Hybridoma cell strain secreting anti-equine salmonella groel protein monoclonal antibody, monoclonal antibody thereof and application thereof

Abstract

The application discloses a hybridoma cell strain secreting an anti-equi salmonella abortusequi groEL protein monoclonal antibody, a monoclonal antibody thereof and application thereof. The hybridoma cell strain is named E11, is preserved in the China General Microbiological Culture Collection Center and has a strain preservation number of CGMCC No. 46349. The application establishes a general cELISA antibody detection method of salmonella which can be applied to different animals by using E11 MAb secreted by the hybridoma cell strain secreting the anti-equi salmonella abortusequi groEL protein monoclonal antibody and equi salmonella groEL protein, and the salmonella includes equi salmonella abortusequi (S. S. ), salmonella typhi (S. S. ), salmonella dublin (S. S. ), salmonella enteritidis (S. S. ), and therefore the method has good specificity and broad spectrum. The application provides an effective technical means for diagnosis and prevention and control of salmonella diseases of different animals in the clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to a hybridoma cell strain secreting anti-Equine abortion Salmonella groEL protein monoclonal antibody, and relates to the monoclonal antibody secreted by the hybridoma cell strain and the application thereof, and belongs to the technical field of medicine. BACKGROUND

[0002] Salmonellosis is a common foodborne zoonosis worldwide, and its pathogen is Salmonella. The pathogen can infect different animals such as birds (chickens, ducks, geese), pigs, cattle, sheep, horses, donkeys and rabbits, and can also cause various symptoms in humans, including gastroenteritis, food poisoning, typhoid fever, diarrhea, bacteremia, etc. It is an important zoonosis pathogen.

[0003] Salmonella includes two species, Enteric Salmonella (Salmonella enterica) and Bongol Salmonella (Salmonella bongor). S. enterica ) and Bongol Salmonella (Salmonella bongor). S. bongori ) and Bongol Salmonella (Salmonella bongor).

[0004] Salmonella gallinarum is one of the important pathogenic bacteria that endanger the poultry industry in China, which has brought great economic losses to the poultry industry. It is of great significance for the breeding farm to purify Salmonella. The serotypes of Salmonella that cause chicken infection are diverse, including Salmonella pullorum, Salmonella typhi, Salmonella enteritidis, Salmonella typhimurium, and Salmonella porsia. Epidemiology shows that chicken flocks on farms are infected with multiple Salmonella species, either singly or in combination. In the past decade, the main serotypes of poultry Salmonella in China are Salmonella pullorum, Salmonella enteritidis, and Salmonella typhimurium. Currently, large-scale farms generally eliminate positive chickens and flocks through pathogenicity and antibody detection to achieve the purpose of purifying Salmonella. Serology commonly uses the slide agglutination method, which is inexpensive but low in sensitivity, making it impossible to completely eradicate the purification work. ELISA is sensitive, and a Chinese patent application with the application number 201811381001.X discloses a competitive ELISA method for detecting Salmonella pullorum based on the IpaJ gene. However, since a certain percentage of Salmonella pullorum lacks the IpaJ gene, the method may miss some detections, leading to the persistence of Salmonella pullorum infection in chicken flocks and the inability to achieve complete purification. In addition, chicken flocks are also infected with other Salmonella species such as Salmonella enteritidis and Salmonella typhimurium. The purification of Salmonella in chicken flocks requires the use of more Salmonella ELISA methods for detection, which greatly increases the workload and cost of purification, making it difficult to carry out the purification work of Salmonella in chicken flocks.

[0005] Bovine salmonellosis is mainly caused by Salmonella typhimurium, Salmonella enteritidis, and Salmonella dublin, characterized by diarrhea and septicemia. The disease is more common in adult cattle and has a high incidence in calves, and can cause pregnant cows to abort. Bovine salmonellosis can occur throughout the year, but is more common in rainy and humid seasons. It is often sporadic or endemic, with adult cattle showing sporadic cases and calves showing endemic cases. Currently, the diagnosis of the disease mainly relies on pathogenic bacterial isolation, and there is a lack of research on ELISA antibody detection and antigen detection.

[0006] Porcine paratyphoid is a conditional porcine infectious disease caused by Salmonella choleraesuis, Salmonella typhimurium, and Salmonella enteritidis, which is a common disease in the pig industry. Porcine paratyphoid is more common in pre-weaned and post-weaned piglets, with a high incidence and mortality rate. Laboratory diagnosis methods include pathogenic bacteria isolation and identification, and fluorescent quantitative PCR. Veterinary clinics commonly use pathogenic bacteria isolation and identification for porcine paratyphoid, while there is a lack of antigen detection methods such as AC-ELISA and colloidal gold, as well as ELISA antibody detection methods.

