Application of SipD recombinant protein in preparation of indirect ELISA (enzyme-linked immuno sorbent assay) detection kit for detecting avian salmonella antibody

Abstract

The invention relates to the technical field of serological detection, in particular to application of SipD recombinant protein in preparation of an indirect ELISA (enzyme-linked immuno sorbent assay) detection kit for detecting an avian salmonella antibody. According to the invention, the specific antigen-SipD protein for detecting the avian salmonella antibody is successfully screened through a large number of researches. According to the method, an avian salmonella III type secretion system SipD subunit (SipD protein) is used as an antigen, a coating antigen (SipD recombinant protein) is obtained after protein expression and purification, and the avian salmonella indirect ELISA detection method which is high in sensitivity, high in specificity and good in repeatability is finally established through exploration and optimization of reaction conditions. Compared with a traditional slide agglutination antigen detection method, the method has the advantage that the detection flux and the detection efficiency are greatly improved. Establishment of the detection method has important public health significance on detection of salmonella in human beings, animals and food.

Description

Technical Field

[0001] The invention relates to the technical field of serological detection, and in particular to application of SipD recombinant protein in preparing an indirect ELISA detection kit for detecting avian salmonella antibodies. Background Art

[0002] Salmonella has a wide range of hosts and can colonize in the stomach and intestines of various livestock, poultry and wild animals. It is usually spread by contaminated water and food, causing group infection. Reports show that Salmonella has caused huge economic losses and medical burdens worldwide. Currently, there are 2,500 different serotypes of Salmonella that have been identified, which are widely distributed in livestock and wild animals. Salmonella can grow and multiply in animals. After infection, it will enter the intestinal mucosal cells, and then enter the lymphatic system and blood circulation, causing systemic infection. Salmonella is the main foodborne and waterborne bacteria that cause health epidemics. It can cause gastroenteritis, bacteremia and focal infection. After infection, it will cause symptoms such as high fever, diarrhea, and abdominal pain.

[0003] The virulence mechanism of Salmonella involves multiple factors, such as the attachment, invasion, growth and survival of bacteria. The pathogenicity of Salmonella mainly comes from virulence factors, such as lipopolysaccharide (LPS), virulence islands and virulence plasmids. The virulence island is an important region for Salmonella to express virulence genes and an important functional region for Salmonella to invade the host. Once Salmonella enters the body, virulence factors begin to regulate expression and activate acid resistance reactions, which helps bacteria survive in acidic environments and allows bacteria to enter the small intestine and colonize in energy-rich areas through flagella. The type III secretion system (T3SS) encoded by the virulence islands SPI-1 and SPI-2 of Salmonella transfers effector proteins to epithelial cells. In host cells, these effector proteins can induce cytoskeletal rearrangements.

[0004] T3SS is a transmembrane channel formed by more than 20 protein complexes, with a size of about 30-40kb, which is widely encoded in Gram-negative bacteria. T3SS can directly transport effector proteins produced by bacteria into the host cell, thereby affecting the internal homeostasis of the host cell. The effector proteins secreted by T3SS play a decisive role in the bacteria's escape from the immune system attack. T3SS is carefully coordinated by regulatory proteins and is highly regulated. It is one of the most complex bacterial secretion systems. T3SS has a unique needle-like structure. SipD protein is a key component of the needle-like structure of the T3SS system. It can deliver more than 30 bacterial virulence proteins such as effector proteins into host cells, thereby interfering with or destroying normal host cell intracellular proteins, causing signal transduction pathway disorders, and causing inflammation and innate immune responses in the host. At present, the sipD gene has been identified in the genomes of more than 30 different serotypes of Salmonella (Typhimurium, Schwarzengrund, Muenster, Agona, Indiana, Tennessee, Kottbus, Enteritidis, London, Braenderup, Hadar, Derby, Rissen, Corvallis, Senftenberg, Gallinarum, Albany, Kedougou, Bareilly, Fanti, Kentucky, Mbandaka, Pullorum, Saintpaul, Anatum, Goldcoast, Aberdeen, Thompson, Stanleyville, Montevideo), and the detection rate is particularly high in the common serotypes of highly pathogenic avian Salmonella (typhoid, enteritis, pullorum, Indiana). The SipD protein is relatively conservative in the genus Salmonella, and the protein has a high homology between different strains, so it can be used as an ideal target antigen for detecting common serotypes of avian Salmonella.

