Universal indirect ELISA antibody detection kit for four pathogenic bacteria of equine abortion Salmonellosis and its application

Abstract

The present invention discloses a universal indirect ELISA antibody detection kit for four pathogenic bacteria of equine abortion salmonellosis and its application. The kit contains an enzyme labeling plate coated with a truncated expressed Salmonella outer membrane recombinant protein OmpA, and the amino acid sequence of the truncated expressed Salmonella outer membrane recombinant protein is shown in SEQ ID NO.3. The present invention successfully screened the common dominant antigens of the four pathogens of equine abortion salmonellosis using the pull-down technology, cloned them into an expression vector, and achieved soluble expression of the OmpA protein through truncated segmented expression. At the same time, a universal indirect ELISA antibody detection kit for the four pathogenic bacteria of equine abortion salmonellosis and a corresponding iELISA antibody diagnostic method were established. Experiments have shown that the method established using the kit has the advantages of strong specificity and high sensitivity, and will become an effective tool for antibody detection of equine abortion salmonellosis, and also lay a methodological foundation for antibody monitoring in future vaccine development.

Description

Technical Field

[0001] The present invention relates to an ELISA antibody detection kit and its application, and in particular to a universal indirect ELISA antibody detection kit capable of detecting four pathogenic bacteria of equine abortion salmonellosis and its application. The present invention belongs to the field of medical technology. Background Art

[0002] Salmonellosis is a major bacterial disease that causes significant economic losses to the pig, poultry, pigeon, horse, and donkey industries. Equine abortus salmonellosis, also known as equine paratyphoid, is an infectious disease in equine species characterized by abortion caused by various bacteria, including Salmonella abortus equi (S. abortusequi), Salmonella typhimurium (S. typhi), Salmonella dublin, and Salmonella enteritidis. Among them, Salmonella equi abortus is the most common pathogen. It has been previously reported that Salmonella typhimurium alone or mixed with Salmonella equi abortus can cause abortion and diarrhea in donkey foals (Cui Silie, Li Chunsheng. Investigation and research report on Salmonella typhimurium infection in donkey foals. Journal of Veterinary Science and Technology, 1983, (02): 10-16). However, abortion in horses and donkeys caused by Salmonella Dublin and Salmonella Enteritidis is rare and has rarely been reported.

[0003] Regarding the serological methods for diagnosing equine Salmonella abortions, there are mainly tube agglutination tests, microagglutination tests, ELISA, etc. The team of the inventors previously established a microagglutination test (MAT) for equine Salmonella abortions and used the microagglutination test method to test 151 horse serum samples. The positive detection rate of equine Salmonella abortions antibodies in the abortion outbreak area was higher than that of the tube agglutination test method, so it has higher sensitivity; foreign researchers used lipopolysaccharide (LPS) to establish the iELISA antibody detection method for equine Salmonella abortions antibody detection, which has good sensitivity, but the specificity still needs further evaluation.

[0004] Therefore, there is currently no universal ELISA serological diagnostic method for equine abortions Salmonellosis. The traditional tube agglutination test has low sensitivity. Although the sensitivity of the microagglutination test has been improved, the agglutination phenomenon itself is limited by the naked eye, which limits the further improvement of the sensitivity. Therefore, there is an urgent need to establish a universal ELISA method with high sensitivity and good specificity that can simultaneously detect antibodies to equine abortions Salmonella, Salmonella typhimurium, Salmonella dublin, and Salmonella enteritidis. Summary of the Invention