[0007] Equine abortus salmonellosis (Equine abortus salmonellosis) Salmonellosis has recently broken out and spread in East China, North China and Northwest China, etc., mainly causing mid-late abortion in pregnant horses and donkeys, with an abortion rate of 30-100%. Therefore, in order to respond to the outbreak, it is urgent to establish a specific, sensitive and high-throughput detection method. In the early stage, we established iELISA (application number CN202010148906.3) and cELISA (application number CN202111444112.2) antibody detection methods specific to equine abortion salmonella, which can effectively detect equine abortion salmonella antibodies and are of great significance for the diagnosis of equine abortion salmonella infection. Since the established iELISA and cELISA methods have high specificity for detecting only equine abortion salmonella antibodies, they cannot detect other animal or other salmonella infections.

[0008] The main transmission route of human salmonella infection is the consumption of contaminated animal products (meat, eggs, and milk). Animal salmonella infection not only causes losses to the livestock industry but also increases the risk of human public health, so it is crucial to carry out pathogen and serological diagnosis of animal salmonella disease. In addition to conventional bacterial isolation and nucleic acid detection, antigen detection methods such as AC-ELISA and colloidal gold are also available. The conventional bacterial isolation method is time-consuming and labor-intensive, with a long cycle and is not suitable for large-scale sample detection. The nucleic acid detection method has high sensitivity, which is of great significance for disease diagnosis, but requires high technical requirements for the detection personnel and is prone to contamination during the detection process. In addition, it also needs to be equipped with a fluorescence quantitative PCR instrument, but the equipment is expensive and not widely used, making the fluorescence quantitative method inconvenient to use. The AC-ELISA method has higher sensitivity than bacterial isolation and is easier to achieve high-throughput detection. ELISA requires less technical requirements for the operator and is more convenient to operate. The use of enzyme-labeled instruments is relatively cheaper than fluorescence quantitative PCR. In addition, the storage and transportation of the reagent kit are also a problem. Generally, the ELISA antibody or antigen products on the market are more affordable than the corresponding nucleic acid detection products, so ELISA has more advantages than fluorescence quantitative methods. Serological detection methods have high sensitivity, mainly including iELISA and cELISA. The cELISA method has great advantages over iELISA and is not limited by the source of serum animals, so it can be applied to antibody detection of different animal sera. Salmonella has a wide variety of species, with more than 2600 serotypes, so establishing a universal cELISA antibody detection method for Salmonella is of great significance for the diagnosis of different animal salmonella diseases. SUMMARY

[0009] The purpose of the present invention is to provide a hybridoma cell line secreting a monoclonal antibody against groEL protein of Salmonella abortus equi, the monoclonal antibody thereof and application thereof.

[0010] In order to achieve the above object, the present invention adopts the following technical means:

[0011] The present invention generates a hybridoma cell line, E11, by immunizing mice with inactivated Salmonella abortus antigen. Pull-down assays using the E11 monoclonal antibody secreted by this cell line and Salmonella abortus antigens revealed that the E11 monoclonal antibody recognizes the Salmonella abortus groEL protein (heat shock protein). This protein is highly conserved among Salmonella species (99.1-100% homology) and has low homology with other bacteria. Antigenically, this protein is an outer membrane protein with a monomer molecular weight of approximately 60 kDa. Two rings composed of seven subunits stack together to form a tetradecamer. This antigenic component accounts for a significant proportion of the total bacterial composition, making it a dominant antigen and therefore suitable as a target for Salmonella antigen or antibody diagnosis.

[0012] A kind of secretion anti-equine abortion Salmonella of the present invention ( Salmonella Abortusequi, abbreviated as S. The hybridoma cell line is named E11 and classified as a hybridoma cell line (Mus musculus). It is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Its culture deposit number is CGMCC No. 46349, and the deposit date is April 22, 2025.

[0013] The monoclonal antibody against groEL protein of Salmonella abortus secreted by the hybridoma cell line is also within the protection scope of the present invention.

[0014] Furthermore, the present invention also proposes the use of the hybridoma cell line and the monoclonal antibody in preparing a reagent for detecting Salmonella.

[0015] Among them, preferably, the Salmonella includes Salmonella abortus, Salmonella typhimurium ( Salmonella Typhi, abbreviated as S. Typhi), Salmonella Dublin ( Salmonella Dublin, abbreviated as S. Dublin) and Salmonella Enteritidis ( Salmonella Enteritidis, abbreviated as S. Enteritidis).

[0016] Further, the application further provides a cELISA kit for detecting Salmonella, wherein the kit comprises the anti-Eq groEL monoclonal antibody.

[0017] Preferably, the kit further comprises a groEL protein coated enzyme labeled plate, a blocking solution, a PBST washing solution, an HRP labeled anti-mouse IgG enzyme labeled secondary antibody, a color developing solution and a termination solution, wherein the amino acid sequence of the groEL protein is shown as SEQ ID NO. 1.

[0018] Preferably, the coating concentration of the groEL protein is 1 μg / ml, the dilution of the anti-Eq groEL monoclonal antibody is 0.2 μg / ml, and the dilution of the HRP labeled anti-mouse IgG enzyme labeled secondary antibody is 1:10000.

[0019] Finally, the application further provides an application of the cELISA kit in preparing a reagent for detecting Salmonella, wherein the Salmonella preferably comprises Salmonella abortusequi, Salmonella typhi, Salmonella dublin, Salmonella enteritidis. S. S. S. S. .

