Eae-Stx2 fusion protein and kit for detecting pathogenic escherichia coli antibody based on indirect ELISA method

Abstract

The invention discloses an Eae-Stx2 fusion protein and a kit for detecting a pathogenic escherichia coli antibody based on an indirect ELISA method, and belongs to the technical field of biology. The Eae-Stx2 fusion protein is formed by connecting an Eae protein and an Stx2 protein through a flexible linker, and the amino acid sequence of the Eae-Stx2 fusion protein is as shown in SEQ ID No. 1. According to the invention, the Eae-Stx2 fusion protein is used as a coating antigen, and an indirect ELISA (enzyme-linked immunosorbent assay) detection method and a kit for the pathogenic Escherichia coli antibody are established. The Escherichia coli Eae-Stx2 fusion protein prepared by the invention is good in antigenicity, and a constructed indirect ELISA antibody detection method has the characteristics of high sensitivity, strong characteristics and good repeatability, is suitable for detecting the condition of carrying / infecting pathogenic Escherichia coli antibodies by pigs, cattle and humans, and is convenient for large-scale sample detection and epidemiological monitoring.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an Eae-Stx2 fusion protein and a kit for detecting pathogenic Escherichia coli antibodies based on an indirect ELISA method. Background Art

[0002] Escherichia coli is divided into different pathotypes according to the characteristics of their virulence factors and pathogenic mechanisms. Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroaggregative Escherichia coli (EAEC), and enteroinvasive Escherichia coli (EIEC) are five very important pathotypes. Most of these pathogenic Escherichia coli produce Shiga toxins, so they are also called Shiga toxin-producing Escherichia coli (STEC). STEC is one of the most important zoonotic pathogens, which can cause serious diseases in humans, such as hemorrhagic enteritis (HC) and hemolytic uremic syndrome (HUS). Some patients may die from acute and chronic renal failure. All warm-blooded animals can be infected. Cattle, sheep and pigs are the main targets of infection and long-term storage hosts, which may cause diarrhea, enteritis and non-intestinal system diseases, or may not cause obvious clinical symptoms, but carry bacteria for a long time. It is circulated and transmitted and infected humans through meat, dairy products and vegetables contaminated by feces and other excreta.

[0003] Human and animal STEC can produce two toxins, Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), which are the main culprits of HC and HUS. STEC carrying Stx2 is significantly higher than Stx1, and Stx2 can be secreted outside the bacteria, which has a more serious pathogenic effect. Stx2 has multiple subtypes, Stx2c, Stx2d, Stx2e, Stx2f and stx2v, which are closely related to the strain serotype and pathogenicity.

[0004] Intimin is another important virulence factor of pathogenic Escherichia coli. Its function is the most clearly studied. It controls the colonization of the host intestinal epithelial cells, promotes actin polymerization and the formation of pedestal-like structures, and causes severe intestinal villus shedding, i.e., A / E damage. Intimin encodes the gene eae, which has 27 subtypes, including α1, α2, β1, β2, β3, ε1, ε2, ε3, ε4, η1, η2, θ, λ, μ, ν, ξ1, ξ3, ο, π, ρ, σ, τ1, τ2, б, γ1, γ2, and κ, and is closely related to the serotype and pathogenicity of the strain. Most STEC O157:H7 and O145 carry the eae-γ subtype, and a few O157:H7 carry the eae-α subtype. Most O26 and O121 carry eae-β, most O103 mainly carry eae-ε, and most O45 and O111 mainly carry eae-θ subtype.

[0005] A fast, specific, and batch detection method suitable for foodborne pathogenic Escherichia coli will provide technical support for the prevention and control of foodborne pathogenic Escherichia coli. With the widespread use of antibiotics, resulting in more variations in the virulence genes of strains, coupled with changes in the human diet structure, more emphasis is placed on the retention of food nutrition, the probability of food being contaminated by pathogenic Escherichia coli increases, and the chance of human infection will become more frequent. Effectively controlling the infection and carriers of animals in the food chain will greatly reduce the harm to humans. The ELISA method has a mature detection process and is easy to master. It does not require high-value instruments and is more suitable for the detection of large-scale samples in farms, disease control centers, and import and export quarantine agencies, as well as epidemiological analysis. Therefore, the present invention selects two protein fragments with good conservatism and immunogenicity in series, uses a prokaryotic expression platform to obtain recombinant antigens in vitro, establishes an ELISA detection technology for pathogenic Escherichia coli detection, and assembles a kit for easy promotion and use. Summary of the invention