[0005] The present invention aims to provide a universal indirect ELISA antibody detection kit capable of detecting four pathogenic bacteria of equine Salmonellosis (Salmonella abortus, Salmonella typhimurium, Salmonella dublin and Salmonella enteritidis) and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] Obtaining in vitro expressed candidate proteins is a prerequisite for establishing an indirect ELISA method for detecting serum antibodies. The present invention first uses positive and negative serum of Salmonella abortus equi and whole-cell antigens of Salmonella abortus equi to conduct immunoprecipitation (pulldown) experiments to screen out the dominant antigen of Salmonella abortus equi - Outer Membrane Protein A (OmpA). Sequence comparison shows that the amino acid sequence of the protein is 99.43-100% homologous to those of Salmonella typhimurium, Salmonella dublin, and Salmonella enteritidis of the same genus, and is highly conserved. Secondly, the present invention performs segmented expression of the dominant antigen encoding gene based on antigenicity analysis, designs three pairs of primers, uses polymerase chain reaction (PCR) to amplify the Salmonella dominant antigen gene, and clones them into the prokaryotic expression vector pET28a, thereby successfully constructing pET28a-ompA1, pET28a-ompA2, and pET28a-ompA3. Three recombinant plasmids. SDS-PAGE results showed that under 24°C and 0.6 mM IPTG induction for 5 h, the recombinant bacteria containing pET28a-ompA1, pET28a-ompA2, and pET28a-ompA3 were all expressed. The recombinant OmpA1 and OmpA2 proteins were expressed in the form of inclusion bodies, and the recombinant OmpA3 protein was expressed in a soluble form. Western blot analysis showed that the recombinant OmpA1 and OmpA2 proteins were expressed in the form of inclusion bodies, and the recombinant OmpA3 protein was expressed in a soluble form. Blot analysis showed that the recombinant protein OmpA3 reacted specifically with positive sera of Salmonella abortus, Salmonella typhimurium, Salmonella dublin, and Salmonella enteritidis, demonstrating that the recombinant OmpA3 protein has good reactivity and can be used as an antigen for detecting Salmonella antisera. An indirect ELISA (iELISA) method was established using OmpA3 protein as the coating antigen, and the optimal reaction conditions were determined as follows: the optimal coating antigen concentration was 1 μg / mL, the test serum (1:200) was incubated at 37°C for 1 hour, the enzyme-labeled anti-equine secondary antibody (1:10,000 dilution), and TMB was incubated at 37°C for 10 minutes. The test serum was considered positive when OD450>0.143. Specificity test results showed that the coating antigen had no cross-reaction with positive sera for common equine infectious diseases. Using serum samples from horses infected intravenously with Salmonella, the iELISA method demonstrated sustained antibody positivity for up to 116 days, 47 days longer than the microagglutination method (69 days), demonstrating its superior sensitivity. The established iELISA method was used to test 180 serum samples from eight different farms, yielding an average antibody positivity rate of 63.3%, 53.9% higher than the microagglutination method.

[0008] Based on the above research, the present invention proposes a universal indirect ELISA antibody detection kit for four pathogenic bacteria of equine abortion Salmonellosis. The kit contains an ELISA plate coated with truncated expressed Salmonella outer membrane recombinant protein A (OmpA). The amino acid sequence of the truncated expressed Salmonella outer membrane recombinant protein is shown in SEQ ID NO.3.

[0009] Among them, preferably, the four pathogenic bacteria are Salmonella abortusequi S. abortus equi, Salmonella typhimurium S. typhi, Salmonella dublin and Salmonella enteritidis.

[0010] Preferably, the coating antigen concentration of the truncated expressed Salmonella outer membrane recombinant protein A is 1 μg / mL, and 100 μl / well is used to coat a 96-well ELISA plate at 4° C. overnight.

[0011] Among them, preferably, the kit further contains HRP-labeled anti-horse IgG secondary antibody, blocking solution, diluent, color developing solution and stop solution.

[0012] Among them, preferably, the blocking solution is 5% w / w skim milk, which is blocked at 37°C for 1.5 hours, the diluent is 5% w / w skim milk or 5% w / w BSA, the serum to be tested is diluted 1:200 with 5% w / w skim milk and incubated at 37°C for 1 hour, the HRP-labeled anti-horse IgG secondary antibody is diluted 1:10000 with 5% w / w BSA and incubated at 37°C for 30 minutes, and the color developing solution is TMB color developing solution, which is incubated at 37°C for 10 minutes.

[0013] Furthermore, the present invention also proposes the use of the kit in preparing reagents for detecting four pathogenic bacteria of equine abortion salmonellosis.

[0014] Among them, preferably, the four pathogenic bacteria are Salmonella abortusequi S. abortus equi, Salmonella typhimurium S. typhi, Salmonella dublin and Salmonella enteritidis.

[0015] Among them, preferably, the reagent is an indirect ELISA antibody detection reagent.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention successfully screened a common dominant antigen for four pathogens of equine abortion Salmonellosis using pull-down technology, clarified the detection specificity range of the antigen, cloned it into an expression vector, and achieved soluble expression of the OmpA protein through truncated segmented expression. A universal indirect ELISA antibody detection kit for the four pathogenic bacteria of equine abortion Salmonellosis and a corresponding OmpA iELISA antibody diagnostic method were established. Experiments have shown that the method established using the kit has the advantages of strong specificity and high sensitivity, and will become an effective tool for equine abortion Salmonellosis antibody detection, and also lay a methodological foundation for antibody monitoring in future vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Screening for dominant antigens of equine Salmonella abortus;

[0019] Wherein, M: protein molecular weight standard; 1: purified IgG antibody from equine Salmonella abortus positive serum; 2: purified IgG antibody from equine Salmonella abortus negative serum; 3: Protein A / G Magnetic Beads + equine Salmonella abortus positive serum IgG antibody + equine Salmonella abortus antigen complex; 4: Protein A / G Magnetic Beads + equine Salmonella abortus negative serum IgG antibody + equine Salmonella abortus antigen complex; 5: equine Salmonella abortus antigen.

