A multi-epitope fusion antigen of multiple toxins and fimbriae of Escherichia coli from porcine diarrhea and its application

Abstract

The present invention provides a multi-epitope fusion antigen of multiple toxins and fimbriae of Escherichia coli causing swine diarrhea and its application. The amino acid sequence of the multi-epitope fusion antigen is shown in SEQ ID NO.1. The multi-epitope fusion antigen MEAET prepared by the present invention can be used to prepare antibodies and can also be used to prepare products for detecting antibodies against pathogenic Escherichia coli. The present invention analyzed the epitopes of multiple toxin / fimbria antigens of Escherichia coli causing swine diarrhea, and after optimizing the arrangement of multiple antigen epitopes, MEAET was obtained, which can simultaneously recognize antibodies against multiple antigens. The specificity test of indirect ELISA shows that MEAET does not cross-react with the positive sera of other common pathogens, only has a good reaction with the positive sera of Escherichia coli causing swine diarrhea, and has a small cross-reaction with antibodies of commensal Escherichia coli, which is suitable for detecting the antibody situation of Escherichia coli causing swine diarrhea, facilitating the detection of large-scale samples and epidemiological monitoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine antigen preparation, and particularly relates to a multi-epitope fusion antigen of various toxins and fimbriae of Escherichia coli from swine diarrhea and its application. Background Art

[0002] There are many pathogenic subtypes of Escherichia coli. Enteropathogenic Escherichia coli (EPEC), Enterohemorrhagic Escherichia coli (EHEC), Enterotoxigenic Escherichia coli (ETEC), Enteroaggregative Escherichia coli (EAEC), and Enteroinvasive Escherichia coli (EIEC) are the five main subtypes causing intestinal infections. Among them, ETEC is one of the main pathogens causing bacterial diarrhea in humans and young livestock, especially the yellow and white scour of neonatal piglets, with high morbidity and mortality.

[0003] The Escherichia coli causing piglet diarrhea mainly causes diseases through two types of virulence factors: various toxins and fimbrial adhesins. Toxins include three enterotoxins (STa, STb, LT), hemolysin (HlyCABD), Shiga toxin (Stx1, Stx2), etc., and adhesins include F4 / K88, F18 fimbriae, etc. In addition, the intimin receptor Tir related to the secretion system is also one of the important pathogenic factors. During the infection process, Escherichia coli adheres to the surface of the intestinal mucosa of piglets through fimbriae and releases various toxins to stimulate the intestine to secrete a large amount of fluid, causing severe watery diarrhea and dehydration.

[0004] With the continuous iteration and update of gene sequencing technology and genomics analysis technology, more and more pathogenic subtypes and hybrid pathogenic strains have been found in swine isolates. Therefore, the current situation of Escherichia coli from swine diarrhea is becoming more and more complex in terms of epidemiology, pathogenic mechanism, quarantine and prevention, etc. There is an urgent need to construct accurate and efficient detection methods. There are many types of toxins and fimbrial adhesins produced by Escherichia coli, and their coding situations in strains are intricate. At present, there is no method to detect most toxins at one time, which brings great difficulties to the health assessment of pig herds. The ELISA method is suitable for laboratory serum diagnosis and large-scale epidemic disease investigation because of its fast speed, low requirements for experimental conditions, no need for aseptic operation, and the ability to detect a large number of samples in a short time. The technical difficulty of the one-step ELISA for evaluating the antibody positive rate of various toxins and fimbriae of Escherichia coli in pig herds lies in obtaining a fusion antigen that can recognize the antibodies of the above-mentioned multiple pathogenic factors. Summary of the Invention

[0005] Objective of the Invention: The objective of the present invention is to provide a multi-epitope fusion antigen of multiple toxins and fimbriae of Escherichia coli causing swine diarrhea and its application, so as to solve the problems existing in the above-mentioned prior art. In the present invention, the antigenic epitopes of the main toxins and fimbrial adhesins of Escherichia coli are optimized and arranged to design a multi-epitope fusion antigen, and based on this, a one-step ELISA detection technology is established and a kit is assembled for easy popularization and use.

[0006] Technical Solution: To achieve the above objective, the present invention provides the following solutions:

[0007] The present invention provides a multi-epitope fusion antigen of multiple toxins and fimbriae of Escherichia coli causing swine diarrhea and its application. The prepared multi-epitope fusion antigen MEAET can be used to prepare antibodies and can also be used as a coating antigen to prepare indirect ELISA detection products.