[0006] The purpose of the present invention is to provide an Eae-Stx2 fusion protein and a kit for detecting pathogenic Escherichia coli antibodies based on the indirect ELISA method to solve the problems existing in the above-mentioned prior art. By using the Eae-Stx2 fusion protein as a coating antigen, an indirect ELISA detection kit for pathogenic Escherichia coli antibodies is established. The kit has the characteristics of high sensitivity, strong characteristics and good repeatability.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an Eae-Stx2 fusion protein. The Eae-Stx2 fusion protein is formed by connecting an Eae protein and a Stx2 protein via a flexible linker. The amino acid sequence of the Eae-Stx2 fusion protein is shown in SEQ ID No.1.

[0009] The present invention also provides a gene encoding the Eae-Stx2 fusion protein, and the nucleotide sequence of the gene encoding is shown in SEQ ID No.2.

[0010] The present invention also provides the use of the Eae-Stx2 fusion protein or the encoding gene in preparing a product for detecting pathogenic Escherichia coli antibodies.

[0011] Preferably, the Eae-Stx2 fusion protein is used as the coating antigen to detect pathogenic Escherichia coli antibodies by establishing an indirect ELISA method.

[0012] The present invention also provides a kit for detecting pathogenic Escherichia coli antibodies based on the indirect ELISA method, the kit comprising an enzyme labeling plate with the Eae-Stx2 fusion protein coated with an antigen, a blocking solution, a washing solution, a diluent, an enzyme labeling reagent, a substrate color developing solution, a positive standard serum and a negative standard serum.

[0013] Preferably, the optimal coating concentration of the Eae-Stx2 fusion protein is 1 μg / mL, and the coating conditions are: coating at 37° C. for 1 hour and then at 4° C. overnight.

[0014] Preferably, the blocking solution is gelatin with a mass concentration of 5%; the washing solution is PBST solution; the diluent contains an aqueous solution with a mass concentration of 5% gelatin, 8.5% sodium chloride, 0.2% potassium chloride, 2.9% disodium hydrogen phosphate and 0.2% dipotassium hydrogen phosphate; the enzyme labeling reagent is staphylococcal protein A labeled with horseradish peroxidase; the substrate color developing solution is 3,3',5,5'-tetramethylbenzidine solution; the positive standard serum is Eae-stx2 antibody positive serum; and the negative standard serum is pathogenic Escherichia coli antibody negative serum.

[0015] Preferably, the optimal dilution ratio of the enzyme-labeled reagent is 1:20000; the optimal reaction time of the substrate color developing solution is 12 min, and the optimal dilution ratio of the positive standard serum and the negative standard serum is 1:100.

[0016] Preferably, when the kit is used to detect serum samples to be tested, the optimal incubation time of the serum samples to be tested is 30 minutes.

[0017] Preferably, the OD of the serum sample to be tested is measured. 450nm The value is used to judge whether the pathogenic Escherichia coli antibody is present. The judgment standard is: when OD 450nm The values ​​are all less than 0.362, then the serum sample to be tested does not contain pathogenic Escherichia coli antibodies. 450nm If the values ​​are all ≥0.362, the serum sample to be tested contains pathogenic Escherichia coli antibodies.

[0018] The serum samples to be tested include but are not limited to sera from pigs, cattle and humans carrying / infected with pathogenic Escherichia coli.

[0019] The present invention discloses the following technical effects:

[0020] The present invention constructs a fusion protein Eae-Stx2 by connecting the Eae protein and the Stx2 protein through a flexible linker, and uses the Eae-Stx2 fusion protein as a coating antigen to establish an indirect ELISA detection method for pathogenic Escherichia coli antibodies. The Escherichia coli Eae-Stx2 fusion protein prepared by the present invention has good antigenicity, and an indirect ELISA antibody detection method is established based on it, which has the characteristics of high sensitivity, strong characteristics, and good repeatability. It is suitable for detecting the antibody situation of pigs, cattle and humans carrying / infected with pathogenic Escherichia coli, clarifying the infection background, and providing basic data for the prevention and control of zoonotic diseases.