[0020] Figure 2 Amplification of target genes;

[0021] Among them, M: DM2000 DNA Marker; 1-2: ompA1; 3-4: ompA2; 5-6: ompA3.

[0022] Figure 3 SDS-PAGE identification of OmpA protein;

[0023] Wherein, M: protein molecular weight standard; 1: supernatant after OmpA1 protein induction; 2: precipitate after OmpA1 protein induction; 3: supernatant after OmpA2 protein induction; 4: precipitate after OmpA2 protein induction; 5: supernatant after OmpA3 protein induction; 6: precipitate after OmpA3 protein induction.

[0024] Figure 4 Western blot identification of OmpA3 protein;

[0025] Wherein, M: protein molecular weight standard; 1: equi abortion Salmonella positive serum; 2: Salmonella typhimurium positive serum; 3: Salmonella dublin positive serum; 4: Salmonella enteritidis positive serum; 5: Salmonella negative serum.

[0026] Figure 5 Results of monitoring of antibodies to Salmonella abortus in the serum of infected and control horses.

[0027] Among them, A. micro agglutination test (MAT) test results; B. indirect ELISA (iELISA) test results. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the present invention, not all of the embodiments. All other embodiments derived by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are also within the scope of protection of the present invention.

[0029] Example 1

[0030] 1 Materials and Methods

[0031] The trial was conducted from March 2018 to March 2022 at the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, State Key Laboratory of Veterinary Biotechnology, by the Equine Infectious Diseases and Lentivirus Diseases Research Innovation Team.

[0032] 1.1 Plasmids, strains, and samples

[0033] Salmonella equi abortus strain 180316.H.AES.G (CGMCC No. 18341, documented in patent application CN111100817A, entitled "Salmonella equi abortus equine strain and its use in the preparation of an inactivated Salmonella equine vaccine") was isolated, identified, and provided by the Equine Infectious Diseases and Lentivirus Innovation Team of the Harbin Veterinary Research Institute. The prokaryotic expression host strain Rosetta (DE3) was purchased from TIANGEN, and the pET28a vector was maintained in our laboratory. 180 clinical samples were obtained from eight different ranches, and 130 Salmonella-negative sera and 58 sera from infected and control horses were prepared and maintained in our laboratory. Positive sera for equine infectious anemia virus (EIAV), equine influenza virus (EIV), equine herpes virus (EHV), equine arteritis virus (EAV), Theileria equi (T.equi), Babesia caballi (B.caballi), Streptococcus equi, Escherichia coli, Salmonella abortus, Salmonella typhimurium, Salmonella dublin, and Salmonella enteritidis, as well as negative sera for Salmonella, are preserved in this laboratory.

[0034] 1.2 Main Reagents

[0035] IPTG was purchased from Takara Biotechnology (Dalian) Co., Ltd.; DNAMarker DL 2000 and T4 DNA ligase were purchased from Shanghai Sangon Biotechnology Co., Ltd.; agarose was from Promega, rabbit serum positive for Salmonella equine abortus was purchased from the China Veterinary Drug Administration; and a bacterial genomic DNA extraction kit was purchased from TIANGEN. Protein A / G Magnetic Beads (MCE) and an HRP-conjugated anti-horse IgG secondary antibody were used.

[0036] 1.3 Screening of dominant antigens of Salmonella abortus equi

[0037] First, IgG antibodies were purified from Salmonella abortus equi positive and negative sera. 2 mL of each serum was filtered through a 0.45 μm filter. Antibody purification was performed according to the HiTrap Protein GHP (GE) instructions. Next, the Salmonella abortus equi antigen complex was prepared. In a biosafety cabinet, 8 mL of fresh logarithmic-phase Salmonella abortus equi culture was centrifuged at 10,000 rpm at 4°C for 2 minutes. The cells were then harvested and resuspended in 2 mL of sterile PBS, centrifuged, and the supernatant discarded. The cells were then washed thoroughly twice. The cells were then resuspended in 1.5-2 mL of sterile PBS and disrupted by sonication at 39% power, 3 seconds on, 5 seconds off, and 5 minutes on. Finally, the bacterial lysate was centrifuged at 10,000 rpm at 4°C for 2 minutes. The supernatant was collected and the protein concentration was determined using the BCA assay. The protein concentration was then diluted to 1 mg / mL for later use. Then, referring to the method published by Xu Zhichao (Xu Zhichao. Molecular mechanism of IFN-β production induced by bacterial ferritin, the dominant antigen of Salmonella pullorum. 2016:125), a pull-down test was performed on antibodies and antigens of Salmonella abortus equi; finally, the mass spectrometry results were analyzed.

