A recombinant LI0902 protein, its preparation method and application, and an indirect ELISA kit.

Abstract

This invention belongs to the field of veterinary diagnostics and discloses a LI0902 recombinant protein, its preparation method and application, and an ELISA kit. The LI0902 recombinant protein used in this invention exhibits good antigenicity. The indirect ELISA method established and optimized based on this protein shows no cross-reactivity with positive sera from common swine pathogens such as porcine circovirus, porcine epidemic diarrhea virus, porcine rotavirus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus. It can detect positive sera diluted to 1:800, with intra- and inter-assay coefficients of variation both less than 10%, indicating that the method has good specificity, sensitivity, and reproducibility, and the results are reliable and stable. Furthermore, the indirect ELISA kit of this invention has clearly defined components, a standardized operating procedure, and a short detection time, making it suitable for large-scale serological screening, epidemiological surveys, and vaccine efficacy evaluation in pig farms, breeding groups, animal health companies, and third-party testing institutions, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of veterinary diagnostic technology, and in particular to a LI0902 recombinant protein, its preparation method and application, and an ELISA kit. Background Technology

[0002] Lawsonia intracellularis is a major pathogen causing proliferative enteritis in pigs, clinically manifesting as acute hemorrhagic enteritis or chronic intestinal adenomatosis. This leads to stunted growth, reduced feed conversion ratios, and even death in affected pigs, causing significant economic losses to the global pig industry. This bacterium is an obligate intracellular parasite, making traditional pathogen isolation and culture difficult. Serological detection, especially the ELISA method, has become a key tool for epidemiological surveillance and disease eradication due to its ease of operation, high throughput, and suitability for large-scale screening.

[0003] Existing commercially available ELISA kits for detecting Lawsonia intracellularis antibodies in porcines mostly utilize whole-cell antigens or specific recombinant proteins. Whole-cell antigens may exhibit cross-reactivity with other pathogens, and their preparation process is complex. Furthermore, some reported recombinant protein antigens (such as LsaA) show high homology with Mycoplasma hyopneumoniae, potentially leading to decreased detection specificity and increased false-positive risk. Therefore, identifying and utilizing target proteins with high specificity and high immunoreactivity to establish superior serological detection methods is of great significance for the precise prevention and control of this disease.

[0004] Therefore, the technical problem to be solved by the present invention is: how to provide an indirect ELISA detection kit with high specificity, high sensitivity and good repeatability. Summary of the Invention

[0005] The purpose of this invention is to provide a LI0902 recombinant protein, the amino acid sequence of which is shown in SEQ ID NO: 1 and the nucleotide sequence of which is shown in SEQ ID NO: 2; using this protein as a coating antigen, an indirect ELISA kit for detecting antibodies against Lawsonia intracellularis of porcine bacteria is prepared, which has the advantages of high specificity, high sensitivity and good reproducibility.

[0006] Meanwhile, this invention discloses the application of the LI0902 recombinant protein, an indirect ELISA kit containing the LI0902 recombinant protein, and an indirect ELISA method.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A recombinant protein LI0902, wherein the amino acid sequence of the recombinant protein LI0902 is shown in SEQ ID NO: 1 and the nucleotide sequence is shown in SEQ ID NO: 2.

[0009] Preferably, the preparation method of the LI0902 recombinant protein is as follows: the nucleotide sequence shown in SEQ ID NO: 2 is amplified by PCR technology, the nucleotide sequence is cloned into the pGEX-6p-1 prokaryotic expression vector, the prokaryotic expression vector is then transformed into Escherichia coli BL21(DE3) competent cells, and then induced expression and purification are performed to obtain the protein.

[0010] Furthermore, this invention discloses the application of the LI0902 recombinant protein as described above in the preparation of antibody reagents for detecting Lawsonia intracellularis in porcine cells.

[0011] Furthermore, this invention discloses the application of the LI0902 recombinant protein as described above in the preparation of an indirect ELISA kit for detecting antibodies against Lawsonia intracellularis in porcine cells.

[0012] Furthermore, this invention discloses an indirect ELISA kit for detecting antibodies against Lawsonia intracellularis in porcine cells, wherein the kit contains the LI0902 recombinant protein as described above as a coating antigen.