[0021] The indirect ELISA kit and method for detecting pathogenic Escherichia coli antibodies provided by the present invention are convenient for large-scale sample detection and epidemiological monitoring, and have the prospect and value of wide promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 Induced expression of recombinant protein Eae-Stx2; M, molecular weight protein marker; 1, BL21 (pCold I-eae-stx2) before induction; 2, whole cell lysis protein after induction; 3, BL21 (pCold I-eae-stx2) whole cell lysis centrifugation supernatant protein; 4, BL21 (pCold I-eae-stx2) whole cell lysis centrifugation precipitate;

[0024] Figure 2 Western blot identification results of recombinant protein Eae-Stx2; M, molecular weight protein marker; 1, before BL21 (pCold I-eae-stx2) induction; 2, whole bacterial lysis protein after induction. 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] The experimental methods in the following examples where specific experimental conditions are not specified are usually carried out under conventional conditions, such as the conditions described in the Sambrook et al. Molecular Cloning; Experimental Manual (New York: Cold Spring Harber Laboratory Press, 1989), or under the conditions recommended by the manufacturer.

[0031] Example 1: Selecting the target fragment to be expressed

[0032] DNASTAR.Lasergene.v7.1 software was used to analyze the sequence diversity and immunogenicity of pathogenic Escherichia coli compactin and Shiga toxin 2, and computer software was used to analyze their conservative fragments, primers were designed to amplify the target fragments, and flexible linker ([G3]S*4) was used to connect Eae and Stx2 and codon optimization was performed, and restriction sites NdeI (CATATG) and XbaI (TCTAGA) were added to the N-terminus and C-terminus, respectively. The amino acid sequence is shown in SEQ ID No.1, and the optimized gene sequence is shown in SEQ ID No.2.

[0033] SEQ ID No.1:

[0034] SGDNTRLGIGEYWRDYFKSSVNGYFRMSGWHESYNKKDYDERPANGFDIRFNGYLPSYPALGAKLMYEQYYGDNVALFNSDKLQSNPGAATVGVNYTPIPLVTMGIDYRHGTGNENDLLYSMQLRYQFDKPWSQQIEPQYVNELRTLSGSRYDLVQRNNNIILEYKKQDILSLNIPHDINGTEHSTQKIQLIVKSKYGLD GSGSGSGS SGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVYTMTPGDVDLTLNWGRINSNVLPEYRGEDGVRVGRISFNNISAILGTVAVILNCHHQGARSVRAVNEDSQPECQITGDRPVI.

[0035] SEQ ID No.2:

[0036] tcaggtgataatacaaggctaggaatagggggcgaatattggcgtgattacttcaaaagctctgtgaacggctatttccgcatgtccggctggcatgaaagctacaataagaaggactacgacgaacggccagctaatggcttcgacatccgtttcaatggttacctcccgagctatccggctttgggtgcgaaactgatgtatgagcaatactacggcgacaatgtagcgttgtttaacagcgataagctgcagagcaatccgggtgcggcaaccgttggtgttaactataccccgattccgctggtaaccatgggaattgattaccgccatggtacaggtaacgagaatgatctgctttactccatgcagctgcgttaccagtttgacaagccgtggagccaacagattgaaccgcaatatgttaacgagttgcgcaccctgtccggtagccgttatgacctggttcagc gtaataacaacatcatcctggagtacaaaaagcaagatatcctctcgctgaacattccgcatgatattaacggtactgagcacagcacccagaaaattcaactgattgttaaatccaaatacggcctggat ggtagtggttctggctctggctcc tcgggcaacaccatgacccgtgatgccagcagagccgtcttgcgcttcgtgacggttactgcggaagcgttgcgctttcgtcaaatccaacgtgaattccgtcaggcgctgtctgaaaccgctccggtttataccatgacgccgggcgacgtggatctgaccctgaattggggtcgtatttctaacgtgctgccggagtatcgtggtgaggacggcgtgcgcgtcggccgtatttcatttaacaacatcagcgcaatcttaggtacggtggcagtgatcttgaactgccaccaccagggtgccagaagcgttcgtgcggtgaacgaggacagccagccggaatgtcagatcaccggtgaccgccccgttatc。

[0037] Example 2 Prokaryotic expression and purification

[0038] The optimized target gene was connected to the pColdI expression vector to construct the recombinant expression plasmid pColdI-eae-stx2; the recombinant plasmid was transferred into BL21(DE3) competent cells; the single colony with the correct bacterial solution PCR and sequencing was selected for expansion culture, and the bacterial solution OD 600nm When the p-value was about 0.6-1.0, IPTG was added to a final concentration of 0.2-1 mM, and the cells were expressed at 16°C overnight. The bacteria were collected and ultrasonically disrupted, and the protein was purified using a Sunrise Anti-His Affinity Resin nickel column and identified by SDS-PAGE and Western blot.