[0038] 1.4 Cloning of the ompA gene

[0039] Based on the reference sequence from mass spectrometry analysis, the ompA gene was amplified using the following primers in a 20 μL reaction system: 2× Taq Master Mix, 10 μL; upstream and downstream primers, 1 μL; ddH₂O, 6 μL; and the Salmonella abortus genome as template, 2 μL. The reaction conditions were: initial denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s (30 s for stretches within 600 bp, 60 s for stretches between 600 and 1000 bp, and 90 s for stretches between 1000 and 1500 bp), for 35 cycles; and a final extension at 72°C for 10 min. After the PCR reaction, 7 μL of the PCR product was analyzed by 1% agarose gel electrophoresis. Specific DNA bands were purified and recovered according to the instructions of the Gel Extraction Kit. The pET28a vector and target fragment were double-digested with BamHI and XhoI, respectively, to recover and purify the gene and linearize pET-28a. The ompA gene and linearized pET-28a were ligated using T4 DNA ligase. The ligation product was transformed into Rosetta, plated onto kanamycin-resistant LB solid medium, and incubated at 37°C for 16 hours. Plasmids were extracted from positive clones, identified by PCR, and sent to Coomix for sequencing. Once sequenced correctly, the plasmids were designated pET28a-ompA1, pET28a-ompA2, and pET28a-ompA3.

[0040] Table 1 PCR amplification primers

[0041]

[0042] 1.5 Expression and purification of OmpA protein

[0043] The positive colonies of pET28a-ompA1, pET28a-ompA2, and pET28a-ompA3 were inoculated into 5 mL of fresh LB liquid medium containing 1 μg / mL Kanamycin (Kan) resistance, and cultured at 37°C and 170 r / min for 16 h. They were then inoculated into Kan / LB liquid medium at a ratio of 1:100 and cultured at 37°C and 170 r / min until the bacterial solution OD reached 0. 600nm When the pH is around 0.3-0.4, add IPTG to a final concentration of 0.6 mM and induce expression at 24°C. After induction, collect 4 mL of bacterial culture and add 1.5 mL of PBS for sonication at 38% power, 5 seconds on, 5 seconds off, for 2 minutes. After sonication, centrifuge at 12,000 rpm at 4°C for 5 minutes and reserve the supernatant. Rinse the pellet twice with an equal volume of PBS and resuspend in an equal volume of PBS. Take 40 μL of the supernatant and resuspended pellet, add 10 μL of 5x SDS-PAGE Loading Buffer to each, mix thoroughly, boil at 95°C for 5 minutes, and perform SDS-PAGE on a 4-12% or 12% gel. After electrophoresis, stain with Coomassie Brilliant Blue to analyze protein expression. The soluble protein was purified according to the experimental method of Liu Cong (Liu Cong. Establishment of horse NLRP3 activation system and its preliminary application in the study of EIAV infection regulation of inflammatory response [D]. Chinese Academy of Agricultural Sciences, 2019.), and the soluble protein OmpA3 was subjected to Western blot analysis. Western blot analysis was performed using equine Salmonella abortus, Salmonella typhimurium, Salmonella dublin, Salmonella enteritidis positive serum and Salmonella negative serum as primary antibodies (1:200 dilution) and HRP-labeled anti-horse IgG as secondary antibody (1:5000) to verify the reactivity and broad spectrum of the protein.

[0044] 1.6 Establishment of the OmpA indirect ELISA (OmpA iELISA) method

[0045] The optimal OmpA3 protein coating concentration and the dilution ratio of serum samples and enzyme-labeled secondary antibodies (HRP-labeled anti-horse IgG secondary antibodies) were determined using the checkerboard method. Recombinant ompA3 protein was diluted to 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL using phosphate buffer. 100 μl / well was coated on a 96-well microplate at 4°C overnight. Equine Salmonella abortus positive and negative sera were serially diluted 2-fold from 1:200 to 1:1600. The enzyme-labeled secondary antibody was diluted 1:8000, 1:10000, 1:12000, and 1:15000. Indirect ELISA was performed, TMB was added, and the OD was measured on a microplate reader for 10 minutes. 450nm Select the maximum P / N value (P / N is the OD value of positive serum and negative serum at the same dilution). 450nm value ratio), and meet the negative serum OD 450nm The optimal reaction conditions were the antigen coating concentration, serum dilution, and enzyme-labeled secondary antibody dilution when the concentration was less than 0.1. The established indirect ELISA method was used to detect 130 negative sera of Salmonella abortus in horses, and the OD values ​​of all negative sera were calculated. 450nm The mean (X) and standard deviation (SD) were calculated, and X+3SD was determined as the critical value for the method to detect positive and negative antibodies to Salmonella abortion in clinical equine samples.