[0020] Compared with the prior art, the application has the following beneficial effects:

[0021] The application establishes a general cELISA antibody detection method for Salmonella which can be applied to different animals by using the E11 MAb secreted by the hybridoma cell strain secreting the anti-Eq groEL monoclonal antibody and the groEL protein of Salmonella abortusequi, Salmonella typhi, Salmonella dublin and Salmonella enteritidis, so that the method has good specificity and broad spectrum. S. S. S. S. Enteritidis Therefore, the application provides an effective technical means for the diagnosis and prevention and control of Salmonella diseases in different animals. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 2 is an SDS-PAGE result diagram of the purified E11 monoclonal antibody;

[0023] ​​​​​​Wherein, M: protein marker; 1-12: monoclonal antibodies in different elution tubes; 13: ascites before purification; 50kDa is the monoclonal antibody heavy chain, 25kDa is the monoclonal antibody light chain;

[0024] Figure 2 This is the result of antibody titer determination after E11 purification;

[0025] Figure 3 This is the identification result of the dominant antigen of Salmonella;

[0026] Among them, M: protein marker; 1: E11 monoclonal antibody; 2: E11 monoclonal antibody + Salmonella antigen; 3: Salmonella antigen;

[0027] Figure 4 This is the amplification result diagram of the groEL target gene;

[0028] Wherein, M: nucleic acid marker; 1-3: PCR amplification products; 4: negative control;

[0029] Figure 5 Figure 1 is the SDS-PAGE analysis result of the soluble expression of recombinant groEL protein;

[0030] Wherein, M: protein marker; 1: supernatant after recombinant bacteria were broken; 2: precipitate after recombinant bacteria were broken;

[0031] Figure 6 Figure 1 is the Western blot analysis result of recombinant groEL protein;

[0032] Wherein, M: protein marker; 1: supernatant after recombinant bacteria were broken; 2: precipitate after recombinant bacteria were broken;

[0033] Figure 7 This is a diagram showing the validation results of the competitive activity of E11 monoclonal antibody;

[0034] Figure 8 Optimize OD for different E11 monoclonal antibody concentrations and anti-mouse secondary antibody dilutions 450nm value;

[0035] Figure 9 This is the N / P value result diagram under different E11 monoclonal antibody concentrations and anti-mouse secondary antibody dilution optimization;

[0036] Figure 10 This is the specificity validation result of the Salmonella cELISA antibody detection method. DETAILED DESCRIPTION

[0037] The experimental methods of the present application are described in detail below in conjunction with the examples in order to more clearly illustrate its technical features and implementation steps. Those skilled in the art should understand that the examples are only typical examples of the present application and do not constitute any limitation on the scope of the present application. Technical details can be adjusted or replaced without departing from the spirit and scope of the present application, and these adjustments and replacements are all within the protection scope of the present application.

[0038] Example 1 Preparation of monoclonal antibody

[0039] 1 Materials

[0040] 1.1 Immunogen Salmonella abortus equi 180316H.AES.G inactivated antigen (its strain preservation number is: CGMCC No. 18341, which has been recorded in the patent application with the publication number CN111100817A and the invention name of "Salmonella abortus equi equine strain and its application in preparing Salmonella abortus equi inactivated vaccine", isolated and identified by the equine infectious disease and slow virus innovation team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, and provided), dilution content 1x10 9 CFU / ml.

[0041] 1.2 Cells Myeloma cell SP2 / 0, provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0042] 1.3 Test animals 6-8 week old female Balb / c mice, provided by the experimental animal center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0043] 1.4 Cell culture medium 1640 culture medium containing 20% fetal bovine serum and 100 U (µg) / ml of ampicillin, stored at 2-8°C. 1640 culture medium, purchased from Sigma company; fetal bovine serum, purchased from Ausbian company.

[0044] 1.5 Cell freezing solution Take 10 ml of dimethyl sulfoxide (DMSO) and dissolve in 90 ml of fetal bovine serum, mix uniformly, and store at 2-8°C.

[0045] 1.6 Kit

[0046] 1.6.1 BCA kit, purchased from Novagen company.

[0047] 1.6.2 SBA ClonotypingTMSystem / HRP, purchased from SouthernBiotech company.

[0048] 1.7 Other reagents PEG4000, Freund's complete adjuvant, Freund's incomplete adjuvant and HRP labeled goat anti-mouse IgG were purchased from Sigma; TMB color developing solution was purchased from Thermo.

[0049] 2 Method

[0050] 2.1 Construction of hybridoma cells

[0051] 2.1.1 Animal immunization

[0052] 2.1.1.1 Primary immunization Equimolar amounts of inactivated Salmonella abortus antigens and Freund's complete adjuvant were emulsified as immunogens, and 4-6 week old Balb / c mice were injected subcutaneously on the back, 200 μl per mouse.

[0053] 2.1.1.2 Secondary immunization Three weeks after the first immunization, equimolar amounts of inactivated antigens and Freund's incomplete adjuvant were mixed and emulsified as immunogens, and the mice were injected intraperitoneally according to the immunization method and dosage of the first immunization.