[0008] In the first aspect, the present invention provides a multi-epitope fusion antigen MEAET of multiple toxins and fimbriae of Escherichia coli causing swine diarrhea, and the amino acid sequence of the multi-epitope fusion antigen is shown as SEQ ID NO:1.

[0009] The multi-epitope fusion antigen MEAET provided by the present invention is designed by performing antigenic epitope analysis on 6 proteins including hemolysin-related protein HlyA, Shiga toxin Stx2e, heat-labile enterotoxin LT, intimin receptor Tir, F18, and K88 / F4 fimbrial subunit of Escherichia coli causing swine diarrhea through bioinformatics software, selecting two optimal antigenic epitopes for each virulence protein, optimizing the arrangement order and adding a linker fragment "GPGPGLRMKLPKS" for connection, and adding a dendritic cell-targeting peptide "FYPSYHSTPQRP" at the C-terminus.

[0010] In the second aspect, the present invention provides a gene encoding the above-mentioned multi-epitope fusion antigen of multiple toxins and fimbriae of Escherichia coli causing swine diarrhea, and the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0011] In the third aspect, the present invention provides an expression vector, and the expression vector contains the above-mentioned gene encoding the multi-epitope fusion antigen of multiple toxins and fimbriae of Escherichia coli causing swine diarrhea.

[0012] In the fourth aspect, the present invention provides a transgenic recombinant engineering bacterium, and the transgenic recombinant engineering bacterium carries the above-mentioned gene or recombinant expression vector.

[0013] In the fifth aspect, the present invention provides the application of the above-mentioned gene, or the above-mentioned expression vector, or the above-mentioned transgenic recombinant engineering bacterium in the preparation of the above-mentioned multi-epitope fusion antigen of multiple toxins and fimbriae of Escherichia coli causing swine diarrhea.

[0014] In a sixth aspect, the present invention provides an application of the multi-epitope fusion antigen of various toxins and fimbriae of Escherichia coli from porcine diarrhea sources in the preparation of products for detecting antibodies against pathogenic Escherichia coli.

[0015] In a seventh aspect, the present invention provides a product for detecting antibodies against pathogenic Escherichia coli, and the product contains the multi-epitope fusion antigen of various toxins and fimbriae of Escherichia coli from porcine diarrhea sources.

[0016] As an implementation scheme, the product is an indirect ELISA detection product, and the multi-epitope fusion antigen of various toxins and fimbriae of Escherichia coli from porcine diarrhea sources is used as the coating antigen.

[0017] As a specific implementation scheme, the coating concentration of the multi-epitope fusion antigen is 0.5 - 1.5 μg / mL; preferably, the optimal coating concentration is 1 μg / mL, and the coating conditions are: coating at 37°C for 1 h and then overnight at 4°C.

[0018] In an eighth aspect, the present invention provides a method for simultaneously detecting antibodies against various toxins and fimbriae of Escherichia coli from porcine diarrhea sources, including using the multi-epitope fusion antigen MEAET of various toxins and fimbriae of Escherichia coli from porcine diarrhea sources, or using the above product for detection.

[0019] As a specific implementation scheme, the antibodies against various toxins and fimbriae of Escherichia coli from porcine diarrhea sources include antibodies against hemolysin-related protein HlyA, Shiga toxin Stx2e, heat-labile enterotoxin LT, intimin receptor Tir, F18, and K88 / F4 fimbria subunit.

[0020] As an implementation scheme, the method is an indirect ELISA detection method, and the multi-epitope fusion antigen MEAET of various toxins and fimbriae of Escherichia coli from porcine diarrhea sources is used as the coating antigen.

[0021] Furthermore, as a preferred scheme, in the indirect ELISA detection method, the coating concentration of the MEAET antigen is 1 μg / mL, the serum dilution is 1:400, the dilution of the secondary antibody is 1:10000, and 5% skim milk is the optimal blocking solution.

[0022] In the indirect ELISA detection method of the present invention, it is determined whether the serum of the sample to be tested contains antibodies against pathogenic Escherichia coli by measuring the OD 450nm value, and the critical values for determining negative and positive are: S / P = 0.203. When S / P ≥ 0.203, it is determined as positive, and when S / P is less than 0.203, it is determined as negative. Preferably, using the multi-epitope fusion antigen MEAET as the coating antigen, an indirect ELISA method is established to detect antibodies against various toxins and fimbriae related to porcine diarrhea at one time to indicate whether it carries or is infected with diarrhea-causing Escherichia coli.