[0013] Preferably, the kit further comprises coating solution, blocking solution, washing solution, enzyme-labeled secondary antibody, chromogenic solution, stop solution, positive control serum and negative control serum.

[0014] More preferably, the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-pig IgG antibody.

[0015] Finally, this invention discloses an indirect ELISA method for detecting antibodies against Lawsonia intracellularis in porcine cells. The specific method is as follows: the serum to be tested is detected using the indirect ELISA kit described above.

[0016] Preferably, the procedure specifically includes the following steps:

[0017] (1) Dilute the LI0902 recombinant protein to the optimal coating concentration with coating buffer, coat it on an ELISA plate, and incubate overnight at 4°C;

[0018] (2) After washing with washing solution, add sealing solution for sealing;

[0019] (3) After washing with washing solution, add the diluted serum sample to be tested and incubate;

[0020] (4) After washing with washing solution, add diluted enzyme-labeled secondary antibody and incubate;

[0021] (5) After washing with washing solution, add colorimetric solution for color development, then add stop solution to terminate the reaction, and measure OD using an enzyme-linked immunosorbent assay (ELISA) reader. 450 nm value;

[0022] (6) Determine the result based on the critical value: when the OD of the sample 450When the nm value is greater than or equal to the critical value, it is determined to be positive for Lawsonia intracellularis antibody; when the OD value is greater than or equal to the critical value, it is determined to be positive for Lawsonia intracellularis antibody; 450 When the nm value is less than the critical value, it is judged as negative.

[0023] More preferably, the optimal coating concentration of the LI0902 recombinant protein in step (1) is 4 μg / mL; the optimal blocking solution in step (2) is 5% skim milk, and the blocking condition is incubation at 37°C for 1.5 hours; the optimal dilution of the serum to be tested in step (3) is 1:100, and the incubation condition is incubation at 37°C for 30 minutes; the optimal dilution of the enzyme-labeled secondary antibody in step (4) is 1:1000, and the incubation condition is incubation at 37°C for 45 minutes; and the optimal color development time of the colorimetric solution in step (5) is 15 minutes.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] The LI0902 recombinant protein selected in this invention exhibits good antigenicity. The indirect ELISA method established and optimized based on this protein shows no cross-reactivity with positive sera from common swine pathogens such as porcine circovirus, porcine epidemic diarrhea virus, porcine rotavirus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus. It can detect positive sera diluted to 1:800, with intra- and inter-assay coefficients of variation both less than 10%, indicating good specificity, sensitivity, and reproducibility, and reliable and stable results. Furthermore, the indirect ELISA kit of this invention has clearly defined components, standardized operating procedures, and a short detection time, making it suitable for large-scale serological screening, epidemiological surveys, and vaccine efficacy evaluation in pig farms, breeding groups, animal health companies, and third-party testing institutions, demonstrating promising application prospects. Attached Figure Description

[0026] Figure 1 This is an SDS-PAGE image of the recombinant LI0902-GST protein. In the image, M: protein molecular weight standard; 1: whole cell protein expressed by empty vector; 2: uninduced whole cell protein; 3: supernatant from cell sonication; 4: cell precipitate from sonication; 5: purified recombinant LI0902-GST protein; 6: recombinant LI0902 protein after GST tag digestion.

[0027] Figure 2 This is a Western blot diagram of the LI0902 recombinant protein. In the diagram, M: protein molecular weight standard; 1: reaction of purified LI0902 recombinant protein with porcine Lawsonia intracellularis-positive porcine serum;

[0028] Figure 3 This study determined the cut-off values ​​for positive and negative results using an indirect ELISA method. The values ​​were calculated by testing 48 negative serum samples. As a criterion for judgment. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. It should be noted that, where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0030] Materials and reagent information:

[0031] E. coli DH5α competent cells were purchased from TransGen Biotech Ltd.

[0032] pGEX-6P-1, E. coli Rosetta (DE3) competent cells, positive porcine circovirus, porcine epidemic diarrhea virus, porcine rotavirus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus, as well as negative and positive porcine sera of Lawsonia intracellularis, are preserved at the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0033] Ampicillin was purchased from Solarbio Biotechnology Co., Ltd.