[0054] 2.1.1.3 Third immunization Three weeks after the second immunization, equimolar amounts of inactivated Salmonella antigens and Freund's incomplete adjuvant were mixed and emulsified as immunogens, and the mice were injected according to the immunization method and dosage of the second immunization.

[0055] 2.1.1.4 Fourth immunization Three days before cell fusion, the mice were injected intraperitoneally with inactivated Salmonella antigens as immunogens, 200 μl per mouse.

[0056] 2.1.2 Preparation of myeloma cells One to two days before fusion, the myeloma cells were expanded and in the logarithmic growth phase, and the growth state was good. On the day of fusion, the culture medium was discarded and the cells were gently washed twice with serum-free 1640, and the cells were gently blown off the bottle wall with 15 ml of 1640 base culture medium. A small amount of myeloma cell suspension was taken and counted with a cell counting plate, and prepared for fusion.

[0057] 2.1.3 Preparation of immune spleen cells

[0058] 2.1.3.1 Before fusion, the mouse was sacrificed by eye blood collection and positive serum was prepared. After soaking in 75% alcohol for 5 minutes, it was placed on the clean bench.

[0059] 2.1.3.2 The mouse was fixed on the mouse stand, the abdominal cavity was opened aseptically, the connective tissue was separated and the spleen was removed, and the spleen was placed in a petri dish containing 15 ml of 1640 base culture medium. The spleen cells were gently blown out with a sterile syringe, and the operation was repeated 3-4 times.

[0060] 2.1.3.3 Transfer the spleen cell suspension into a 50 ml centrifuge tube, add about 30 ml of 1640 basal medium, and mix well. Count the cell suspension with a cell counting chamber, and reserve it for use.

[0061] 2.1.4 Fusion of Spleen Cells with Myeloma Cells

[0062] 2.1.4.1 Preheat the HAT medium, 1640 medium, and 1 ml PEG4000 in a water bath, and prepare 500 ml of sterilized water preheated at 42°C.

[0063] 2.1.4.2 Add the logarithmically growing SP2 / 0 cells and the spleen cells from the immunized mice into a 50 ml centrifuge tube at a ratio of 1:8, mix gently, centrifuge at 800 r / min for 10 minutes, and remove the supernatant to avoid affecting the fusion efficiency. Gently tap the bottom of the centrifuge tube to make the cells evenly spread on the bottom.

[0064] 2.1.4.3 Perform the fusion process in a fusion cup containing 42°C water. Add 1 ml of 37°C preheated PEG4000 solution drop by drop into the 50 ml centrifuge tube, slowly rotate the tube while adding, and complete the addition within 90 seconds. Incubate at 37°C for 1-2 minutes.

[0065] 2.1.4.4 Slowly add the 1640 basal medium to terminate the reaction, add 1 ml of 1640 at the first minute, 1 ml of 1640 at the second minute, 3 ml of 1640 at the third minute, 10 ml of 1640 at the fourth minute, and 10 ml of 1640 at the fifth minute. Incubate for 2 minutes, gently invert twice, incubate for 7 minutes, centrifuge at 800 r / min for 10 minutes, and discard the supernatant. Resuspend the cells with 110 ml of HAT medium, and inoculate 200 μl per well into a 96-well culture plate containing feeder cells, and incubate in a 37°C, 5% CO2 incubator.

[0066] 2.1.4.5 Half-volume medium replacement after 3 days, and half-volume medium replacement after 6 days. When the cells grow to cover 1 / 4-1 / 3 of the well area, collect the supernatant for screening and detection, and replace it with HT medium.

[0067] 2.1.5 Screening and Cloning of Positive Hybridoma Cell Strains Use the indirect ELISA method to detect the culture supernatant of hybridoma cells, and screen positive clones through 2-3 consecutive limited dilution cloning methods. Expand the culture and freeze the cell strains. The indirect ELISA method is as follows:

[0068] 2.1.5.1 Coating Dilute the inactivated antigen to 1 µg / ml with carbonate buffer (0.05 mol / L, pH 9.6), add 100 μl per well into a 96-well enzyme-linked reaction plate, and incubate overnight at 2-8°C.

[0069] 2.1.5.2 Washing Discard the liquid in the wells, wash the plate with PBST (0.01 mol / L, pH 7.4) 3 times, 250 μl / well, and pat dry the liquid on the dry filter paper after each washing by inverting the plate.

[0070] 2.1.5.3 Blocking Add PBS (0.01 mol / L, pH 7.4) containing 5% skim milk, 200 μl / well, and incubate at 37°C for 2 hours.

[0071] 2.1.5.4 Washing The method is the same as item 2.1.5.2.

[0072] 2.1.5.5 Sample addition Add the sample to be tested, 100 μl / well, and incubate at 37°C for 1 hour.

[0073] 2.1.5.6 Washing The method is the same as item 2.1.5.2.

[0074] 2.1.5.7 Addition of secondary antibody Add HRP-labeled goat anti-mouse IgG diluted 1:10,000, 100 μl / well, and incubate at 37°C for 30 minutes.