[0023] In a ninth aspect, the present invention provides an application of the multi-epitope fusion antigen of various toxins and fimbriae of Escherichia coli from porcine diarrhea sources in the preparation of a vaccine for preventing pathogenic Escherichia coli.

[0024] As a specific embodiment, the pathogenic Escherichia coli includes Escherichia coli containing hemolysin-related protein HlyA, Shiga toxin Stx2e, heat-labile enterotoxin LT, intimin receptor Tir, F18 or K88 / F4 fimbrial subunits.

[0025] In a tenth aspect, the present invention provides a vaccine for preventing pathogenic Escherichia coli, and the vaccine contains the above multi-epitope fusion antigen.

[0026] As a specific embodiment, the pathogenic Escherichia coli includes Escherichia coli containing hemolysin-related protein HlyA, Shiga toxin Stx2e, heat-labile enterotoxin LT, intimin receptor Tir, F18 or K88 / F4 fimbrial subunits.

[0027] Advantageous effects: Compared with the prior art, the present invention has the following advantages:

[0028] (1) The multi-epitope fusion antigen MEAET constructed by the present invention can be recognized by antibodies against a total of 6 toxins or fimbrial antigens, namely HlyA, Stx2e, LT, intimin Tir, F18, and K88 / F4. The antibodies produced after immunization can also effectively recognize the above 6 antigens.

[0029] (2) Among the 6 toxins or fimbrial antigens selected by the present invention, porcine diarrhea-source Escherichia coli can carry one or more of them, and the antibodies produced after infection can all be recognized by the multi-epitope fusion antigen MEAET.

[0030] (3) The present invention uses the multi-epitope fusion antigen MEAET as a coating antigen to establish an indirect ELISA detection method for antibodies against Escherichia coli from porcine diarrhea sources, and can detect antibodies against 6 toxins or fimbriae at one time, simplifying the detection steps, saving the cost of monitoring pathogen carriage / infection, and providing basic data for the prevention and control of porcine bacterial diarrhea.

[0031] The indirect ELISA kit and method for detecting antibodies against multiple toxins and fimbriae of pathogenic Escherichia coli provided by the present invention are convenient for detecting large-scale samples and epidemiological monitoring, and have broad prospects and value for popularization and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. However, the drawings described below are only some embodiments of the present invention.

[0033] Figure 1For the optimized arrangement of epitopes in the multi-epitope fusion antigen MEAET.

[0034] Figure 2 For the DiscoTope prediction and analysis of MEAET.

[0035] Figure 3 For the tertiary structure analysis of MEAET and the docking model with the porcine immune recognition receptor SAL1 molecule.

[0036] Figure 4 For the electrophoretic detection map of the multi-epitope fusion antigen MEAET after expression, purification and excision of the GST tag with thrombin.

[0037] Figure 5 For the Western-blot detection map of the MEAET hyperimmune serum recognizing 6 Escherichia coli toxins / pili antigens.

[0038] Figure 6 For the determination map of the indirect ELISA positive critical value with MEAET as the coating antigen.

[0039] Figure 7 For the determination map of the indirect ELISA sensitivity with MEAET as the coating antigen. Specific embodiments

[0040] The following will combine the accompanying drawings in the embodiments of the present invention to detail various exemplary embodiments of the present invention. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments, and should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0041] Regarding the use of "comprising", "including", "having", "containing", etc. in the present invention, they are all open-ended terms, that is, they are intended to include but not limited to. For the test methods without specifying specific experimental conditions in the embodiments, they are usually carried out under conventional conditions, such as the conditions described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harber Laboratory Press, 1989), or the conditions recommended by the manufacturer.

[0042] Example 1: Selection of common toxins and pili antigens of Escherichia coli causing porcine diarrhea, and epitope screening

[0043] Escherichia coli that causes diarrhea in pigs mainly colonizes the pig intestine through K88 / F4 or F18 fimbriae. In addition, the colonized bacteria cause intestinal inflammation and lesions by releasing various toxins. The main toxins include heat-labile enterotoxin LT, heat-stable enterotoxin ST, hemolysin, Shiga toxin Stx2e, and type III secretion system-related effectors (including tight adhesion receptor Tir, etc.). Strains that cause diarrhea in pigs may encode one or more of the above-mentioned pathogenic factors, which brings great complexity to the detection of pathogens and infection antibodies. Except for the poor immunogenicity of ST toxin not being considered, the present invention selects the above-listed 6 main toxin or fimbria antigens for subsequent research.