[0039] The test results showed that the recombinant fusion protein Eae-Stx2 was expressed in a soluble form and was consistent with the expected size ( Figure 1 ), which can react specifically with pathogenic Escherichia coli positive serum ( Figure 2 ).

[0040] Example 3 Optimization of ELISA reaction conditions

[0041] 1. Determination of the optimal coating concentration of recombinant protein antigen and the dilution concentration of the serum to be tested

[0042] The optimal coating concentration and serum dilution of the recombinant fusion protein Eae-Stx2 antigen were determined using the chessboard method. The recombinant protein was diluted in multiples using carbonate buffer to a coating concentration of 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL, 100 μL / well, and incubated at 4°C overnight. The coated ELISA plate was aspirated with a plate washer and washed 3 times with PBST. 5% gelatin blocking solution was added at 300 μL / well and incubated in a 37°C wet box. Incubate for 2 hours; after blocking, wash as above, wash 3 times with PBST; dilute the E. coli positive serum and negative serum with PBS at a ratio of 1:50, 1:100, 1:150, and 1:200, respectively, to form a square array, 100 μL / well, and incubate in a 37°C wet box for 1 hour; after washing 3 times with PBST, add SPA-HRP (working concentration is 1:20,000), 100 μL / well; after washing 3 times, add substrate 100 μL / well, and color at 37°C for 5 minutes; add 2M H2SO4 to terminate the reaction, 50 μL / well; read the value with an enzyme reader; then compare the OD values ​​of positive and negative sera 450The optimal coating concentration of the antigen and the optimal dilution of the serum were determined by the OD value and P / N value. When the positive serum OD value was around 1.0 and the negative value was low, the antigen concentration and serum working concentration with a high P / N value were the optimal coating concentration of the antigen and the optimal dilution concentration of the serum to be tested. The results showed that the optimal coating concentration of the antigen was 1 μg / mL and the optimal dilution multiple of the positive and negative serum was 1:100.

[0043] Table 1 Determination of optimal antigen coating concentration and optimal serum dilution (chessboard method)

[0044]

[0045]

[0046] 2. Determination of recombinant antigen coating conditions

[0047] The ELISA plate was coated with the optimal antigen concentration, 100 μL / well, and divided into 4 groups. Group I, coated at 37℃ for 2h; Group II, coated at 37℃ for 3h; Group III, coated at 37℃ for 1h and 4℃ overnight; Group IV, coated at 4℃ overnight. The coated ELISA plate was washed and blocked in a 37℃ wet box for 2h. After blocking, the optimal serum dilution was added to each well and incubated in a 37℃ wet box for 1h. After washing, HRP-SPA diluted 1:20,000 was added to each well, 100 μL / well, and incubated in a 37℃ wet box for 1h. After washing, 100 μL / well of substrate was added, color developed at 37℃ for 5min, 2M H2SO4 was added to terminate the reaction, and the OD of the ELISA reader was recorded. 450 Value, compare positive and negative serum OD 450 The best coating conditions were determined by the antigen coating time with high negative value and low negative value and high P / N value. The results showed that group III (coating at 37℃ for 1h and 4℃ overnight) had the highest P / N value.

[0048] Table 2 Optimal incubation conditions for antigens

[0049] Grouping Group I Group II Group III Group IV P / N 3.239 2.967 5.002 4.568

[0050] 3. Determination of blocking solution

[0051] The ELISA plate was coated with the optimal antigen concentration and the best antigen coating conditions, and after washing, it was divided into 4 groups. Group I, 5% skim milk powder; Group II, 5% calf serum; Group III, 5% BSA (bovine serum albumin); Group IV, 5% gelatin. The blocking time in the wet box was 2.0h, and 3 parallel wells were made for each group. After blocking, wash, add the optimal concentration of positive and negative serum, 100uL / well, and incubate in a wet box at 37℃ for 1h. After washing, add 1:20,000 diluted HRP-SPA, 100μL / well, and incubate in a wet box at 37℃ for 1h. After washing, add 100μL / well of substrate, develop color at 37℃ for 5min, add 2M H2SO4 to terminate the reaction, and record the OD of the ELISA reader. 450 The results showed that the P / N value of group IV (5% gelatin) was the highest.

