min; add washing solution at 200 μL / well, wash 4 times in succession, and pat dry;

[0191] (6) Color development: Add 100 μL of TMB color development solution to each well and develop the color for 15 min at room temperature in the dark. After color development, add 50 μL of stop solution to each well to terminate the reaction.

[0192] (7) Reading: Read OD in a microplate reader 450nm The absorbance value of

[0193] (8) Calculate the positive serum OD 450nm Value / negative serum OD 450nm , i.e., the P / N ratio, was used to determine the optimal incubation conditions for the serum samples. A blank control without sample was included in the experiment. The results are shown in Tables 6 and 7.

[0194] Table 6 ELISA test results of different incubation conditions for serum samples to be tested

[0195]

[0196] Table 7 P / N values ​​of ELISA reactions

[0197]

[0198] The results showed that according to the P / N value analysis of the test results, when the P / N was the largest, the incubation conditions of the serum sample to be tested were 37°C, 30 min or room temperature for 30 min. Taking into account the negative serum background value, the preferred incubation conditions for the serum sample to be tested were room temperature for 30 min.

[0199] 3.2 Optimization of incubation conditions for mouse anti-hybrid snakehead IgM monoclonal antibody (primary antibody)

[0200] The specific operation is as follows:

[0201] (1) Coating protein: Dilute SLPn to 8 μg / mL and coat the polystyrene reaction plate at 100 μL / well. Incubate at 37°C for 1 h or 4°C overnight. The next day, add washing solution at 200 μL / well, wash three times, and pat dry.

[0202] (2) Blocking: Add 200 μL / well of PBS solution containing 5% BSA and block at 37°C for 2 h. Discard the blocking solution; add 200 μL / well of washing solution, wash three times in a row, and pat dry.

[0203] (3) Add serum samples: dilute positive and negative serum at 1:80, add 100 μL / well, and incubate at room temperature for 30 min. Add 200 μL / well of washing solution, wash three times, and pat dry.

[0204] (4) Incubation with primary antibody: Add 1:4000 diluted mouse anti-hybrid snakehead IgM monoclonal antibody, 100 μL / well; set incubation conditions as ①37°C, 50 min, ②37°C, 30 min, ③room temperature, 50 min, ④room temperature, 30 min. Add washing solution at 200 μL / well, wash three times in a row, and pat dry;

[0205] (5) Incubation with secondary antibody: Add 1:15000 diluted HRP-labeled goat anti-mouse IgG, 100 μL / well, incubate at 37°C for 45 min; add washing solution at 200 μL / well, wash 4 times in succession, and pat dry;

[0206] (6) Color development: Add 100 μL of TMB color development solution to each well and develop the color for 15 min at room temperature in the dark. After color development, add 50 μL of stop solution to each well to terminate the reaction.

[0207] (7) Reading: Read OD in a microplate reader 450nm The absorbance value of

[0208] (8) Calculate the positive serum OD 450nm Value / negative serum OD 450nm , also known as the P / N ratio, was used to determine the optimal incubation conditions for the primary antibody. A blank control was established without sample addition. The results are shown in Tables 8 and 9.

[0209] Table 8 ELISA test results of different incubation conditions of the first antibody

[0210]

[0211]

[0212] Table 9 P / N values ​​of ELISA reactions

[0213]

[0214] The results showed that according to the P / N value analysis of the test results, when the P / N was the largest, the optimal incubation conditions for mouse anti-hybrid snakehead IgM monoclonal antibody were 37°C and 50 min.

[0215] 3.3 Optimization of incubation conditions for HRP-labeled goat anti-mouse IgG (secondary antibody)

[0216] The specific operation is as follows:

[0217] (1) Coating protein: Dilute SLPn to 8 μg / mL and coat the polystyrene reaction plate at 100 μL / well. Incubate at 37°C for 1 h or 4°C overnight. The next day, add washing solution at 200 μL / well, wash three times, and pat dry.

[0218] (2) Blocking: Add 200 μL / well of PBS solution containing 5% BSA and block at 37°C for 2 h. Discard the blocking solution; add 200 μL / well of washing solution, wash three times in a row, and pat dry.