[0044] Predict the B-cell epitopes of the above 6 antigens through tools such as BCPred and IEDB TEPITOP, and conduct secondary analysis on their antigenicity, sequence composition, and physicochemical properties. Finally, 2 optimal epitopes are selected for each antigen (Table 1).

[0045] Table 1 Candidate epitope information table

[0046] 。

[0047] Example 2: Design and evaluation of multi-epitope antigen MEAET

[0048] Analyze the hydrophilicity and hydrophobicity analysis results of each candidate epitope obtained in Example 2 through Expasy. According to the hydrophilicity index, link the epitope peptides through "Linker" (GPGPGLRMKLPKS), and tandem the dendritic cell targeting peptide (FYPSYHSTPQRP) after the sequence. After analyzing and evaluating the characteristics such as immunogenicity, antigenicity, sensitization, and physicochemical properties of the polypeptides connected in different orders, an optimized arrangement plan is selected ( Figure 1 ), use DiscoTope to predict discontinuous B-cell epitopes ( Figure 2 ), predict the three-dimensional structure of the multi-epitope antigen and the molecular docking model with the porcine immune recognition receptor SAL1 ( Figure 3 ), determine that the above plan meets the design requirements, and name it the epitope fusion antigen of multiple toxins of Escherichia coli (Multiple-epitope antigen of Escherichia coli Toxins, MEAET), and its amino acid sequence is shown in SEQ ID NO.1.

[0049] Example 3: Synthesis, expression, and protein purification of the multi-epitope antigen MEAET encoding gene

[0050] Entrust Nanjing Qingke Biotechnology Co., Ltd. to optimize and synthesize the nucleic acid sequence encoding MEAET described in Example 2, as shown in SEQ ID NO.2, construct the recombinant plasmid pGEX-4T-MEAET of pGEX-4T-1, and transfer the recombinant plasmid into BL21(DE3) competent cells; select the single colony with correct colony PCR and sequencing results and transfer it to 200 mL of LB liquid medium containing 100 μg / mL Amp + for enlarged culture. When the OD 600 of the bacterial solution is about 0.4 - 0.6, add IPTG with a final concentration of 500 μM, and induce at 16°C and 120 rpm for 18 h. Collect the bacterial cells and disrupt them by sonication. Purify the MEAET antigen using a glutathione agarose gel chromatography column, incubate with thrombin to cleave the GST tag to obtain the purified MEAET protein. SDS-PAGE electrophoresis analysis shows that the purified 42 kDa target antigen protein is obtained ( Figure 4 ).

[0051] Example 4: Preparation and evaluation of MEAET hyperimmune serum

[0052] Obtain the purified MEAET protein according to the method in Example 3, mix it with ISA 206 VG at a volume ratio of 1:1, and fully vortex and emulsify for standby. Use 30-day-old SPF piglets as the immunization object, inject intramuscularly at multiple points. The primary immunization dose is 1 mg / animal, and the subsequent immunization dose is 2 mg / animal. Boost the immunization every 10 days for three consecutive times. Take ear blood to separate serum before immunization as the pre-immunization serum control. Collect blood after each immunization, analyze the serum to measure the antibody titer by ELISA. After the antibody titer reaches a relatively high level (>1:102400), collect blood from the anterior vena cava, collect the serum as the subsequent positive serum, aliquot it into sterile EP tubes at a volume of 0.5 mL / tube, seal it, and store it at -40°C.

[0053] PCR amplify the nucleic acid coding sequences of the 6 Escherichia coli toxins / pili antigens described in Example 1, specifically hlyA (SEQ ID NO.3), stx2A (SEQ ID NO.4), lta (SEQ ID NO.5), tir (SEQ ID NO.6), k88 (SEQ ID NO.7) and f18 (SEQ ID NO.8). Connect the above PCR products into the pET28a(+) vector respectively, and transfer the recombinant plasmids into BL21(DE3) and culture until the OD 600Values from 0.4 to 0.6, add IPTG with a final concentration of 500 μM, induce at 16 °C and 120 rpm for 18 h. Collect the bacteria and disrupt them by sonication. Purify the target protein using His-tag Ni-NTA affinity chromatography. Using the purified HlyA, Stx2A, LT_A, Tir, K88, and F18 protein fragments, detect by Western-blot method. The results show that the MEAET hyperimmune serum can specifically recognize the above 6 antigen fragments and present single blot bands respectively, and the band positions are in line with the designed sizes ( Figure 5 )). The above results indicate that after the multi-epitope fusion antigen is injected into pigs, it can stimulate the body to produce antibodies against the above 6 antigens respectively.