[0005] The detection methods for Salmonella typhimurium from chickens that have been reported so far are all pathogen detection technologies, and no serological detection technology is involved. Enzyme-linked immunosorbent assay (ELISA) is an enzyme-labeled solid immunoassay technology. Its basic principle is to bind the antigen (or antibody) to a solid phase carrier and connect the antigen (or antibody) with a certain enzyme to form an enzyme-labeled antigen (or antibody). During the test, the sample to be tested and the enzyme-labeled antigen (or antibody) are reacted with the antigen (or antibody) on the solid phase carrier according to a certain procedure, and then the unreacted part is removed by washing. After the substrate is added, the substrate is catalyzed by the enzyme bound to the solid phase carrier to produce a colored substance. By qualitatively or quantitatively detecting the amount of colored products, the content of the substance to be tested in the sample can be determined. This method has high sensitivity, strong specificity, a large amount of samples can be tested at one time, and the results can be read directly, which is convenient and fast. It is one of the hot spots of serological testing at present.

[0006] A literature search of the prior art revealed that no reports have been found on an indirect ELISA kit for detecting avian Salmonella antibodies using SipD protein as a target antigen and its application. Summary of the invention

[0007] The purpose of the present invention is to provide an application of SipD recombinant protein in the preparation of an indirect ELISA detection kit for detecting avian Salmonella antibodies, so as to solve the problems existing in the above-mentioned prior art. The SipD recombinant protein provided by the present invention is used as a coating antigen to effectively detect avian Salmonella, and has the advantages of high sensitivity and strong specificity.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides the use of a SipD recombinant protein in preparing an indirect ELISA detection kit for detecting avian Salmonella antibodies. The amino acid sequence of the SipD recombinant protein is shown in SEQ ID NO.3.

[0010] Preferably, the avian Salmonella includes Salmonella typhimurium, Salmonella pullorum, Salmonella enteritidis and Salmonella indiana.

[0011] The invention provides an indirect ELISA kit for detecting avian salmonella antibodies. The indirect ELISA kit comprises an ELISA plate coated with a SipD recombinant protein. The amino acid sequence of the SipD recombinant protein is shown in SEQ ID NO.3.

[0012] Preferably, the coating concentration of the SipD recombinant protein is 1 μg / mL.

[0013] Preferably, when the SipD recombinant protein is coated, the blocking solution used is 5% BSA, and the coating solution used is 0.05 mol / L carbonate buffer.

[0014] Preferably, the indirect ELISA kit further comprises HRP-labeled goat anti-rabbit IgG, a washing solution, a color developing solution and a stop solution.

[0015] Preferably, the dilution factor of the HRP-labeled goat anti-rabbit IgG is 2000 times.

[0016] Preferably, the stop solution is a 2M H2SO4 solution.

[0017] Further preferably, the indirect ELISA kit further comprises positive serum, negative serum and concentrated washing solution.

[0018] Preferably, the avian Salmonella includes Salmonella typhimurium, Salmonella pullorum, Salmonella enteritidis and Salmonella indiana.

[0019] Preferably, the preparation of the SipD recombinant protein comprises the following steps:

[0020] The SipD gene of avian Salmonella LT2 strain was cloned into a prokaryotic expression vector to obtain a recombinant vector;

[0021] Transforming the recombinant vector into Escherichia coli to obtain recombinant bacteria;

[0022] The recombinant bacteria are sequentially subjected to IPTG induction, centrifugation, ultrasonic disruption, purification and renaturation to obtain the SipD recombinant protein.