[0219] (3) Add serum samples: dilute positive and negative serum at 1:80, add 100 μL / well, and incubate at room temperature for 30 min. Add 200 μL / well of washing solution, wash three times, and pat dry.

[0220] (4) Incubation with primary antibody: Add 1:4000 diluted mouse anti-hybrid snakehead IgM monoclonal antibody, 100 μL / well, and incubate at 37°C for 50 min. Add 200 μL / well of washing solution, wash three times in a row, and pat dry.

[0221] (5) Incubation with secondary antibody: Add 1:15000 diluted HRP-labeled goat anti-mouse IgG, 100 μL / well; set incubation conditions as ①37°C, 45 min, ②37°C, 30 min, ③room temperature, 45 min, ④room temperature, 30 min. Add washing solution at 200 μL / well, wash four times in a row, and pat dry;

[0222] (6) Color development: Add 100 μL of TMB color development solution to each well and develop the color for 15 min at room temperature in the dark. After color development, add 50 μL of stop solution to each well to terminate the reaction.

[0223] (7) Reading: Read OD in a microplate reader 450nm The absorbance value of

[0224] (8) Calculate the positive serum OD 450nm Value / negative serum OD 450nm , i.e., the P / N ratio, was used to determine the optimal incubation conditions for the secondary antibody. A blank control was set up without sample. The results are shown in Tables 10 and 11.

[0225] Table 10 ELISA test results of different incubation conditions of the second antibody

[0226]

[0227] Table 11 P / N values ​​of ELISA reactions

[0228]

[0229] The results showed that according to the P / N value analysis of the test results, when the P / N was the largest, the optimal incubation conditions for HRP-labeled goat anti-mouse IgG were 37°C, 45 min, 37°C, 30 min or room temperature for 45 min; considering the detection time, the preferred incubation conditions for the second antibody were 37°C, 30 min.

[0230] 4. Construction of an indirect ELISA method for detecting specific antibodies against Aeromonas Schubert

[0231] Based on the above parameter exploration, the operation and determination method of the indirect ELISA detection method for specific antibodies to Schubert's Aeromonas are as follows:

[0232] (1) Solid phase coating: SLPn was diluted to 8 μg / mL and coated on a polystyrene reaction plate at 100 μL / well. The plate was incubated at 37°C for 1 h and then at 4°C overnight. The next day, washing solution was added at 200 μL / well, and the plate was washed three times in succession. The plate was then patted dry.

[0233] (2) Blocking: Add 200 μL / well of PBS solution containing 5% BSA and block at 37°C for 2 h. Discard the blocking solution; add 200 μL / well of washing solution, wash three times in a row, and pat dry.

[0234] (3) Add the serum samples to be tested: dilute the serum to be tested, positive serum, and negative serum at a ratio of 1:80, add 100 μL / well, and incubate at room temperature for 30 minutes. Add 200 μL / well of washing solution, wash three times in a row, and pat dry.

[0235] (4) Incubation with primary antibody: Add 1:4000 diluted mouse anti-hybrid snakehead IgM monoclonal antibody, 100 μL / well, and incubate at 37°C for 50 min. Add 200 μL / well of washing solution, wash three times in a row, and pat dry.

[0236] (5) Incubation with secondary antibody: Add 1:15000 diluted HRP-labeled goat anti-mouse IgG, 100 μL / well, incubate at 37°C for 30 min; add 200 μL / well of washing solution, wash 4 times in a row, and pat dry;

[0237] (6) Color development: Add TMB color development solution (100 μL / well) and develop at room temperature in the dark for 15 min. After color development, add 50 μL of stop solution to each well to terminate the reaction.

[0238] (7) Reading: Read OD in a microplate reader 450nm The absorbance value of

[0239] (8) Result determination method: Calculate the OD of positive serum 450nm Value / negative serum OD 450nm , that is, P / N value; OD of the sample to be tested 450nm Value / negative serum OD 450nm , that is, the S / N value.

[0240] ①When P / N>3, the test result is valid; ②When S≥2.1×N, it is judged as positive.