[0054] Example 5: Screening of 6 toxin / fimbria antigen clinical positive sera

[0055] The HlyA, Stx2A, LT_A, Tir, K88, and F18 protein fragments purified in Example 4 were detected by Western-blot method to screen more than 100 clinical sera from diarrhea-recovered pigs. If an immunoblot band of the corresponding size was detected, it was determined to be antibody positive. After detection, a total of 16 clinical positive sera were screened, including 2 positive for anti-HlyA antibody, 2 positive for anti-LT_A antibody, 1 positive for anti-Stx2e antibody, 1 positive for anti-Tir antibody, 2 positive for anti-K88 antibody, 1 positive for anti-F18 antibody, 1 positive for anti-HlyA&Stx2e antibody, 3 positive for anti-LT_A&K88 antibody, 2 positive for anti-LT&F18 antibody, and 1 positive for anti-LT&K88&Stx2e antibody. The specific information is shown in Table 2 below and stored at -40 °C for later use.

[0056] Table 2 Information table of 16 clinical sera

[0057] 。

[0058] Example 6: Establishment and evaluation of an indirect ELISA detection method with MEAET as the coating antigen

[0059] 1. Optimization of antigen coating concentration and primary antibody serum dilution

[0060] Determination of the coating concentration of the multi-epitope fusion antigen MEAET and the dilution of the primary antiserum by checkerboard titration. The purified MEAET epitope polypeptide was coated on the ELISA plate at 4 °C and 37 °C for 1 h at 4 concentrations of 2 μg / L, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL, and then incubated overnight at 4 °C. After washing three times with 1× PBST, it was blocked with 5% skim milk at 37 °C for 2 h. After washing three times with PBST, the positive serum and negative serum were added to the ELISA plate at 6 dilutions of 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, and reacted at 37 °C for 1 h. The ELISA plate was washed with 1× PBST, and HRP-labeled goat anti-pig IgG antibody (diluted 1:10000) was added and incubated at 37 °C for 1 h. After washing three times with 1× PBST, it was developed with 100 μL of TMB solution. The reaction was terminated with 50 μL of ELISA termination solution. The absorbance was measured at OD450 using an ELISA reader. The optimal serum dilution and antigen coating concentration were determined according to the maximum P / N value. The results showed (Table 3) that when the antigen coating concentration was 1 μg / mL and the serum dilution was 1:800, the P / N value was 14.795, which was the largest. Therefore, this condition was selected as the reaction condition for the subsequent experiment.

[0061] Table 3 Determination table of the coating concentration of the multi-epitope fusion antigen MEAET, serum dilution, and the optimal dilution of the secondary enzyme-labeled antibody

[0062] 。

[0063] 2. Determination of the ELISA blocking solution

[0064] The ELISA plate was coated with the optimal antigen concentration and blocked with 1% BSA, 2% BSA, 5% skim milk, and 10% skim milk at 37 °C for 2 h respectively. After washing three times with 1× PBST, the positive serum and negative serum at the optimal dilution were used as the primary antibody and reacted at 37 °C for 1 h. After washing three times with 1× PBST, HRP-labeled goat anti-pig IgG antibody was incubated at 1:10000 at 37 °C for 1 h. After washing three times with 1× PBST, 100 μL of TMB substrate chromogenic solution was added, and the reaction was terminated with 50 μL of ELISA termination solution. The absorbance was measured at OD450 using a general ELISA reader. The optimal ELISA blocking solution was determined according to the maximum P / N value. Comparing the ELISA test results (Table 4), it can be seen that when the ELISA plate was blocked with 5% skim milk, the P / N value was 14.894, which was the largest. Therefore, 5% skim milk was determined as the optimal blocking solution.