[0023] The present invention discloses the following technical effects:

[0024] The present invention has successfully screened out a specific antigen-SipD protein for detecting avian Salmonella antibodies through a large number of studies. The present invention uses the SipD subunit (SipD protein) of the type III secretion system of avian Salmonella as an antigen, obtains a coating antigen (SipD recombinant protein) after protein expression and purification, and finally establishes an indirect ELISA detection method for avian Salmonella with high sensitivity, strong specificity and good repeatability through exploration and optimization of reaction conditions. Each antigen-coated plate can detect 96 samples at a time. Compared with the traditional slide agglutination antigen detection method, the indirect ELISA detection method for avian Salmonella provided by the present invention greatly improves the detection throughput and detection efficiency. The establishment of this detection method has important public health significance for the detection of Salmonella in humans, animals and food. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example 1

[0031] 1. Amplification of target gene

[0032] According to the gene sequence of the chicken-derived Salmonella typhimurium LT2 strain (Genbank: NC_003197), a pair of cloning primers F1 / F2 targeting the sipD gene were designed. The 5' ends of the primers contained restriction endonuclease sites Nde I and HindIII, respectively. The primers were designed using Primer Premier 5.0 software and synthesized by GenScript. The primer sequences are as follows:

[0033] F1: 5'-CATATGCTAAATATACAAAAC-3', SEQ ID NO.1;

[0034] F2: 5'-AAGCTTTCATTAGTGGTGATG-3', SEQ ID NO. 2.

[0035] The genome of Salmonella enterica serovar Typhimurium LT2 (M McClelland, KE Sanderson, J Spieth, etc. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2, Nature. 413 (6858): 852-856) was used as a template and subjected to 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 56°C annealing for 30 s; 72°C extension for 70 s; a total of 35 cycles, and finally 72°C extension for 10 min to obtain a target gene band with a size of 1062 bp.

[0036] 2. Construction of prokaryotic expression plasmid

[0037] After the PCR product was subjected to agarose gel electrophoresis, the target band was cut under ultraviolet light, and the target fragment in the gel was recovered using a DNA rapid purification kit. The recovered PCR product was double-digested with Nde I and HindIII, and the plasmid pET-30a(+) was extracted and double-digested, and the digested product was ligated with T4 DNA ligase. The ligation product was transformed into DH5ɑ Escherichia coli competent cells, and the prokaryotic expression plasmid (pET-30a(+)-sipD vector) was successfully constructed through PCR identification and double-digestion identification.

[0038] 3. Induced expression of target protein

[0039] 1 μL of the recombinant pET-30a(+)-sipD vector was used to transform Escherichia coli BL21 (DE3), and the cells were heat-shocked at 42°C for 60 s and placed on ice for 90 s. The cells were then spread on an LB solid plate containing ampicillin at a final concentration of 100 μg / mL and cultured at 37°C overnight.

[0040] Pick a single colony of the expression bacteria containing the recombinant plasmid pET-30a(+)-sipD in 5mL LB medium (ampicillin concentration is 100μg / mL), and culture overnight at 37℃ and 180rpm. Inoculate the overnight cultured bacteria into 10mL LB medium at a ratio of 1:100, add ampicillin at a final concentration of 100μg / mL, and culture at 37℃ and 180rpm. When the OD value reaches 0.4, add IPTG at a final concentration of 1mM, 150rpm, 16℃ for induction overnight, and the sample without IPTG inducer is used as a negative control.

[0041] The cells were collected by centrifugation at 5000 rpm for 8 min, the supernatant was discarded, the cells were suspended in 1 mL of PBS (pH 7.4) buffer, ultrasonically disrupted for 5 min, ultrasonicated for 1 s and then stopped for 4 s, the supernatant and precipitate were collected by centrifugation, the precipitate was dissolved with 1 mL of inclusion body dissolution solution (8 M Urea, 50 mM Tris-HCl, 300 mM NaCl, pH 8.0), 40 μL of sample and 10 μL of 5×protein loading buffer were mixed, and the mixture was boiled in a boiling water bath for 15 min. SDS-PAGE was performed to determine the optimal induction conditions and protein expression form.

[0042] After determining the optimal induction conditions, induce the expression of fusion protein in large quantities. Inoculate the cultured bacterial solution into 2L LB liquid medium at a ratio of 1:50, add ampicillin at a final concentration of 100 μg / mL, and culture at 37°C and 180 rpm. When the OD value reaches 0.4, add IPTG at a final concentration of 1 mM, induce overnight at 16°C and 150 rpm, and collect bacterial cells by centrifugation.