[0075] 2.1.5.8 Washing The method is the same as item 2.1.5.2.

[0076] 2.1.5.9 Color development Add TMB color developing solution, 100 μl / well, and develop at room temperature (15-25°C) for 5 minutes (avoid light incubation).

[0077] 2.1.5.10 Termination Add termination solution, 50 μl / well, mix gently, and then read the OD450nm value at a wavelength of 450 nm using an enzyme marker (the reading should be completed within 5 minutes after the addition of the termination solution), and record the results.

[0078] 2.1.5.11 Judgment

[0079] Conditions for a valid test: when the OD450nm value of the sample to be tested is >0.5 and the P / N value (P / N = OD450nm value of the sample to be tested / OD450nm value of the negative control) is >2.1, it is determined to be positive, and the maximum dilution corresponding thereto is its titer. 450nm

[0080] 2.1.6 Subcloning of positive hybridoma cell lines Subclone the positive hybridoma cells by limited dilution, clone 3 times, until the positive rate of the antibody in the cloned wells reaches 100%, and then store the obtained positive hybridoma cells in liquid nitrogen after expanding the culture.

[0081] 2.2 Preparation and purification of monoclonal antibodies

[0082] ​2.2.1 Preparation of ascites fluid 6-8 week old Balb / c mice were injected intraperitoneally with 0.5 ml Freund's incomplete adjuvant. After 7-10 days, each mouse was injected intraperitoneally with 1-2.5 x 10 6 The ascites fluid was collected after the abdomen of the mouse was visibly distended. The ascites fluid was centrifuged at 10,000 rpm for 10 minutes at 4°C, and the supernatant was collected and stored for later use.

[0083] 2.2.2 Purification of monoclonal antibody

[0084] 2.2.2.1 Sample preparation The prepared ascites fluid was dissolved at room temperature, mixed with 4-5 times the volume of binding / washing buffer (20 mmol / L NaH2P04, pH 7.0), and filtered through a 0.45 μm filter.

[0085] 2.2.2.2 Column equilibration The HiTrap protein G packing (about 2 ml bed volume) was loaded into a suitable chromatography column, and equilibrated with 10 times the column volume of binding / washing buffer (20 mmol / L NaH2P04, pH 7.0) at a flow rate of 1 ml / min.

[0086] 2.2.2.3 Sample loading The sample was added to the equilibrated chromatography column at a flow rate of 0.2-1 ml / min, and the effluent was collected. The sample was passed through the column 3-5 times.

[0087] 2.2.2.4 Washing The non-specifically adsorbed impurities were removed by washing with 10-15 times the column volume of binding / washing buffer (20 mmol / L NaH2P04, pH 7.0).

[0088] 2.2.2.5 Elution The purified monoclonal antibody was eluted 5-6 times with elution buffer (0.1 mol / L glycine, pH 2.7) at a volume of 1 ml each time, and the effluent was collected. The eluted effluent was immediately neutralized with neutralization buffer (1 mol / L Tris-HCl, pH 9.0) at a ratio of about 120 μl of neutralization buffer (1 mol / L Tris-HCl, pH 9.0) per 1 ml of eluted effluent.

[0089] 2.2.2.6 SDS-PAGE analysis The purified monoclonal antibody in different elution tubes was mixed with 1 / 4 volume of 5x SDS loading buffer, and subjected to SDS-PAGE analysis at 100°C for 10 minutes in a metal bath. 10 μL of the treated sample and a protein molecular weight marker were subjected to 12% SDS-PAGE electrophoresis, and the gel image was analyzed using software BandScan 5.0 to determine the purity of the monoclonal antibody.

[0090] 2.3 Identification of monoclonal antibodies

[0091] 2.3.1 Identification of monoclonal antibody subclasses The monoclonal antibody subclasses screened were identified using the SBA Clonotyping™ System / HRP kit according to the instructions.

[0092] 2.3.2 Determination of protein concentration The protein concentration of the purified monoclonal antibodies was determined according to the detection method in the BCA kit instructions.

[0093] 2.3.3 Titer determination The purified antibody (1 mg / mL) from the hybridoma cell line was diluted 2-fold in gradient from 1:200 with PBS (0.01 mol / L, pH 7.4), and detected by indirect ELISA to determine the titer.

[0094] 3 Results

[0095] 3.1 Screening of monoclonal antibody cell lines

[0096] Through 3 rounds of purification, 2 hybridoma cell lines that could recognize Salmonella abortus equi antigens were screened.

[0097] 3.2 Identification of monoclonal antibodies

[0098] 3.2.1 Identification of monoclonal antibody subclasses The monoclonal antibody subclasses screened were identified according to the SBA Clonotyping™ System / HRP kit instructions, and the results showed (Table 1) that the E11 monoclonal antibody subclass was IgG2a, and the 2D2 monoclonal antibody subclass was IgM. For the convenience of subsequent antibody purification, only the IgG subtype antibody E11 was preserved and studied. The hybridoma cell line E11 that secretes monoclonal antibodies against Salmonella abortus equi screened was preserved in the China General Microbiological Culture Collection Center, located at No. 1 Yard of the Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road, and the strain preservation number is CGMCC No. 46349, and the preservation date is April 22, 2025.