[0034] HRP-labeled goat anti-pig IgG was purchased from Jackson Immuno Research Laboratories, USA.

[0035] The GST affinity chromatography column was purchased from Cytiva, USA.

[0036] The BCA protein concentration assay kit and ELISA antigen-coated plate were both purchased from Thermofisher.

[0037] Homologous recombination kit and plasmid extraction kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0038] The SDS-PAGE kit was purchased from Shanghai Yamei Biomedical Technology Co., Ltd.

[0039] The ultrafiltration tubes were purchased from Millipore.

[0040] PreScission Protease was prepared in our laboratory.

[0041] 780 clinical swine serum samples were collected from 9 pig farms in 5 regions of Guangdong, 1 pig farm in 1 region of Guangxi, and 2 pig farms in 2 regions of Yunnan.

[0042] Example 1: Expression and purification of recombinant protein LI0902

[0043] 1. Based on the genomic sequence of the reference strain *L. suis* (AM180252.1), specific primers for the gene LI0902 (SEQ ID NO: 2) were designed and synthesized, introducing BamHI and XhoI restriction sites. PCR amplification was performed using *L. suis* genomic DNA as a template, and the amplification products were recovered. The pGEX-6p-1 vector was double-digested with BamHI and XhoI, recovered, and ligated using a seamless cloning ligase to construct the recombinant expression plasmid pGEX-6p-1-LI0902. The correctly identified recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and inoculated into LB broth containing ampicillin and cultured at 37°C until OD2000. 600 When the bacterial cell density was approximately 0.6–0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 16°C for 16–18 hours. The cells were collected, sonicated, and the supernatant was collected by centrifugation. The recombinant protein was purified using a GST affinity chromatography column to obtain high-purity LI0902-GST recombinant protein, approximately 61.9 kDa. After adding PreScission Protease to cleave the GST tag, the LI0902 recombinant protein, approximately 35.1 kDa, was obtained. Figure 1 Western blot analysis confirmed that the purified protein specifically reacted with porcine Lawsonia intracellularis positive serum. Figure 2 ).

[0044] The amino acid sequence of the LI0902 recombinant protein is shown in SEQ ID NO: 1:

[0045] MKIIHSAIFAVTLLTAWSTVCFAAEVTASCTKRVESYNYLVDYSGSMMMKHVAVREPKIELAKEAILKINAAMPKMSYQGGLYTFAPYSVIIPQGSWNSCVAECAVNTIKSDLEIFGRLTPMGDGIKMHETVINQMPPQAAVILLTDGHNNLGMNPVEEVKSIYQTNPNV CFHVVSFADDAEGKAIIDQIVALNSGSVLVDGLQLLQNPAVCQEFVNSVFCQEQILVTEEVVVLRGVNFAFDSFALDDTAKAILEETVRLIRANPDFNVRLLGWTDSTGPDAYNLRLSQERADAVKNFLVKMGIPSNRLFAKGMGKSYQYNNATKEGRYMNRRTELVFFD;

[0046] The nucleotide sequence of the LI0902 recombinant protein is shown in SEQ ID NO: 2:

[0047]

[0048] Example 2: Establishment and Optimization of Indirect ELISA Detection Method

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

[0050] Matrix titration was used. The LI0902 recombinant protein was diluted with coating buffer to eight concentrations (8, 7, 6, 5, 4, 3, 2, and 1 μg / mL) to coat ELISA plates. Positive and negative control sera were diluted with sample diluent at ratios of 1:20, 1:50, 1:100, 1:200, 1:500, and 1:1000, respectively. The optimal conditions were determined based on maximizing the P / N ratio (positive serum OD value / negative serum OD value), as shown in Table 1. The optimal conditions were: antigen coating concentration of 4 μg / mL and serum dilution of 1:100.

[0051] Table 1 Determination of Optimal Antigen Coating Concentration and Serum Dilution

[0052]

[0053] 2. Optimization of other conditions

[0054] Determining the optimal sealing solution and optimal sealing time:

[0055] Based on the screening criteria, 1% BSA, 5% skim milk, and 2% fish skin gelatin were used for blocking at 37℃ for 1, 1.5, and 2 hours, respectively. The optimal blocking solution was determined to be 5% skim milk, with the highest possible P / N ratio (positive serum OD value / negative serum OD value). The results are shown in Table 2. The optimal blocking solution was determined to be 5% skim milk, and the optimal blocking time was 2 hours.