[0065] Table 4 Determination table of the blocking conditions after coating with the multi-epitope fusion antigen MEAET

[0066] 。

[0067] 3. Determination of the dilution ratio of the enzyme-labeled secondary antibody

[0068] Coat the enzyme-labeled plate with the optimal antigen concentration. After washing, add 5% skim milk and incubate in a humid box at 37°C for 2 h for blocking. After blocking, wash as above, add the optimal concentrations of negative and positive sera, and react at 37°C for 1 h. Dilute the HRP-labeled goat anti-pig IgG antibody at ratios of 1:5000, 1:10000, 1:15000, and 1:20000. Make 3 parallels for each dilution ratio, and the reaction time is 60 min for all. After washing, add 100 μL of substrate per well, develop color at 37°C for 5 min, add 2M H2SO4 to terminate the reaction, and record the OD value of the microplate reader 450 value to determine the optimal dilution ratio of the secondary antibody. The results show (Table 5) that when the dilution ratio of the secondary antibody is 1:10000, the P / N value is the highest

[0069] Table 5 Determination of the blocking conditions after coating with the multi-epitope fusion antigen MEAET

[0070] 。

[0071] 4. Determination of the reaction time of the primary antibody and the secondary antibody

[0072] Coat the enzyme-labeled plate with the optimal antigen concentration, and block with 5% skim milk at 37°C for 2 h. After washing three times with 1× PBST, use the positive and negative sera at the optimal dilution ratio as the primary antibody and incubate at 37°C for 0.5, 1 h, and 2 h respectively. After washing three times with 1× PBST, dilute the HRP-labeled goat anti-pig IgG antibody at a ratio of 1:10000 and incubate at 37°C for 0.5 h, 1 h, and 2 h respectively. After washing three times with 1× PBST, add 100 μL of TMB substrate chromogenic solution and terminate the reaction with 50 μL of ELISA termination solution. Measure the absorbance at OD 450 to determine the reaction time of the primary antibody serum and the enzyme-labeled secondary antibody according to the maximum P / N value

[0073] The results show that when the primary antibody serum is incubated for 1 h and the reaction time of the enzyme-labeled antibody is 1 h, the P / N value is the largest at 14.741 (Table 6). Therefore, the optimal incubation time of the primary antibody and the reaction time of the enzyme-labeled antibody are determined to be 1 h

[0074] Table 6 Determination of the reaction time of the primary antibody and the secondary antibody

[0075] 。

[0076] 5. Indirect ELISA negative and positive determination criteria

[0077] Select 24 pig negative sera screened in Example 5, set positive controls, and measure the OD values of the negative and positive sera according to the optimized indirect ELISA detection method450 values, and calculate the average value (X) and standard deviation (SD) of the N / P value. Calculate the SP value: (sample value - negative control value) / (positive control value - negative control value). When S / P ≥ X + 3SD, it is determined as positive; when S / P < X + 3SD, it is determined as negative. Statistically analyze the OD 450 values of 24 negative sera at a dilution factor of 1:800, and calculate the OD 450 At 450 nm, X = 0.125 and SD = 0.026. After conversion, the critical value for determining positive and negative is: S / P = 0.203. When S / P ≥ 0.203, it is determined as positive; when S / P < 0.203, it is determined as negative ( Figure 6 ).

[0078] 6. Repeatability test

[0079] Perform within-batch repeatability tests using 3 batches of enzyme-linked immunosorbent assay (ELISA) plates coated with the same batch of antigen; perform between-batch repeatability tests using ELISA plates coated with antigens from different batches. Each time, use 3 sera for indirect ELISA detection, set 3 replicates for each serum, and read the OD 450 values, calculate X, SD, and coefficient of variation (CV), and statistically analyze the results. The results show that the minimum within-batch CV of 3 positive sera in the repeatability test is 1.82%, and the maximum is 4.26%; while the minimum between-batch CV is 2.78%, and the maximum is 5.57%, both less than 10% (Table 7).

[0080] Table 7 Results of within-batch and between-batch repeatability

[0081] .

[0082] 7. Specificity test

[0083] Use the established indirect ELISA method to detect serum samples 4, 8, and 13 of the positive sera screened in Example 5, as well as positive sera of Salmonella (Sal), Pasteurella (PAS), Clostridium perfringens (CP), Listeria monocytogenes (LM), Campylobacter jejuni (CJ), Streptococcus (SS), non-pathogenic Escherichia coli K12 (K-12), PEDV, and PDCoV. Dilute them at 1:200, and simultaneously set MEAET positive serum and negative serum controls. Repeat each sample 3 times to determine whether there is cross-reaction in this method.