[0052] Table 3 Determination of the best blocking solution

[0053] Grouping Group I Group II Group III Group IV P / N 4.003 3.671 5.21 5.577

[0054] 4. Determination of the dilution multiple of enzyme-labeled secondary antibody

[0055] Coat the ELISA plate with the most appropriate antigen concentration and the best antigen coating conditions. After washing, add 5% gelatin blocking solution and block in a 37°C wet box for 2 hours. After blocking, wash as above, add the most appropriate concentration of positive and negative serum, and react in a 37°C wet box for 1 hour. HRP-SPA was diluted at 1:10,000, 1:20,000, 1:25,000 and 1:30,000, and each dilution was replicated 3 times, with an action time of 60 minutes. After washing, add 100 μL / well of substrate, develop color at 37°C for 5 minutes, add 2M H2SO4 to terminate the reaction, and record the OD of the ELISA reader. 450 The results showed that when the secondary antibody dilution was 1:20,000, the P / N value was the highest.

[0056] Table 4 Determination of the optimal secondary antibody dilution

[0057] Grouping 1:10,000 1:20,000 1:25,000 1:30,000 P / N 4.886 5.659 5.113 4.347

[0058] 5. Determination of the optimal action time of serum

[0059] The ELISA plates were coated with the optimal antigen concentration and the best antigen coating conditions and divided into 4 groups. The coated ELISA plates were washed and blocked in a 37°C wet box for 2 hours. After blocking, the optimal serum dilution was added to each well and the plates were incubated in a 37°C wet box for 20 minutes, 40 minutes, 60 minutes and 90 minutes. After washing, HRP-SPA diluted 1:20,000 was added in a 37°C wet box for 1 hour. After washing, fresh substrate solution was added to each well and the plates were color developed at 37°C for 5 minutes. 2M H2SO4 was added to terminate the reaction and the OD of the ELISA reader was recorded. 450 The results showed that the P / N value was the highest when the serum was incubated for 30 minutes.

[0060] 6. Determination of substrate reaction time

[0061] The ELISA plates were coated with the optimal antigen concentration and the best antigen coating conditions and divided into 4 groups. The coated ELISA plates were washed and blocked in a 37°C wet box for 2 hours. After blocking, the optimal serum dilution was added to each well and the plates were incubated in a 37°C wet box for 1 hour. After washing, HRP-SPA diluted 1:20,000 was incubated in a 37°C wet box for 1 hour. After washing, the substrate solution was added to each well. The color development time at 37°C was 5 minutes, 8 minutes, 12 minutes and 15 minutes, respectively. 2M H2SO4 was added to terminate the reaction and the OD of the ELISA reader was recorded. 450 The results showed that when the incubation time was 12 minutes, the P / N reached a higher value, and the negative control was no higher than 0.3.

[0062] Table 6 Optimal incubation time for serum

[0063] Grouping 5min 8min 12min 15min P / N 3.998 5.007 5.567 5.572

[0064] 7. Determination of critical value

[0065] The indirect ELISA antibody detection kit of the present invention was used to detect 30 known standard positive sera and 30 negative sera, and the critical value was determined by ROC curve analysis. The results showed that when OD 450nm When OD <0.362, the sensitivity (100%) and specificity (98.6%) were high, and this OD value was selected as the critical value; when OD 450nm ≥0.362, it was judged as positive; when OD 450nm When <0.362, it was judged as negative.

[0066] 8. Specific crossover test

[0067] The purified recombinant protein was used to coat the ELISA plate under the optimal coating conditions. After washing and blocking, the positive sera of Salmonella (Sal), Listeria monocytogenes (LM), Campylobacter jejuni (CJ), Streptococcus (SS2), Pasteurella (PAS) and non-pathogenic Escherichia coli K12 (K-12) were diluted 1:100, and pathogenic Escherichia coli positive serum control and negative serum control were set at 100 μL / well. The plate was incubated in a humidified box at 37°C for 1 hour. After washing, HRP-SPA diluted 1:20,000 was added and incubated in a humidified box at 37°C for 1 hour. After washing, the substrate was added to each well for color development for 10 minutes. The reaction was terminated with the stop solution and the OD on the ELISA reader was recorded. 450 The results showed that after the Eae-stx2 fusion protein antigen reacted with different pathogen-positive sera, OD 450nm The values ​​were all <0.362, indicating that the Eae-stx2 fusion protein antigen did not non-specifically bind to the positive sera of the above pathogens (Table 7), indicating that the established ELISA method has good specificity.