[0043] 4. Purification and refolding of target protein (SipD recombinant protein)

[0044] The collected cells were dissolved in disruption buffer (50 mM Tris, 150 mM NaCl, 7 mM Gua-HCl, 0.1% TritonX-100, pH 8.0), and the cells were disrupted by ultrasound in an ice bath at a power of 400 W for 30 min (ultrasound for 2 s, pause for 5 s as one cycle). After ultrasound, centrifuge at 12000 rpm and 4°C for 30 min, and collect the supernatant for the next purification step.

[0045] Take 10mL Ni-NTA and wash the equilibrated column with 5 column volumes of Binding buffer at a flow rate of 4mL / min. Add the sample to be filtered to the Ni column at a flow rate of 1mL / min and collect the penetrated liquid. Wash the column with 5 column volumes of Binding buffer at a flow rate of 4mL / min. Wash the impurities with Washbuffer at a flow rate of 4mL / min and collect the eluate. Finally, elute with Elutionbuffer at a flow rate of 1mL / min and collect the eluate.

[0046] Confirm that the purified protein needs to be further renatured, and the component with the best purity is dialyzed into a mixed solution (2M urea, 50mM Tris, 300mM NaCl, pH 8.0). After 18 hours, the buffer is changed to (1×PBS, 2mM DTT, pH 8.5), and the dialysis is continued for 8 hours. After ultrafiltration and concentration, the solution is filtered through a 0.45μm filter membrane to obtain the target protein solution, which is divided into 1mL / tube and stored at -80°C; the amino acid sequence of the SipD recombinant protein in the protein solution is shown in SEQ ID NO.3, specifically:

[0047] MLNIQNYSASPHPGIVAERPQTPSASEHVETAVVPSTTEHRGTDIISLSQAATKIHQAQQTLQSTPPISEENNDERTLARQQLTSSLNALAKSGVSLSAEQNENLRSAFSAPTSALFSASPMAQPRTTISDAEIWDMVSQNISAIGDSYLGVYENVVAVYTDFYQAFSDILS KMGGWLLPGKDGNTVKLDVTSLKNDLNSLVNKYNQINSNTVLFPAQSGSGVKVATEAEARQWLSELNLPNSCLKSYGSGYVVDLTPLQKMVQDIDGLGAPGKDSKLEMDNAKYQAWQSGFKAQEENMKTTLQTLTQKYSNANSLYDNLVKVLSSTISSSLETAKSFLQG.

[0048] Example 2 Establishment of indirect ELISA detection method

[0049] 1. Determination of the optimal coating concentration of antigen and the optimal dilution of serum

[0050] The square array titration method was used to dilute the solution in the dialysis bag collected in step "4. Purification and refolding of target protein" of Example 1 by 2 times with a coating solution (0.05 mol / L carbonate buffer at pH 9.6), and the final concentrations were 0.5, 1, 2, 4, 8, and 16 μg / mL, respectively. 100 μL was added to each well, and each dilution was repeated in two rows, and the wells were placed at 4°C for overnight coating; then washed 5 times, 5 min each time; 200 μL of blocking solution (5% BSA) was added to each well, and the wells were blocked at 37°C for 2 h; washed 5 times, 5 min each time; the chicken Salmonella negative serum and positive serum were diluted with the blocking solution at 1:50, 1:100, 1:200, and 1:300, respectively. 1:400, 1:800 and 1:1600 dilutions were taken, 100 μL was added to each well, and incubated at 37°C for 1.5 h; washed 5 times, 5 min each time; HRP-labeled goat anti-rabbit IgG secondary antibody (1:2000 dilution) diluted in blocking solution was added to each well, 100 μL per well, incubated at 37°C for 1 h; washed 5 times, 5 min each time; finally, 100 μL of TMB color development solution was added to each well, and color was developed at 37°C in the dark for 10 min, and 50 μL of reaction stop solution (2 mol / L H2SO4 solution) was added to each well, and the absorbance value (OD) at a wavelength of 450 nm was read with an enzyme reader. 450 ). Select the OD of the positive serum well 450 The value is around 1.0, and the OD of the negative serum well 450 The antigen concentration and serum dilution when the value is around 0.1 and the P / N value is the largest are taken as the optimal antigen coating concentration and serum dilution.