[0084] The results showed that the S / P values of the 3 clinically positive sera were all higher than the critical value, while the S / P values of other sera to be tested were all lower than the critical value of 0.203 (Table 8). Thus, it can be seen that the multi-epitope fusion antigen MEAET can be specifically recognized by each of the 6 toxin / pilus antibodies and does not non-specifically bind to the positive sera of the above pathogens, indicating that the above indirect ELISA assay can detect multiple toxins and pilus antibodies of Escherichia coli at one time, exclude the interference of Escherichia coli-related antibodies in the intestinal flora, improve the detection efficiency of pathogenic Escherichia coli infection or carriage, and has good detection specificity.

[0085] Table 8 Specificity test results

[0086] 。

[0087] 8. Sensitivity test

[0088] Take serum samples 4, 8, and 13 of the clinically positive sera screened in Example 5, and dilute them 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800 times with the sample diluent respectively, and then perform indirect ELISA detection on the positive sera of different dilution degrees. The results showed that when the serum dilution ratio was 1:3200, the detection results were all higher than the determination standard S / P value of 0.203, indicating that the method had high sensitivity. Figure 7 ), when the serum dilution ratio was 1:3200, the detection results were all higher than the determination standard S / P value of 0.203, indicating that the method had high sensitivity.

[0089] Example 7: Detection of clinical samples by the indirect ELISA assay

[0090] Using the indirect ELISA antibody kit, 47 serum samples of diarrheal pigs (excluding major concentrated virus infections) and 106 serum samples of asymptomatic pigs collected clinically were detected. The results showed that the positive detection rate of the diarrheal samples was 74.5%, and that of the asymptomatic samples was 34% (Table 9), indicating that 34% of the asymptomatic pigs had been infected with Escherichia coli carrying at least one toxin or pilus adhesin, suggesting that the latent or occult infections caused by the above Escherichia coli were very common in the pig population.

[0091] Table 9 Clinical sample results

[0092] 。

[0093] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A multi-epitope fusion antigen of multiple toxins and fimbriae of Escherichia coli causing swine diarrhea, characterized in that, The amino acid sequence of the multi-epitope fusion antigen of multiple toxins and fimbriae of the porcine diarrhea-causing Escherichia coli is shown in SEQ ID NO.

1.

2. A gene encoding a multi-epitope fusion antigen of multiple toxins and fimbriae of Escherichia coli causing swine diarrhea as claimed in claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. An expression vector, characterized in that, The expression vector contains the gene described in claim 2.

4. A genetically modified recombinant engineering bacterium, characterized in that, The transgenic recombinant engineering bacteria contain the gene described in claim 2, or the expression vector described in claim 3.

5. Use of the gene described in claim 2, or the expression vector described in claim 3, or the transgenic recombinant engineering bacteria described in claim 4 in the preparation of the multi-epitope fusion antigen of multiple toxins and fimbriae of the porcine diarrhea-causing Escherichia coli described in claim 1.

6. Use of the multi-epitope fusion antigen of multiple toxins and fimbriae of Escherichia coli from porcine diarrhea sources according to claim 1 in the preparation of a product for detecting antibodies against pathogenic Escherichia coli, characterized in that, The pathogenic Escherichia coli is Escherichia coli containing hemolysin-related protein HlyA, Shiga toxin Stx2e, heat-labile enterotoxin LT, intimin receptor Tir, F18 or K88 / F4 fimbrial subunit.

7. A product for detecting pathogenic Escherichia coli antibodies, characterized in that, The product contains the multi-epitope fusion antigen of multiple toxins and fimbriae of the porcine diarrhea-causing Escherichia coli described in claim 1; the pathogenic Escherichia coli is Escherichia coli containing hemolysin-related protein HlyA, Shiga toxin Stx2e, heat-labile enterotoxin LT, intimin receptor Tir, F18 or K88 / F4 fimbrial subunit.

8. A method for simultaneously detecting multiple toxins and fimbrial antibodies of Escherichia coli causing swine diarrhea, characterized in that, It includes detecting by using the multi-epitope fusion antigen of multiple toxins and fimbriae of the porcine diarrhea-causing Escherichia coli described in claim 1, or by using the product described in claim 7; the antibodies against multiple toxins and fimbriae of the porcine diarrhea-causing Escherichia coli include antibodies against hemolysin-related protein HlyA, Shiga toxin Stx2e, heat-labile enterotoxin LT, intimin receptor Tir, F18 and K88 / F4 fimbrial subunit.

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