[0056] Table 2 Determination of Optimal Sealing Solution and Optimal Sealing Time

[0057]

[0058] Determining the optimal serum incubation time:

[0059] Based on the screening criteria, serum was added and incubated at 37°C for 0.5, 1, and 1.5 hours, respectively. Using the maximum P / N ratio (OD value of positive serum / OD value of negative serum) as the guiding principle, the results are shown in Table 3, indicating that the optimal serum incubation time was 0.5 hours.

[0060] Table 3 Determination of Optimal Serum Incubation Time

[0061] P 1.319 1.788 2.321 N 0.127 0.230 0.306 P / N 10.360 7.760 7.594

[0062] Determination of optimal enzyme-labeled secondary antibody dilution and optimal enzyme-labeled secondary antibody incubation time:

[0063] Based on the screening criteria, goat anti-porcine IgG-HRP was diluted at ratios of 1:1000, 1:2000, 1:4000, and 1:8000 and added to the solution, respectively, and incubated at 37℃ for 30, 45, and 60 min, respectively. Using the highest P / N value (positive serum OD value / negative serum OD value) as the criterion, the results are shown in Table 4. The optimal enzyme-labeled secondary antibody dilution was determined to be 1:1000, and the optimal incubation time was 45 min.

[0064] Table 4 Determination of Optimal Enzyme-Labeled Secondary Antibody Dilution and Incubation Time

[0065]

[0066] Determining the optimal TMB color development time:

[0067] Based on the screening criteria, the TMB chromogenic solution was added and incubated at 37℃ for 3, 5, 10, and 15 min, respectively. Using the maximum P / N ratio (positive serum OD value / negative serum OD value) as the guiding principle, the results are shown in Table 5, indicating that the optimal TMB chromogenic time was 15 min.

[0068] Table 5 Determination of Optimal TMB Developing Time

[0069] P 0.414 0.525 0.897 1.508 N 0.078 0.093 0.142 0.209 P / N 5.289 5.645 6.307 7.201

[0070] Determining the critical value:

[0071] 48 negative serum samples were tested under optimized reaction conditions. Figure 3 ), calculate its OD 450 average value( The value was 0.206, and the standard deviation (s) was 0.052. According to... The +3s calculation determined the cut-off value to be 0.362. Therefore, when the sample OD... 450 A value ≥ 0.362 is considered positive, and a value < 0.362 is considered negative.

[0072] Example 3: Evaluation of the specificity, sensitivity, and repeatability of indirect ELISA

[0073] Specificity tests for indirect ELISA:

[0074] The optimized iELISA method was used to detect positive swine serum antibodies against porcine circovirus (PCV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), and African swine fever virus (ASFV). Negative and positive swine serum antibodies against Lawsonia intracellularis (LI) were used as positive and negative controls to evaluate the specificity of the method. Three replicate controls were set up for each serum sample. The results are shown in Table 6. The results show that, except for LI antibody-positive swine serum, the detection results for the other viruses in positive swine serum were negative, indicating that the method has high specificity.

[0075] Table 6 Specificity tests for indirect ELISA

[0076]

[0077] Sensitivity testing for indirect ELISA:

[0078] Porcine serum positive for Lawsonia intracellularis (LI) antibodies was serially diluted 2-fold in the range of 1:100 to 1:12800, and then detected using the established indirect ELISA method to verify its sensitivity. Three replicate controls were set up for each dilution. The results are shown in Table 7. The results show that the detection result remained positive even when the LI antibody-positive porcine serum was diluted to 1:800, indicating that the iELISA method has high sensitivity.

[0079] Table 7 Sensitivity test of indirect ELISA

[0080]

[0081] Repeatable tests of indirect ELISA:

[0082] Four positive serum samples and four negative serum samples were randomly selected for repeatability testing, with each test performed in triplicate. Three ELISA plates coated from the same batch were used for intra-batch repeatability testing; three ELISA plates coated from different batches were used for inter-batch repeatability testing. The coefficient of variation (CV) was calculated based on the test results to assess the repeatability of the method. The results are shown in Table 8. The results show that the intra-batch CV was less than 5%, the inter-batch CV was less than 7%, and the intra-batch and inter-batch CVs were all less than 10%, indicating that the method has good repeatability.