[0046] 1.7 Specificity and sensitivity of OmpA iELISA

[0047] The established iELISA was used to detect positive sera for Salmonella abortus, equine infectious anemia virus, equine influenza viruses (H7N7 and H3N8), equine arteritis virus, equine herpesviruses (types I, II, III, IV, and VII), Theileria equi, Babesia spp., Streptococcus equi, Salmonella typhimurium, Salmonella dublin, and Salmonella enteritidis. Negative serum controls were also included, and duplicate wells were used to verify the specificity of the method. Positive sera for Salmonella abortus, Salmonella typhimurium, Salmonella dublin, and Salmonella enteritidis were serially diluted two-fold, and the sensitivity of the OmpA iELISA was evaluated for the four sera. The highest dilution at which the serum tested positive was defined as the assay titer.

[0048] 1.8OmpA iELISA monitors antibody levels in horse serum after infection with Salmonella abortus equi.

[0049] Salmonella-negative horses were intravenously infected with 10 billion / mL Salmonella abortus. OmpA antibody levels were monitored dynamically after infection. A control group of horses was injected with 1 mL of PBS in the same manner. Blood was collected before infection, daily for the first 12 days after infection, and then every other week. Antibody levels were also monitored using an OmpA iELISA with 58 sera from infected and control horses.

[0050] 1.9 Clinical application of the universal OmpA iELISA method for equine abortive Salmonellosis

[0051] The established OmpA iELISA method was used to test serum clinical samples from four abortion epidemic areas and four healthy area ranches, and the test results were compared with the MAT results. The MAT was operated with reference to the paper published by Guo Kui et al. (Guo Kui, Wang Ning, Wang Jinhui, Chu Xiaoyu, Zhao Yuting, Guo Wei, Liu Dihuo, Hu Zhe, Wang Xiaojun. Isolation and identification of equine abortion Salmonella and establishment and application of its microagglutination antibody detection method [J]. Chinese Agricultural Science, 2020, 53(10): 2112-2121.).

[0052] 2 Results

[0053] 2.1 Pull-down search for dominant antigens of Salmonella abortus equi

[0054] Through the pull-down test, compared with the control group of "Protein A / G Magnetic Beads+equine Salmonella abortions negative serum IgG antibody+equine Salmonella abortions antigen complex", the experimental group of "Protein A / G Magnetic Beads+equine Salmonella abortions positive serum IgG antibody+equine Salmonella abortions antigen complex" showed a specific band (such as Figure 1 , marked in the box). The specific pull-down band was sent to Beijing BGI Protein Research Center Co., Ltd. for mass spectrometry identification, which confirmed the band to be the OmpA protein. The dominant antigen identified through pull-down technology will lay the foundation for the development of a diagnostic kit for equine Salmonellosis abortus.

[0055] 2.2 Cloning of the Salmonella ompA gene

[0056] Using the three pairs of primers designed, ompA was amplified into fragments of different sizes ( Figure 2 ), digested with enzymes, ligated into pET28a, and transformed into Rosetta Stone. Sequencing confirmed the Salmonella ompA gene sequence. The three positive plasmids were named pET28a-ompA1, pET28a-ompA2, and pET28a-ompA3.

[0057] 2.3 Induced expression of target protein

[0058] The three expression bacteria containing pET28a-ompA1, pET28a-ompA2, and pET28a-ompA3 plasmids were induced at 24°C, and the induced bacteria were sonicated and then subjected to SDS-PAGE electrophoresis, Coomassie Brilliant Blue staining, and decolorization analysis. The results showed that OmpA1 and OmpA2 proteins were expressed in the form of inclusion bodies, and OmpA3 protein was expressed in a soluble form (such as Figure 3 ), the amino acid sequences of OmpA1, OmpA2 and OmpA3 proteins are shown in SEQ ID NO.1-3, respectively.