[0099] ;

[0100] 3.2.2 Titer determination Antibody purification was performed according to the instructions for HiTrap Protein G HP (GE). The SDS-PAGE results showed that after SDS-PAGE electrophoresis, two specific bands appeared, with a heavy chain of about 50 kDa and a light chain of about 25 kDa. The gel image was analyzed using the software BandScan 5.0, and the purity of the purified monoclonal antibodies (lanes 3-10) was greater than 98%, obtaining relatively pure antibodies, as shown in detail inFigure 1 The purified antibody was mixed (lanes 3-10) and diluted to 1 mg / ml, and the titer of the purified antibody of the hybridoma cell strain E11 was determined by indirect ELISA. The results showed that the titer of the E11 monoclonal antibody was 6.4 x 10 6 Figure 2

[0101] Example 2 Identification of the antigen recognized by the E11 monoclonal antibody, expression and verification of the competitive activity of the E11 monoclonal antibody

[0102] 1. Materials and methods

[0103] 1.1 Strains

[0104] Salmonella abortus equi strain 180316H.AES.G (its strain preservation number is: CGMCC No. 18341)

[0105] 1.2 Samples and reagents

[0106] The agarose gel DNA extraction kit (Gel Extraction Kit), high-purity plasmid mini extraction kit (Pure plasmid Mini Kit), etc. were purchased from Kangwei Century Co., Ltd.

[0107] 1.3 Identification of antigens

[0108] Preparation of Salmonella abortus equi antigen complex, 8 mL of fresh Salmonella abortus equi in the logarithmic phase was taken in a biological safety cabinet, centrifuged at 4 °C and 10 000 r / min for 2 min to collect the bacterial bodies, then resuspended with 2 mL of sterile PBS, centrifuged to discard the supernatant, and the bacterial bodies were washed twice. Then resuspend with 1.5-2 mL of sterile PBS, and perform ultrasonic disruption. Power 39%, work 3 s, intermittent 5 s, work 5 min. Finally, centrifuge the bacterial lysate at 4 °C and 10 000 r / min for 2 min, take the supernatant, measure the protein concentration by BCA, and adjust the protein concentration to 1 mg / mL for standby. Then perform pull-down test of E11 monoclonal antibody and Salmonella abortus equi antigen, and finally analyze the mass spectrometry results.

[0109] 1.4 Amplification and sequence analysis of target genes

[0110] According to the groEL sequence (GenBank: AB033231), a pair of primers was designed:

[0111] ;

[0112] ​​The gorEL gene was amplified by PCR using Salmonella abortus (180316H.AES.G) as a template and cloned into the pET28a vector to generate the recombinant vector pET28a-gorEL. The amplified gorEL was sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing. Following sequencing, the amino acid sequence of the S. abortus groEL sequence was compared with that of Salmonella pullorum, Salmonella Enteritidis, Salmonella Galli, Salmonella Dublin, Salmonella Typhimurium, Salmonella Choleraesuis, and Salmonella Paratyphi A, as well as groELs from other bacteria (including Escherichia coli, Streptococcus, Helicobacter pylori, Staphylococcus aureus, Brucella, Bacillus, Pseudomonas, Riemerella anatipestifer, and Pasteurella multocida).

[0113] 1.5 Induced expression, purification and identification of target protein

[0114] Transform the recombinant plasmid pET28a-gorEL into BL21 and culture at 37℃ for 12-14 hours. Inoculate the positive colonies into 5mL of fresh LB liquid medium containing 1µg / mL Kanamycin resistance and culture at 37℃ with shaking at 170r / min for 16h. Inoculate the colonies into Kan / LB liquid medium at a ratio of 1:100 and culture at 37℃ with shaking at 170r / min until the OD value of the bacterial solution reaches 0. 600nm When the pH value was approximately 0.6-0.8, IPTG was added to a final concentration of 0.6 mmol / L at 25°C for induction. After 8 hours of induction, 4 ml of bacterial culture was collected for enrichment and ultrasonically disrupted in 1.5 ml of PBS. After disruption, the pellet was centrifuged at 12,000 rpm at 4°C for 5 minutes. The pellet was rinsed twice with an equal volume of PBS and resuspended in an equal volume of PBS. 40 µL of the supernatant and resuspended pellet were each added with 10 µL of 5x SDS-PAGE Loading Buffer, mixed thoroughly, and boiled at 98°C for 10 minutes. A 20 µL sample was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. Western blot analysis was performed using E1 monoclonal antibody as the primary antibody (1:5000 dilution) and fluorescently labeled anti-mouse IgG as the secondary antibody (1:5000 dilution).

[0115] 1.6 Verification of the competitive activity of E11 mAb

[0116] (1) The purified groEL protein was coated at 1 μg / ml (100 μl / well) at 4°C for 16 h.