[0051] The results showed that when the recombinant antigen coating concentration was 1 μg / mL and the serum dilution was 1:100, the positive serum OD 450 Values ​​close to 1, negative serum OD 450 The value is close to 0.1, and the P / N value is the largest. Therefore, the optimal antigen coating concentration is determined to be 1 μg / mL, and the optimal serum dilution is 1:100.

[0052] 2. Determination of blocking solution

[0053] The ELISA plate was coated with the optimal antigen concentration, incubated at 4°C overnight and washed, and then 5 groups of different blocking solutions were added: 1% BSA for group 1, 5% BSA for group 2, 1% FBS for group 3, 5% skim milk powder for group 4, and 2% gelatin for group 5. After blocking, the optimal serum dilution was used for reaction, and HRP-labeled goat anti-rabbit IgG secondary antibody was added after washing. Finally, TMB was added for color development and the stop solution was added. OD was read. 450 The readings and P / N values ​​of each group were compared, and the blocking solution with the largest P / N value was selected as the optimal blocking solution.

[0054] The results showed that there was no granular precipitate after washing the ELISA plate blocked with 5% BSA, and the P / N value was the largest compared with the control plate. Therefore, 5% BSA was selected as the best blocking solution.

[0055] 3. Determination of the optimal serum reaction time

[0056] The ELISA plate was coated with the optimal antigen concentration, coated overnight at 4°C and washed, then blocked with the optimal blocking solution for 2 hours, and negative and positive serum diluted with the optimal dilution multiples were added respectively. The plates were divided into 4 groups according to the different incubation times at 37°C: Group 1 for 30 minutes, Group 2 for 45 minutes, Group 3 for 60 minutes, and Group 4 for 90 minutes. After washing, HRP-labeled goat anti-rabbit IgG secondary antibody was added, and finally TMB was added for color development and stop solution was added. OD was read. 450 Values ​​were obtained, and the readings and P / N values ​​of each group were compared. The serum reaction time when the P / N value was the largest was selected as the optimal serum reaction time.

[0057] The results showed that the P / N value was the highest when the primary antibody serum was exposed to 37°C for 90 minutes. Therefore, the optimal exposure time for the serum to be tested was determined to be 90 minutes.

[0058] 4. Determination of enzyme-labeled secondary antibody reaction concentration and time

[0059] The ELISA plate was coated at the optimal antigen concentration, coated overnight at 4°C and washed, then blocked for 2 hours with the optimal blocking solution, and negative and positive sera diluted at the optimal dilution were added for the optimal reaction time. The plates were divided into 4 groups according to the dilution multiples of the HRP-labeled goat anti-rabbit IgG secondary antibody: Group 1 was 1:5000, Group 2 was 1:3000, Group 3 was 1:2000, and Group 4 was 1:1000. Keeping other conditions unchanged, read and compare the OD values ​​of the positive and negative sera in each group. 450 The dilution factor with the maximum P / N value was selected as the optimal reaction concentration of the enzyme-labeled secondary antibody.

[0060] The ELISA plate was coated at the optimal antigen concentration, coated overnight at 4°C and washed, then blocked for 2 hours with the optimal blocking solution, and the negative and positive sera diluted at the optimal dilution were added for the optimal reaction time. The plate was divided into 4 groups according to the reaction time of HRP-labeled goat anti-rabbit IgG secondary antibody at 37°C: Group 1 was 15 minutes, Group 2 was 30 minutes, Group 3 was 45 minutes, and Group 4 was 60 minutes. Keeping other conditions unchanged, the OD values ​​of the positive and negative sera in each group were read and compared. 450 The dilution factor with the maximum P / N value was selected as the optimal reaction time for the enzyme-labeled secondary antibody.

[0061] The results showed that when the dilution of the HRP-labeled goat anti-rabbit IgG secondary antibody was 1:2000, the P / N value was the largest. Therefore, 1:2000 was determined as the optimal dilution of the secondary antibody. When the reaction time of the HRP-labeled goat anti-rabbit IgG secondary antibody was 60 min, the P / N value was the largest. Therefore, the optimal reaction time of the HRP-labeled goat anti-rabbit IgG secondary antibody was determined to be 60 min.

[0062] 5. The operation procedure of indirect ELISA is shown in Table 1.