[0059] 2.4 Reactogenicity analysis of recombinant OmpA3 protein

[0060] Western blot analysis was performed using Salmonella abortus, Salmonella typhimurium, Salmonella dublin, Salmonella enteritidis positive and Salmonella negative sera. Figure 4 As shown, the results showed that the recombinant OmpA3 protein could react with the positive sera of Salmonella abortus, Salmonella typhimurium, Salmonella dublin and Salmonella enteritidis, confirming that the OmpA3 protein has good reactogenicity and conservation, and can be used as a universal antigen for the diagnosis of equine Salmonellosis.

[0061] 2.5 Determination of reaction conditions

[0062] By optimizing the various conditions of the OmpA iELISA method, the optimal reaction conditions were determined as follows: antigen 1 μg / mL was coated overnight at 4°C, 5% skim milk was blocked at 37°C for 1.5 hours, serum was diluted 1:200 (diluted with 5% skim milk) at 37°C for 1 hour, secondary antibody was diluted 1:10000 (diluted with 5% BSA) at 37°C for 30 minutes, and TMB color development was performed for 10 minutes (37°C).

[0063] 2.6 Determination of OmpA iELISA cut-off value

[0064] According to the test results of 130 Salmonella negative sera, the mean value (X = 0.097, and the standard deviation (SD = 0.018) were calculated by biological statistical analysis method. The critical value (X + 3SD) of positive and negative serum in clinical serum test was 0.143. 450nm A value greater than the critical value is considered positive, and a value lower than the critical value is considered negative.

[0065] 2.7 Specificity Identification Results

[0066] The established OmpA iELISA method was used to detect positive sera for equine Salmonella abortus, equine infectious anemia virus, equine influenza virus (H7N7, H3N8), equine arteritis virus, equine herpesvirus (type I, type II, type III, type IV, type VII), Streptococcus equi, Salmonella Typhimurium, Salmonella Dublin, and Salmonella Enteritidis. The results showed that only the positive sera for equine Salmonella abortus, Salmonella Typhimurium, Salmonella Dublin, and Salmonella Enteritidis tested positive, while the positive sera for other pathogens tested negative.

[0067] 2.8 Sensitivity identification results

[0068] The established OmpA iELISA method was used to determine the titer of standard positive sera of equine Salmonella abortions, Salmonella typhimurium, Salmonella dublin, and Salmonella enteritidis. The results showed that the titer of the positive sera of equine Salmonella abortions, Salmonella typhimurium, Salmonella dublin, and Salmonella enteritidis could reach 16.

[0069] 2.9 Antibody Monitoring in Serum of Artificially Infected Animals

[0070] Antibody detection was performed on infected horses and control horses using MAT and the established OmpA iELISA method. The MAT results showed that ( Figure 5 A) In the vein 10 10 When CFU / mL is used to infect horses, antibodies can be positive on the fourth day of MAT, reaching a peak on the 7th to 12th day. After 12 days, the antibody level gradually decreases and remains positive until the 69th day, after which the antibody turns negative. The results of OmpA iELISA are as follows: Figure 5 B) Antibody expression turned positive on day 7, 3 days later than the MAT. It peaked between days 13-20 and then declined. OmpAi ELISA remained positive until day 116.

[0071] 2.10 Clinical Sample Testing

[0072] 180 sera from eight farms were tested. MAT results showed that the antibody-positive detection rate for the four farms with abortion prevalence was 7%-42.9%, while the antibody-positive detection rate in the non-abortion-prevalent areas was 0%. The OmpAiELISA method developed by the present invention showed an antibody-positive detection rate of 42.9%-100% for the four farms with abortion prevalence, while the antibody-positive detection rate in the non-abortion-prevalent areas was 0%. The results are shown in Table 2. Statistics of the test results also showed that 14 samples tested positive by both microagglutination and OmpAiELISA, while 66 samples tested negative for antibodies. Of these, the OmpAiELISA detected 97 more samples than the microagglutination method, as shown in Table 3.

[0073] Table 2 Testing of clinical samples

[0074]