[0117] (2) After blocking, add Salmonella positive and negative serum (100 μL / well) and incubate at 37°C for 1 h;

[0118] (3) After washing twice with PBST, E11 hybridoma cell supernatant was added, and the mixture was incubated at 37°C for 30 min;

[0119] (4) After washing twice with PBST, 1:20000 diluted HRP-labeled anti-mouse IgG secondary antibody was added, and the mixture was incubated at 37°C for 30 min;

[0120] (5) After washing twice with PBST, substrate (100 μl / well) was added, and the mixture was incubated at 37°C for 10 min;

[0121] (6) 2M H2SO4 was added to terminate the reaction, and OD 450nm was read.

[0122] 2. Results

[0123] 2.1 Pull-down to find Salmonella antigen

[0124] Compared with the E11 monoclonal antibody group (lane 1), the test group (lane 2) showed a specific band of about 60 KD (Fig. 1) by pull-down experiment. Figure 3 The specific band pulled down was sent to Beijing Huada Protein Research and Development Center Co., Ltd. for mass spectrometry identification, and the results showed that the band was groEL protein. The antigen recognized by E11 monoclonal antibody was determined to be groEL protein by pull-down technology. By comparing the amino acid sequences of groEL from other Salmonella and other bacteria, the protein is highly conserved in Salmonella, with a homology of 99.1-100%, and has a large difference from other bacteria, which lays a foundation for the establishment of a general diagnostic kit for salmonellosis.

[0125] 2.2 Cloning of Salmonella groEL gene

[0126] The groEL gene was amplified by PCR using the designed primers, and a band of about 1647 bp was obtained (Fig. 2). Figure 4 After digestion and ligation, the positive plasmid was sent for sequencing, and the results showed that the obtained sequence was the groEL sequence of Salmonella. The positive plasmid was named pET28a-groEL.

[0127] 2.3 Expression and identification of groEL protein

[0128] SDS-PAGE analysis showed that a protein band of about 60 KDa appeared in the supernatant after the recombinant pET28a-groEL bacteria were broken, indicating that the recombinant protein was expressed and mainly existed in a soluble form. Figure 5 Western blot analysis showed that the recombinant protein specifically reacted with E11 monoclonal antibody.Figure 6 ), which was consistent with SDS-PAGE results, confirming that the recombinant protein had good reactivity. The amino acid sequence of groEL protein is shown in SEQ ID NO. 1.

[0129] 2.4 Verification of E11 monoclonal antibody competitive activity

[0130] E11 competitive activity was determined by ELISA method, and the results showed that when the serum contained Salmonella antibodies, it could effectively block the binding of E11 monoclonal antibody to coated groEL antigen, thus proving that E11 monoclonal antibody had good competitive activity ( Figure 7 ), which could be used for subsequent research on the establishment of Salmonella competitive ELISA method.

[0131] Example 3 Establishment and application of Salmonella cELISA antibody detection method

[0132] 1. Screening of optimal antibody dilution and enzyme-labeled antibody working concentration

[0133] The purified groEL protein was coated at 4°C for 16h at 1μg / ml (100μl / well), and after blocking, Salmonella positive and negative (100μL / well) serum was added and reacted at 37°C for 1h; after PBST washing for 2 times, different concentrations of E11 monoclonal antibody were added and reacted at 37°C for 30min; after PBST washing for 2 times, different dilutions of HRP-labeled anti-mouse IgG enzyme-labeled secondary antibody were added and reacted at 37°C for 30min; after PBST washing for 2 times, substrate (100μl / well) was added and color developed at 37°C for 10min; 2M H2SO4 was added for termination, and OD4 50nm was read. The reaction conditions were optimized and screened for antibody dilution and enzyme-labeled antibody working concentration, and the optimal reaction conditions were determined, as shown in Figure 8 . According to the OD 450nm values and N / P values under different monoclonal antibody concentrations and anti-mouse secondary antibody dilutions, the optimal conditions were determined, as shown in Figure 9 . The final conditions were determined as follows: coating antigen concentration was 1μg / ml, antibody dilution was 0.2μg / ml, and anti-mouse secondary antibody concentration was 1:10000 dilution.

[0134] 2. Determination of critical value of Salmonella cELISA antibody detection method

[0135] The optimized cELISA antibody detection method was used to detect 30 Salmonella negative sera, and the inhibition rate (PI=1-S / N) was calculated using the following formula. According to the PI values of the 30 Salmonella negative sera, the cutoff value was 45% (average value+3SD). When the PI value of the sample to be detected was greater than 45%, the Salmonella antibody was determined to be positive, and when it was less than 45%, the Salmonella antibody was determined to be negative.

[0136] 3. Specificity test

[0137] The optimized reaction conditions were used to detect equine infectious anemia virus (EIAV), equine arteritis virus (EAV), equine herpes virus (EHV), equine influenza virus (EIV), Theileria equi ( T.equi ), Babesia equi ( B.caballi ), Streptococcus equine ( S.equi ), Salmonella abortus equi ( S. Abortusequi), Salmonella typhimurium ( S. Typhi), Salmonella Dublin ( S. Dublin), Salmonella Enteritidis ( S. Enteritidis) and other positive sera were tested to evaluate the specificity of the Salmonella cELISA antibody detection method. The results are as follows Figure 10 The results showed that all Salmonella-positive sera were positive, while the test results of other pathogen-positive sera were negative, thus proving that the method has good specificity and broad spectrum.