[0063] Table 1 Operation procedure of indirect ELISA

[0064]

[0065] Example 3 Determination of the cut-off value for positive and negative results of the indirect ELISA detection method

[0066] Using the indirect ELISA method established under the optimal conditions determined in Example 2, 20 chicken-derived Salmonella negative sera were detected. According to the obtained OD 450 values, the overall mean (X) and standard deviation (S) were calculated. According to statistical principles, when the OD 450 value ≥ X + 3S, it can be judged as positive; when the OD 450 value ≤ X + 2S, it can be judged as negative; when X + 2S < OD 450 value < X + 3S, the sample is judged as suspicious.

[0067] According to the calculated average OD 450 value of 0.203 and standard deviation of 0.028. According to the test results, when the OD 450 value of the serum sample ≥ 0.287, it is judged as positive; when the OD 450 value ≤ 0.259, it is judged as negative; when 0.259 < OD 450 value < 0.287, it is judged as suspicious.

[0068] Example 4 Specificity of the indirect ELISA detection method

[0069] Using the established method, 3 positive sera of 4 common Salmonella serotypes such as Salmonella typhimurium, Salmonella pullorum, Salmonella enteritidis, and Salmonella indiana, 3 positive sera of 3 common pathogenic bacteria of non-Salmonella genera such as Escherichia coli, Pasteurella multocida, and Haemophilus paragallinarum, and 3 positive sera of 3 common viral diseases such as avian influenza H5 subtype, Newcastle disease, and infectious bursal disease of chickens (all the above sera were chicken-derived and prepared by ourselves) were detected respectively. To determine whether there was cross-reaction to test the specificity of the ELISA method, the results are shown in Table 2. The results showed that the results of 3 non-Salmonella genus bacteria and 3 viral sera were all judged as negative. While the sera prepared by the method of artificial infection with 4 chicken Salmonella were all positive.

[0070] It can be seen from this that the indirect ELISA detection method for Salmonella typhimurium established in the present invention has high specificity.

[0071] Table 2 Specificity of indirect ELISA detection method

[0072]

[0073]

[0074] Example 5 Sensitivity of indirect ELISA detection method

[0075] Randomly select 1 portion each of the positive chicken serum of Salmonella typhimurium, the positive chicken serum of Salmonella pullorum, the positive chicken serum of Salmonella enteritidis and the positive chicken serum of Salmonella Indiana, and dilute them to 1:50-1:5000 respectively. The indirect ELISA method established by the optimal conditions determined in Example 2 was used for detection to verify its sensitivity, and the results are shown in Table 3. The results show that the indirect ELISA kit has a detection limit of 1:1000 for the chicken serum infected with Salmonella typhimurium and the chicken serum infected with Salmonella Indiana, a detection limit of 500 for the chicken serum infected with Salmonella enteritidis, a detection limit of 1:500 for the chicken serum infected with Salmonella pullorum, and a detection limit of 1:1000 for the chicken serum infected with Salmonella enteritidis, and a detection limit of 1:1000 for the chicken serum infected with Salmonella pullorum, and a detection limit of 1:1000 for the other two strains.

[0076] Table 3 Sensitivity of indirect ELISA detection method

[0077] Serial number Serum type result 1 Salmonella typhimurium infected chicken serum 1：1000(3 / 3) 2 Chicken serum infected with Salmonella pullorum 1：500(1 / 3)，1：1000(2 / 3) 3 Salmonella Enteritidis infected chicken serum 1：500(3 / 3) 4 Salmonella Indiana infected chicken serum 1：1000(3 / 3)

[0078] Example 6 Repeatability of the indirect ELISA detection method

[0079] 1. Intra-batch reproducibility test

[0080] The same batch of antigen-coated plates were tested with the established ELISA method for 5 consecutive days for the same 5 random serum samples. The intra-batch repeatability test showed that the coefficient of variation of the test results was between 1.06% and 3.82%.

[0081] 2. Batch-to-batch reproducibility test

[0082] Plates coated with different batches were used to test another 5 random serum samples under the same conditions using the established ELISA method for batch repeatability testing. The coefficient of variation of the test results was between 4.52% and 6.49%.

[0083] Example 7 Assembly of Indirect ELISA Detection Kit

[0084] The assembly of the indirect ELISA detection kit is shown in Table 4.