[0068] Table 7 Specificity detection

[0069]

[0070] 9. Sensitivity

[0071] The positive control serum of pathogenic Escherichia coli O157:H7 was diluted 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800 times with sample diluent, and then ELISA test was performed on the positive serum of different dilutions. The results showed that when the serum dilution ratio was 1:3200, the test result was still positive (OD 450nm >0.362), indicating that the method had a high sensitivity (Table 8).

[0072] Table 8 Sensitivity test

[0073]

[0074] Example 4

[0075] Assembly, use, result judgment and specificity, sensitivity and stability of indirect ELISA kit for detecting antibodies to pathogenic Escherichia coli

[0076] 1. Kit Assembly

[0077] According to the optimization of the detection conditions in Example 3, an indirect ELISA kit for detecting pathogenic large intestine was assembled, and its components were as follows:

[0078] (1) ELISA plate: This ELISA plate has been coated with claudin protein and is prepared as follows:

[0079] ① Antigen coating: Add 100 μL of 1.0 μg / mL recombinant eae-stx2 protein to each well of the ELISA plate and coat at 37°C for 1 hour and 4°C overnight.

[0080] ② Washing: After taking out and patting dry, wash with PBST 3 times, 3 min / time, and pat dry the liquid.

[0081] ③ Blocking: Block with 5% gelatin-PBST, 300 μL / well, in a 37°C wet box for 2 h, and wash as above.

[0082] (2) 10× washing solution: pH 7.4, volume 100 mL; 10× washing solution refers to an aqueous solution containing 5% Tween-20, 8.5% sodium chloride, 0.2% potassium chloride, 2.9% disodium hydrogen phosphate and 0.2% dipotassium hydrogen phosphate by mass concentration.

[0083] (3) 10× serum diluent: pH 7.4, volume 100 mL; 10× serum diluent is an aqueous solution containing 5% gelatin, 8.5% sodium chloride, 0.2% potassium chloride, 2.9% disodium hydrogen phosphate and 0.2% dipotassium hydrogen phosphate.

[0084] (4) Enzyme labeling reagent: Staphylococcus aureus protein A labeled with horseradish peroxidase (Boster Company), diluted 1:25000 with PBST, 25 mL.

[0085] (5) Substrate colorimetric solution: single-component 3,3',5,5'-tetramethylbenzidine solution (Nanjing Research Institute Co., Ltd.), 100 mL.

[0086] (6) Stop solution: 2M sulfuric acid solution, 50 mL.

[0087] (7) Positive serum: Eae-stx2 antibody positive serum, 0.5 mL. The purified Eae-stx2 recombinant protein was obtained according to the method in Example 2, and after being fully emulsified with an equal volume of ISA201 (complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for the booster immunization), it was subcutaneously injected into rabbits (Jinling Rabbit Breeding Farm, male, 2.0 kg) at multiple points for immunization. Blood was collected from the ear before immunization to separate the serum, which was used as the pre-immunization serum control. The amount of antigen for the initial immunization was 500 μg / rabbit, and booster immunization was performed 21 days later. Blood was collected from the ear vein on the 10th day after the booster immunization, and the antibody titer was determined by ELISA. After the antibody titer reached a relatively high level (≥1:12800), blood was collected from the heart, and the serum was collected as the pathogenic Escherichia coli positive serum, which was divided into sterile glass bottles in 0.5 mL bottles, sealed, and stored at -20°C.

[0088] (8) Negative serum: 0.5 mL of pathogenic E. coli antibody negative serum. Blood was collected from the heart of pathogenic E. coli negative rabbits (Jinling Rabbit Breeding Farm). The serum was separated as pathogenic E. coli negative serum and dispensed into sterile glass bottles in 0.5 mL bottles, sealed, and stored at -20°C.

[0089] 2. The operating steps for using the kit of the present invention to detect pathogenic Escherichia coli are as follows:

[0090] Each kit contains two 96-well ELISA plates vacuum-packed in foil, each containing a removable ELISA microwell strip.

[0091] (1) Preparation: Take the kit out of the refrigerator and place it at room temperature for 30 minutes. Dilute 10× Washing Solution and 10× Serum Diluent into 1× Washing Solution and Serum Diluent respectively with distilled water.