[0075] Table 3 Comparison of detection results of the two methods

[0076] Sequence Listing <110> Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin Branch of China Animal Health and Epidemiology Center) <120> Universal indirect ELISA antibody detection kit for four pathogenic bacteria of equine abortion salmonellosis and its application <141> 2022-04-18 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 360 <212> PRT <213> Salmonella abortus equi S.abortus equi <400> 1 Met Glu Thr Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly 1 5 10 15 Phe Ala Thr Val Ala Gln Ala Ala Pro Lys Asp Asn Thr Trp Tyr Ala 20 25 30 Gly Ala Lys Leu Gly Trp Ser Gln Tyr His Asp Thr Gly Phe Ile Asn 35 40 45 Asn Asp Gly Pro Thr His Glu Asn Gln Leu Gly Ala Gly Ala Phe Gly 50 55 60 Gly Tyr Gln Val Asn Pro Tyr Val Gly Phe Glu Met Glu Thr Gly Tyr 65 70 75 80 Asp Trp Leu Gly Arg Met Glu Thr Pro Tyr Lys Gly Asp Asn Ile Asn 85 90 95 Gly Ala Tyr Lys Ala Gln Gly Val Gln Leu Thr Ala Lys Leu Gly Tyr 100 105 110 Pro Ile Thr Asp Asp Leu Asp Val Tyr Thr Arg Leu Gly Gly Met Glu 115 120 125 Thr Val Trp Arg Ala Asp Thr Lys Ser Asn Val Pro Gly Gly Pro Ser 130 135 140 Thr Lys Asp His Asp Thr Gly Val Ser Pro Val Phe Ala Gly Gly Ile 145 150 155 160 Glu Tyr Ala Ile Thr Pro Glu Ile Ala Thr Arg Leu Glu Tyr Gln Trp 165 170 175 Thr Asn Asn Ile Gly Asp Ala Asn Thr Ile Gly Thr Arg Pro Asp Asn 180 185 190 Gly Leu Leu Ser Val Gly Val Ser Tyr Arg Phe Gly Gln Gln Glu Ala 195 200 205 Ala Pro Val Val Ala Pro Ala Pro Ala Pro Ala Pro Glu Val Gln Thr 210 215 220 Lys His Phe Thr Leu Lys Ser Asp Val Leu Phe Asn Phe Asn Lys Ser 225 230 235 240 Thr Leu Lys Pro Glu Gly Gln Gln Ala Leu Asp Gln Leu Tyr Ser Gln 245 250 255 Leu Ser Asn Leu Asp Pro Lys Asp Gly Ser Val Val Val Leu Gly Phe 260 265 270 Thr Asp Arg Ile Gly Ser Asp Ala Tyr Asn Gln Gly Leu Ser Glu Lys 275 280 285 Arg Ala Gln Ser Val Val Asp Tyr Leu Ile Ser Lys Gly Ile Pro Ser 290 295 300 Asp Lys Ile Ser Ala Arg Gly Met Glu Thr Gly Glu Ser Asn Pro Val 305 310 315 320 Thr Gly Asn Thr Cys Asp Asn Val Lys Pro Arg Ala Ala Leu Ile Asp 325 330 335 Cys Leu Ala Pro Asp Arg Arg Val Glu Ile Glu Val Lys Gly Val Lys 340 345 350 Asp Val Val Thr Gln Pro Gln Ala 355 360 <210> 2 <211> 337 <212> PRT <213> Salmonella abortus equi S.abortus equi <400> 2 Ala Pro Lys Asp Asn Thr Trp Tyr Ala Gly Ala Lys Leu Gly Trp Ser 1 5 10 15 Gln Tyr His Asp Thr Gly Phe Ile Asn Asn Asp Gly Pro Thr His Glu 20 25 30 Asn Gln Leu Gly Ala Gly Ala Phe Gly Gly Tyr Gln Val Asn Pro Tyr 35 40 45 Val Gly Phe Glu Met Glu Thr Gly Tyr Asp Trp Leu Gly Arg Met Glu 50 55 60 Thr Pro Tyr Lys Gly Asp Asn Ile Asn Gly Ala Tyr Lys Ala Gln Gly 65 70 75 80 Val Gln Leu Thr Ala Lys Leu Gly Tyr Pro Ile Thr Asp Asp Leu Asp 85 90 95 Val Tyr Thr Arg Leu Gly Gly Met Glu Thr Val Trp Arg Ala Asp Thr 100 105 110 Lys Ser Asn Val Pro Gly Gly Pro Ser Thr Lys Asp His Asp Thr Gly 115 120 125 Val Ser Pro Val Phe Ala Gly Gly Ile Glu Tyr Ala Ile Thr Pro Glu 130 135 140 Ile Ala Thr Arg Leu Glu Tyr Gln Trp Thr Asn Asn Ile Gly Asp Ala 145 150 155 160 Asn Thr Ile Gly Thr Arg Pro Asp Asn Gly Leu Leu Ser Val Gly Val 165 170 175 Ser Tyr Arg Phe Gly Gln Gln Glu Ala Ala Pro Val Val Ala Pro Ala 180 185 190 Pro Ala Pro Ala Pro Glu Val Gln Thr Lys His Phe Thr Leu Lys Ser 195 200 205 Asp Val Leu Phe Asn Phe Asn Lys Ser Thr Leu Lys Pro Glu Gly Gln 210 215 220 Gln Ala Leu Asp Gln Leu Tyr Ser Gln Leu Ser Asn Leu Asp Pro Lys 225 230 235 240 Asp Gly Ser Val Val Val Leu Gly Phe Thr Asp Arg Ile Gly Ser Asp 245 250 255 Ala Tyr Asn Gln Gly Leu Ser Glu Lys Arg Ala Gln Ser Val Val Asp 260 265 270 Tyr Leu Ile Ser Lys Gly Ile Pro Ser Asp Lys Ile Ser Ala Arg Gly 275 280 285 Met Glu Thr Gly Glu Ser Asn Pro Val Thr Gly Asn Thr Cys Asp Asn 290 295 300 Val Lys Pro Arg Ala Ala Leu Ile Asp Cys Leu Ala Pro Asp Arg Arg 305 310 315 320 Val Glu Ile Glu Val Lys Gly Val Lys Asp Val Val Thr Gln Pro Gln 325 330 335 Ala <210> 3 <211> 136 <212> PRT <213> Salmonella abortus equi S.abortus equi <400> 3 Lys His Phe Thr Leu Lys Ser Asp Val Leu Phe Asn Phe Asn Lys Ser 1 5 10 15 Thr Leu Lys Pro Glu Gly Gln Gln Ala Leu Asp Gln Leu Tyr Ser Gln 20 25 30 Leu Ser Asn Leu Asp Pro Lys Asp Gly Ser Val Val Val Leu Gly Phe 35 40 45 Thr Asp Arg Ile Gly Ser Asp Ala Tyr Asn Gln Gly Leu Ser Glu Lys 50 55 60 Arg Ala Gln Ser Val Val Asp Tyr Leu Ile Ser Lys Gly Ile Pro Ser 65 70 75 80 Asp Lys Ile Ser Ala Arg Gly Met Glu Thr Gly Glu Ser Asn Pro Val 85 90 95 Thr Gly Asn Thr Cys Asp Asn Val Lys Pro Arg Ala Ala Leu Ile Asp 100 105 110 Cys Leu Ala Pro Asp Arg Arg Val Glu Ile Glu Val Lys Gly Val Lys 115 120 125 Asp Val Val Thr Gln Pro Gln Ala 130 135