[0085] Table 4 Composition of indirect ELISA test kit

[0086]

[0087]

[0088] Example 8 Compliance of Indirect ELISA Detection Kit

[0089] Three randomly selected chicken sera positive for Salmonella typhimurium, three chicken sera positive for Salmonella pullorum, three chicken sera positive for Salmonella enteritidis, and three chicken sera positive for Salmonella Indiana were tested, and the indirect ELISA test kit established in Example 7 was used for comparison with the serum agglutination method;

[0090] The coincidence rate of the detection results between the indirect ELISA detection kit established by the recombinant coated antigen protein of this embodiment and the serum agglutination method is compared and analyzed, and the results are shown in Table 5 and Table 6. The results show that the detection results of the serum antibodies of four Salmonella-positive chickens by the two methods are all positive, with a coincidence rate of 100%.

[0091] Table 5 Compliance results of the indirect ELISA test kit established in Example 7

[0092] Serial number Serum type result 1 Salmonella typhimurium infected chicken serum +(3 / 3) 2 Chicken serum infected with Salmonella pullorum +(3 / 3) 3 Salmonella Enteritidis infected chicken serum +(3 / 3) 4 Salmonella Indiana infected chicken serum +(3 / 3)

[0093] Table 6 Compliance results of serum agglutination method

[0094] Serial number Serum type result 1 Salmonella typhimurium infected chicken serum +(3 / 3) 2 Chicken serum infected with Salmonella pullorum +(3 / 3) 3 Salmonella Enteritidis infected chicken serum +(3 / 3) 4 Salmonella Indiana infected chicken serum +(3 / 3)

[0095] Example 9 Clinical application of indirect ELISA detection method

[0096] The indirect ELISA test kit established in Example 7 was used for testing, and chicken serum was tested on chicken farms where Salmonella typhimurium, Salmonella pullorum, Salmonella enteritidis or Salmonella Indiana had occurred. A total of 22 clinical chicken sera were tested, and the results are shown in Table 7. The results show that the detection positive rate was 40.9% (9 / 22). It is closely related to factors such as the time of occurrence of Salmonella infection, the antibody level of the chickens, and the sensitivity of the detection method.

[0097] Table 7 Test results of indirect ELISA test kit

[0098]

[0099]

[0100] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of SipD recombinant protein in the preparation of an indirect ELISA test kit for detecting avian Salmonella antibodies, characterized in that: The amino acid sequence of the SipD recombinant protein is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: The avian Salmonella include Salmonella typhimurium, Salmonella pullorum, Salmonella enteritidis and Salmonella indiana.

3. An indirect ELISA kit for detecting avian Salmonella antibodies, characterized in that: The indirect ELISA kit comprises an ELISA plate coated with a SipD recombinant protein; the amino acid sequence of the SipD recombinant protein is shown in SEQ ID NO.

3.

4. The indirect ELISA kit according to claim 3, characterized in that The coating concentration of the SipD recombinant protein is 1 μg / mL.

5. The indirect ELISA kit according to claim 3, characterized in that When the SipD recombinant protein was coated, the blocking solution used was 5% BSA, and the coating solution used was 0.05 mol / L carbonate buffer.

6. The indirect ELISA kit according to claim 3, characterized in that The indirect ELISA kit also includes HRP-labeled goat anti-rabbit IgG, washing solution, color developing solution and stop solution.

7. The indirect ELISA kit according to claim 6, characterized in that The dilution factor of the HRP-labeled goat anti-rabbit IgG was 2000 times.

8. The indirect ELISA kit according to claim 6, characterized in that The stop solution is 2M H2SO4 solution.

9. The indirect ELISA kit according to claim 3, characterized in that: The avian Salmonella include Salmonella typhimurium, Salmonella pullorum, Salmonella enteritidis and Salmonella indiana.

10. The indirect ELISA kit according to claim 3, characterized in that: The preparation of the SipD recombinant protein comprises the following steps: The SipD gene of avian Salmonella LT2 strain was cloned into a prokaryotic expression vector to obtain a recombinant vector; Transforming the recombinant vector into Escherichia coli to obtain recombinant bacteria; The recombinant bacteria are sequentially subjected to IPTG induction, centrifugation, ultrasonic disruption, purification and renaturation to obtain the SipD recombinant protein.

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