[0241] Example 7 Specificity Experiment

[0242] The optimized indirect ELISA method for detecting specific antibodies to Aeromonas Schubert (Example 6) was used to test for antibodies against (1) Nocardia serrata, (2) Flavobacterium columnaris, (3) Hybrid snakehead rhabdovirus, and (4) Neuronecrosis virus to evaluate the specificity of the assay. A blank control was used as a control without sample. The results are shown in Table 12 below.

[0243] Table 12 ELISA detection specificity analysis

[0244]

[0245]

[0246] The above results show that, except for the serum positive for Aeromonas schubertii antibodies, the coating protein does not react with other common pathogens of hybrid snakehead. The results indicate that the indirect ELISA detection method for Aeromonas schubertii-specific antibodies in Example 6 has good specificity and can be used for the specific detection of Aeromonas schubertii.

[0247] Example 8 Sensitivity Test

[0248] According to the experimental conditions of the optimized indirect ELISA method for detecting specific antibodies to Aeromonas schubertii (Example 6), the serum positive for Aeromonas schubertii (Example 3) was tested. The serum to be tested was diluted at 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, and 1:5120. The results are shown in Table 13.

[0249] Table 13 Sensitivity test results

[0250]

[0251] Results: When the serum to be tested was diluted at 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280, the test results were judged to be positive; when the serum to be tested was diluted at 1:2560 and 1:5120, the results were judged to be negative, indicating that the sensitivity of the indirect ELISA detection method for the detection of hybrid snakehead serum by the Schubert's Aeromonas specific antibody established in Example 6 is 1:1280.

[0252] Example 9 Repeatability Experiment

[0253] Three batches of the kit (laboratory product) were assembled according to Example 5. Eight hybrid snakehead serum samples (clear background) were tested using the optimized indirect ELISA method for Aeromonas Schubert-specific antibodies from Example 6. Three replicates were set for each serum sample. The test results were statistically analyzed to assess both intra- and inter-batch reproducibility of the kit, expressed as the coefficient of variation (CV). The purified proteins used in this method were from the same batch. The results are shown in Table 14.

[0254] Table 14 Repeatability test results

[0255]

[0256] The results showed that the maximum coefficient of variation of the kit test within the batch was 4.3%, which was lower than 5%; the maximum coefficient of variation of the inter-batch test was 6.9%, which was lower than 10%, indicating that the kit of Example 5 has good repeatability and stability, and also that the indirect ELISA detection method for the specific antibody to Aeromonas Schubert of Example 6 has good repeatability and stability.

[0257] Example 10 Practical Application Case

[0258] 100 healthy hybrid snakeheads (30±5g) were randomly selected and divided into 2 groups on average. They were temporarily raised in two 1000L buckets and marked as Group 1 and Group 2. The inactivated Schubert's Aeromonas was used as the antigen and the ISA 201 adjuvant was emulsified to prepare the vaccine. The hybrid snakeheads in Group 1 were injected intraperitoneally at 100μl / tail. The hybrid snakeheads in Group 2 were used as blank controls and fed normally. 28 days after immunization, blood was collected from the tail veins of the two groups of fish. After serum separation, the hybrid snakehead serum samples were detected using the indirect ELISA detection method for Schubert's Aeromonas specific antibodies established in Example 6. Three positive serum controls and three negative serum controls were set up, and 44 serum samples were tested in each group. The test results are shown in Tables 15 and Figure 7 shown.

[0259] Table 15 Results of serum sample Aeromonas Schubert antibody detection

[0260]

[0261] Note: In Table 15, columns 1 to 11 are for the testing of two groups of serum samples, and column 12 is for the positive controls (A, B, C) and negative controls (E, F, G).

[0262] The test results showed that the positive control serum OD 450nm The average value was 0.807±0.016, and the negative control serum OD 450nmThe average value was 0.085±0.006, and the cut-off value for positive and negative results was 0.179. 450nm The average value was 0.407±0.140, of which 41 samples were judged to be serum antibody positive, accounting for 93.2%. 450nm The average value was 0.099±0.039, of which 42 samples were determined to be serum antibody negative, accounting for 95.5%. The T-test showed that the difference between the two was significant, indicating that the indirect ELISA detection method for specific antibodies to Aeromonas Schubert established in Example 6 can be used to evaluate the changes in the level of specific antibodies to Aeromonas Schubert in the serum of hybrid snakehead after immunization.