Claims

1. A universal indirect ELISA antibody detection kit for four pathogenic bacteria of equine abortion salmonellosis, characterized in that: The kit contains an ELISA plate coated with truncated expression of Salmonella outer membrane recombinant protein A (OmpA), the amino acid sequence of which is shown in SEQ ID NO.

3. The four pathogenic bacteria are Salmonella abortus ( Salmonella abortus equi S. abortus equi ), Salmonella typhimurium ( S.typhimurium S.typhi ), Salmonella Dublin ( S. dublin ) and Salmonella Enteritidis ( S. enteritidis ).

2. The indirect ELISA antibody detection kit according to claim 1, wherein The coating antigen concentration of the truncated expressed Salmonella outer membrane recombinant protein A was 1 μg / mL, and 100 μl / well was used to coat a 96-well ELISA plate at 4°C overnight.

3. The indirect ELISA antibody detection kit according to claim 1, wherein The kit also contains HRP-labeled anti-horse IgG secondary antibody, blocking solution, diluent, color developing solution and stop solution.

4. The indirect ELISA antibody detection kit according to claim 3, wherein The blocking solution is 5% w / w skim milk, which is blocked at 37°C for 1.5 hours. The diluent is 5% w / w skim milk or 5% w / w BSA. The serum to be tested is diluted 1:200 with 5% w / w skim milk and incubated at 37°C for 1 hour. The HRP-labeled anti-horse IgG secondary antibody is diluted 1:10000 with 5% w / w BSA and incubated at 37°C for 30 minutes. The colorimetric solution is TMB colorimetric solution and incubated at 37°C for 10 minutes.

5. Use of the kit according to any one of claims 1 to 4 in the preparation of reagents for detecting four pathogenic bacteria of equine abortion Salmonella, wherein the four pathogenic bacteria are Salmonella abortus ( Salmonella abortus equi S. abortus equi ), Salmonella typhimurium ( S.typhimurium S.typhi ), Salmonella Dublin ( S. dublin ) and Salmonella Enteritidis ( S. enteritidis ).

6. The use according to claim 5, characterized in that The reagent is an indirect ELISA antibody detection reagent.

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