[0092] (2) Sample addition: Take out the ELISA plate, fix it on the frame, set up the positive and negative serum wells, the test sample wells and the blank control wells respectively, and record the position of each well. The test sample, positive serum and negative serum are diluted 1:40 with serum diluent respectively, and added to the sample wells and standard wells respectively, 100 μL / well. No blank control is added.

[0093] (3) Incubation: Incubate in a 37°C wet box for 1 h.

[0094] (4) Washing the plate: discard the liquid, pat dry with absorbent paper, add 250 μL of washing solution to each well, let stand for 3 min, discard the washing solution, and repeat this process 3 times.

[0095] (5) Add enzyme labeling reagent: Add 100 μL of enzyme labeling reagent to each well.

[0096] (6) Washing the plate: Same as step (4).

[0097] (7) Color development: Add 100 μL of substrate color development solution to each well, shake gently for 30 seconds, and react at room temperature in the dark for 15 minutes.

[0098] (8) Termination: Add 50 μL of stop solution to each well to terminate the reaction.

[0099] (9) Measurement: Measure the optical density absorbance (OD) of each well in an automatic enzyme-linked detector. 450nm ).

[0100] (10) Result determination

[0101] Example 5 Clinical Application

[0102] The pathogenic Escherichia coli indirect ELISA antibody kit was used to test 56 pig serum samples collected from the clinic. The results showed that the positive detection rate of the samples was 12.5% ​​(Table 9).

[0103] Table 9 Clinical sample test results

[0104] Number of samples Positive Negative 56 12.5％(7 / 56) 87.5％(49 / 56)

[0105] 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. An Eae-Stx2 fusion protein, characterized in that The Eae-Stx2 fusion protein is formed by connecting the Eae protein and the Stx2 protein via a flexible linker. The amino acid sequence of the Eae-Stx2 fusion protein is shown in SEQ ID No.

1.

2. A gene encoding the Eae-Stx2 fusion protein according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID No.

2.

3. Use of the Eae-Stx2 fusion protein as claimed in claim 1 or the encoding gene as claimed in claim 2 in the preparation of a product for detecting antibodies against pathogenic Escherichia coli.

4. The use according to claim 3, characterized in that The Eae-Stx2 fusion protein is used as the coating antigen, and an indirect ELISA method is established to detect pathogenic Escherichia coli antibodies.

5. A kit for detecting pathogenic Escherichia coli antibodies based on indirect ELISA method, characterized in that: The kit comprises an ELISA plate coated with the Eae-Stx2 fusion protein of claim 1, a blocking solution, a washing solution, a diluent, an ELISA reagent, a substrate color developing solution, a positive standard serum and a negative standard serum.

6. The kit according to claim 5, characterized in that The optimal coating concentration of the Eae-Stx2 fusion protein was 1 μg / mL, and the coating conditions were: coating at 37° C. for 1 hour and then at 4° C. overnight.

7. The kit according to claim 5, characterized in that The blocking solution is gelatin with a mass concentration of 5%; the washing solution is a PBST solution; the diluent contains an aqueous solution with a mass concentration of 5% gelatin, 8.5% sodium chloride, 0.2% potassium chloride, 2.9% disodium hydrogen phosphate and 0.2% dipotassium hydrogen phosphate; the enzyme labeling reagent is staphylococcal protein A labeled with horseradish peroxidase; the substrate color developing solution is a 3,3',5,5'-tetramethylbenzidine solution; the positive standard serum is Eae-stx2 antibody positive serum; and the negative standard serum is pathogenic Escherichia coli antibody negative serum.

8. The kit according to claim 5, characterized in that The optimal dilution ratio of the enzyme-labeled reagent is 1:20000; the optimal reaction time of the substrate color developing solution is 12 min, and the optimal dilution ratio of the positive standard serum and the negative standard serum is 1:

100.

9. The kit according to claim 5, characterized in that When the kit is used to detect the serum of a sample to be tested, the optimal incubation time of the serum of the sample to be tested is 30 minutes.

10. The kit according to claim 9, characterized in that By measuring the OD of the serum sample to be tested 450nm The value is used to judge whether the pathogenic Escherichia coli antibody is present. The judgment standard is: when OD 450nm The values ​​are all less than 0.362, then the serum sample to be tested does not contain pathogenic Escherichia coli antibodies. 450nm If the values ​​are all ≥0.362, the serum sample to be tested contains pathogenic Escherichia coli antibodies.

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