[0138] 4. Comparison of detection of Salmonella cELISA and equine Salmonella abortus specific ELISA antibodies

[0139] The Salmonella cELISA of the present invention was used to test 20 Salmonella-positive sera and 20 Salmonella-negative sera, and compared with the equine Salmonella abortus iELISA (patent application number CN202010148906.3) and cELISA (patent application number CN202111444112.2). The results showed that the Salmonella cELISA of the present invention achieved an overall concordance rate of 100% with the other two equine Salmonella abortus iELISA / cELISA methods (Table 2), demonstrating excellent concordance.

[0140] ;

[0141] Note: “+” represents a positive result, “-” represents a negative result

[0142] 5. Application of Salmonella cELISA in the detection of Salmonella antibodies in equines

[0143] The cELISA antibody detection method of Salmonella established by the application was used to detect 7 farm serum samples, and the results were compared with those of the iELISA (CN202010148906.3) and cELISA (CN202111444112.2) methods of Equine Salmonella abortus, as shown in Table 3. For the iELISA (CN202010148906.3) and cELISA (CN202111444112.2) methods of Equine Salmonella abortus, the positive detection rate of the cELISA of Salmonella established by the application was also 100%, which proved that the cELISA of the application had good sensitivity and would not produce false negative results; for the iELISA (CN202010148906.3) and cELISA (CN202111444112.2) methods of Equine Salmonella abortus, the detection results of the cELISA of Salmonella established by the application were also all negative, which proved that the method had good specificity and would not cause false positive results. For the iELISA (CN202010148906.3) and cELISA (CN202111444112.2) methods of Equine Salmonella abortus, the detection rate of the cELISA (CN202111444112.2) method of Salmonella was 30% and 75%, respectively, and the sensitivity was also higher than that of the iELISA (CN202010148906.3) method of Equine Salmonella abortus. Since the iELISA (CN202010148906.3) and cELSIA (CN202111444112.2) methods of Equine Salmonella abortus are only specific to the detection of Equine Salmonella abortus antibodies, the cELISA (CN202111444112.2) method of Salmonella established by the application can detect different Salmonella antibodies, and the early detection of clinical samples using the iELISA antibody detection method of Salmonella typhimurium established by us also confirmed that some farms had single or mixed infections of Equine Salmonella abortus and Salmonella typhimurium, therefore, for the iELISA (CN202010148906.3) and cELISA (CN202111444112.2) methods of Equine Salmonella abortus, the detection rate of the cELISA (CN202111444112.2) method of Salmonella established by the application was 50%-62.5% positive, which was also reasonable, and further proved that the method had higher sensitivity and better broad-spectrum in detecting different Salmonella antibodies, and was suitable as a general diagnostic method for Salmonella.

[0144] .

Claims

1. Secretion of anti-equine Salmonella abortus ( Salmonella A hybridoma cell line producing a monoclonal antibody against groEL protein, characterized in that: The hybridoma cell line was named E11 and deposited at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Its culture deposit number is CGMCC No. 46349, and the deposit date is April 22, 2025.

2. A monoclonal antibody against groEL protein of Salmonella abortus equi, characterized in that: The monoclonal antibody is secreted by the hybridoma cell line according to claim 1.

3. Use of the hybridoma cell line according to claim 1 in preparing a reagent for detecting Salmonella, wherein the Salmonella is Salmonella abortus equi, Salmonella typhimurium ( Salmonella Typhi), Salmonella Dublin ( Salmonella Dublin) or Salmonella Enteritidis ( Salmonella Enteritidis).

4. Use of the monoclonal antibody according to claim 2 in the preparation of a reagent for detecting Salmonella, wherein the Salmonella is Salmonella abortus equi, Salmonella typhimurium, Salmonella dublin or Salmonella enteritidis.

5. A cELISA kit for detecting Salmonella, characterized in that: The kit comprises the monoclonal antibody against groEL protein of Salmonella abortus equi according to claim 2.

6. The cELISA kit according to claim 5, wherein The kit also includes a groEL protein-coated enzyme-labeled plate, a blocking solution, a PBST washing solution, an HRP-labeled anti-mouse IgG enzyme-labeled secondary antibody, a color development solution, and a stop solution. The amino acid sequence of the groEL protein is shown in SEQ ID NO.

1.

7. The cELISA kit according to claim 5 or 6, wherein The coating concentration of the groEL protein is 1 μg / ml, the dilution of the monoclonal antibody against the equine Salmonella abortus groEL protein is 0.2 μg / ml, and the concentration of the HRP-labeled anti-mouse IgG enzyme-labeled secondary antibody is 1:10,000.

8. Use of the cELISA kit according to any one of claims 5 to 7 in preparing a reagent for detecting Salmonella, wherein the Salmonella is Salmonella abortus equi, Salmonella typhimurium, Salmonella dublin or Salmonella enteritidis.

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