[0263] In summary, the indirect ELISA detection method for detecting Aeromonas schubertii antibodies constructed by the present invention has the advantages of strong specificity, good repeatability and high accuracy, and provides a detection method for the pathogenesis and immune prevention and control of Aeromonas schubertii in hybrid snakehead aquaculture.

[0264] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A protein, wherein the protein is any one of a1) to a3): a1) a protein with an amino acid sequence as shown in SEQ ID NO: 7; a2) a protein comprising SEQ ID NO: 7, wherein one or more amino acids are substituted and / or deleted and / or added, and which has the same or similar function as SEQ ID NO: 7; a3) A protein that has at least 95% homology to SEQ ID NO: 7 and has the same or similar function as SEQ ID NO:

7.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The biomaterial related to the nucleic acid molecule according to claim 2, characterized in that The biological material comprises at least one of b1) to b8): b1) The nucleic acid molecule according to claim 2; b2) an expression cassette containing the nucleic acid molecule described in b1); b3) a recombinant vector containing the nucleic acid molecule described in b1); b4) a recombinant vector containing the expression cassette described in b2); b5) a recombinant cell containing the nucleic acid molecule described in b1); b6) a recombinant cell containing the expression cassette described in b2); b7) a recombinant cell containing the recombinant vector described in b3); b8) A recombinant cell containing the recombinant vector described in b4).

4. Use of the protein according to claim 1, the nucleic acid molecule according to claim 2, or the biomaterial according to claim 3 in at least one of c1) to c6): c1) Detection of Aeromonas Schubert; c2) preparing a product for detecting Aeromonas schubertii; c3) detecting whether the sample to be tested is infected with Aeromonas schubertii; c4) preparing and testing whether the sample to be tested is infected with Aeromonas schubertii; c5) Evaluate the immune efficacy of the Aeromonas Schubert vaccine; c6) preparing a product for evaluating the immune effect of the Aeromonas Schubert vaccine; The above applications are for non-disease diagnosis.

5. A reagent or kit comprising the protein according to claim 1.

6. The reagent or kit according to claim 4, characterized in that The reagent or kit further comprises at least one of a capture antibody, an enzyme-labeled secondary antibody detection antibody, a solid phase carrier, a coating solution, a washing solution, a substrate color development solution, a stop solution, a positive standard substance, and a negative standard substance.

7. A method for detecting whether a sample is infected with Aeromonas schubertii for non-diagnostic purposes or a method for evaluating the immune effect of a vaccine against Aeromonas schubertii, comprising the step of detecting the sample using the protein of claim 1 or the reagent or kit of claim 5 or 6.

8. The method according to claim 7, characterized in that ELISA was used to detect the samples to be tested; Preferably, the ELISA includes direct ELISA, indirect ELISA, competitive ELISA, and double antibody ELISA sandwich method.

9. The method according to claim 8, characterized in that The method specifically comprises the following steps: coating: coating the protein according to claim 1 onto a solid phase carrier; Closed; Sample addition: Add the sample to be tested, incubate, and set up negative and positive controls at the same time; Primary antibody reaction: adding the capture antibody described in claim 6 and incubating; Secondary antibody reaction: adding the enzyme-labeled secondary antibody detection antibody described in claim 6 and incubating; Color development; Stop: Add stop solution and measure OD 450 nm value; Result determination: OD of the sample to be tested 450nm Value / negative serum OD 450nm ≥2.1 is positive, otherwise it is negative.

10. The method according to claim 9, characterized in that The concentration of the protein coating is 0.5-20 μg / mL; and / or the effective concentration of the capture antibody is 1:(1000-8000); and / or, the working concentration of the enzyme-labeled secondary detection antibody is 1:(10,000-30,000); and / or, the incubation condition of the sample to be tested is 20-40° C. for 30-50 min; and / or, the incubation condition in the primary antibody reaction is 20-40° C. for 30-70 min; And / or, the incubation condition in the secondary antibody reaction is 20-40° C. for 30-50 min.

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