[0083] Table 8. Repeatability tests of indirect ELISA

[0084]

[0085] Example 4: Clinical Serum Sample Detection

[0086] The indirect ELISA method established in this study was used to detect LI antibody in 780 clinical swine serum samples from Guangdong, Guangxi, and Yunnan provinces. The results showed that the LI antibody positivity rate was 19.7% (130 / 660) in Guangdong, 60% (6 / 10) in Guangxi, and 55.5% (61 / 110) in Yunnan, with an overall positivity rate of 27.4% (214 / 780).

[0087] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A LI0902 recombinant protein, characterized in that, The amino acid sequence of the LI0902 recombinant protein is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO:

2.

2. The LI0902 recombinant protein according to claim 1, characterized in that, The preparation method of the LI0902 recombinant protein is as follows: the nucleotide sequence shown in SEQ ID NO: 2 is amplified by PCR technology, the nucleotide sequence is cloned into the pGEX-6p-1 prokaryotic expression vector, the prokaryotic expression vector is then transformed into Escherichia coli BL21(DE3) competent cells, and then obtained by induction expression and purification.

3. The use of the LI0902 recombinant protein as described in any one of claims 1 or 2 in the preparation of antibody reagents for detecting Lawsonia intracellularis in pigs.

4. The use of the LI0902 recombinant protein as described in any one of claims 1 or 2 in the preparation of an indirect ELISA kit for detecting antibodies against Lawsonia intracellularis.

5. An indirect ELISA kit for detecting antibodies against Lawsonia intracellularis in porcine bacteria, characterized in that, The kit contains the LI0902 recombinant protein as described in any one of claims 1 to 2 as a coating antigen.

6. The indirect ELISA kit according to claim 5, characterized in that, The kit also includes coating solution, blocking solution, washing solution, enzyme-labeled secondary antibody, chromogenic solution, stop solution, positive control serum and negative control serum.

7. The indirect ELISA kit according to claim 6, characterized in that, The enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-pig IgG antibody.

8. An indirect ELISA method for detecting antibodies against Lawsonia intracellularis in porcine bacteria, characterized in that, The specific method is as follows: the serum to be tested is detected using the indirect ELISA kit as described in any one of claims 5 to 7.

9. The indirect ELISA method according to claim 8, characterized in that, Specifically, the following steps are included: (1) Dilute the LI0902 recombinant protein to the optimal coating concentration with coating buffer, coat it on an ELISA plate, and incubate overnight at 4°C; (2) After washing with washing solution, add sealing solution for sealing; (3) After washing with washing solution, add the diluted serum sample to be tested and incubate; (4) After washing with washing solution, add diluted enzyme-labeled secondary antibody and incubate; (5) After washing with washing solution, add colorimetric solution for color development, then add stop solution to terminate the reaction, and measure OD using an enzyme-linked immunosorbent assay (ELISA) reader. 450 nm value; (6) Determine the result based on the critical value: when the OD of the sample 450 When the nm value is greater than or equal to the critical value, it is determined to be positive for Lawsonia intracellularis antibody; when the OD value is greater than or equal to the critical value, it is determined to be positive for Lawsonia intracellularis antibody; 450 When the nm value is less than the critical value, it is judged as negative.

10. The indirect ELISA method according to claim 9, characterized in that, The optimal coating concentration of the LI0902 recombinant protein in step (1) is 4 μg / mL; the optimal blocking solution in step (2) is 5% skim milk, and the blocking condition is incubation at 37°C for 1.5 hours; the optimal dilution of the serum to be tested in step (3) is 1:100, and the incubation condition is incubation at 37°C for 30 minutes; the optimal dilution of the enzyme-labeled secondary antibody in step (4) is 1:1000, and the incubation condition is incubation at 37°C for 45 minutes; the optimal color development time of the colorimetric solution in step (5) is 15 minutes.

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