CN119161493A

CN119161493A - An Eae-Stx2 fusion protein and a kit for detecting pathogenic Escherichia coli antibodies based on indirect ELISA method

Info

Publication number: CN119161493A
Application number: CN202411588796.7A
Authority: CN
Inventors: 张雪寒; 李彬; 汪伟; 周俊明; 祝昊丹; 李运川; 王丹丹; 杨珊珊; 何孔旺
Original assignee: Jiangsu Yanjiang Agricultural Science Research Institute
Current assignee: Jiangsu Yanjiang Agricultural Science Research Institute
Priority date: 2024-11-08
Filing date: 2024-11-08
Publication date: 2024-12-20

Abstract

The invention discloses an Eae-Stx2 fusion protein and a kit for detecting pathogenic escherichia coli antibodies based on an indirect ELISA method, and belongs to the technical field of biology. The Eae-Stx2 fusion protein is formed by connecting Eae protein and Stx2 protein through a flexible connector, and the amino acid sequence of the Eae-Stx2 fusion protein is shown as SEQ ID No. 1. The invention utilizes the Eae-Stx2 fusion protein as a coating antigen to establish a pathogenic escherichia coli antibody indirect ELISA detection method and a kit. The prepared escherichia coli Eae-Stx2 fusion protein has good antigenicity, and the constructed indirect ELISA antibody detection method has the characteristics of high sensitivity, strong characteristics and good repeatability, is suitable for detecting the conditions of pigs, cattle and human carrying/infecting pathogenic escherichia coli, and is convenient for large-scale sample detection and epidemiological monitoring.

Description

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

Technical Field

The invention relates to the technical field of biology, in particular to an Eae-Stx2 fusion protein and a kit for detecting pathogenic escherichia coli antibodies based on an indirect ELISA method.

Background

Coli are classified into different pathotypes according to their virulence factor characteristics and pathogenic mechanisms. Enteropathogenic escherichia coli (EPEC), enterohemorrhagic escherichia coli (EHEC), enterotoxigenic escherichia coli (ETEC), enteroaggregating escherichia coli (EAEC), enteroinvasive escherichia coli (EIEC) are five of the very important pathotypes. Most of these pathogenic E.coli produce shiga toxins, so they are also called shiga toxin-producing E.coli (STEC). STEC is one of the most important zoonotic pathogens, and can cause serious diseases in humans, such as hemorrhagic enteritis (HC) and Hemolytic Uremic Syndrome (HUS), and individual patients can die from acute and chronic renal failure. All warm-blooded animals can be infected. Cattle, sheep and pigs are the primary infectious subjects and long-term storage hosts, or cause diarrhea, enteritis and diseases of the parenteral system, or do not cause obvious clinical symptoms, but are chronically infected. Meat, dairy products, vegetables and other foods contaminated by excrement such as feces are circulated and infested with human beings.

STEC of human and animal origin can produce two toxins, shiga toxin 1 (Stx 1) and shiga toxin 2 (Stx 2), respectively, which are the main muxes responsible for the initiation of HC and HUS. STEC carrying Stx2 is significantly higher than Stx1, and Stx2 can be secreted outside the bacterial body, with more serious pathogenic effects. Stx2 has multiple subtypes Stx2c, stx2d, stx2e, stx2f and Stx2v, and has a close relationship with strain serotypes and pathogenicity.

Compactin (intimin) is another important virulence factor of pathogenic escherichia coli, the function research is the most clear, and the dominant bacteria are fixed in host intestinal epithelial cells, promote actin polymerization and base-like structure formation, and cause serious intestinal villus shedding, namely A/E injury. Intimin encodes gene eae, and 27 subtypes of alpha 1, alpha 2, beta 1, beta 2, beta 3, epsilon 1, epsilon 2, epsilon 3, epsilon 4, eta 1, eta 2, theta, lambda, mu, gamma, zeta 1, zeta 3, omicron, pi, rho, sigma, tau 1, tau 2, sigma, gamma 1, gamma 2 and kappa are closely related with the serotype and pathogenicity of the strain. Most STEC O157H 7 and O145 carry the eae-gamma subtype, and a few O157H 7 carry the eae-alpha subtype. Most of O26 and O121 carry eae-. Beta.and most of O103 carry primarily eae-. Epsilon.and most of O45 and O111 carry primarily eae-. Theta.subtypes.

The method is rapid and specific, and is suitable for batch detection, and provides technical support for prevention and control of food-borne pathogenic escherichia coli. Along with the overuse of antibiotics, the virulence genes of the strains are more changeable, and the dietary structure of human beings is changed, the retention of food nutrition is more important, the probability of food contamination by pathogenic escherichia coli is increased, and the probability of human infection is also more frequent. Effectively controlling infection and bacteria of animals in the food chain will greatly reduce harm to human beings. The ELISA method has mature detection flow, is easy to master, does not need high-value instruments, and is more suitable for detection and epidemiological analysis of standard quantity samples of farms, disease control centers and import and export quarantine institutions. Therefore, the invention selects two fragments with good protein conservation and immunogenicity to connect in series, uses the prokaryotic expression platform to obtain recombinant antigen in vitro, establishes ELISA detection technology for detecting pathogenic escherichia coli, assembles the kit, and is convenient for popularization and use.

Disclosure of Invention

The invention aims to provide an Eae-Stx2 fusion protein and a kit for detecting pathogenic escherichia coli antibodies based on an indirect ELISA method, so as to solve the problems in the prior art, and the Eae-Stx2 fusion protein is taken as a coating antigen to establish the pathogenic escherichia coli antibody indirect ELISA detection kit, which has the characteristics of high sensitivity, strong characteristics and good repeatability.

In order to achieve the above object, the present invention provides the following solutions:

The invention provides an Eae-Stx2 fusion protein, wherein the Eae-Stx2 fusion protein is formed by connecting Eae protein and Stx2 protein through a flexible connector, and the amino acid sequence of the Eae-Stx2 fusion protein is shown as SEQ ID No. 1.

The invention also provides a coding gene of the Eae-Stx2 fusion protein, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 2.

The invention also provides application of the Eae-Stx2 fusion protein or the coding gene in preparing products for detecting pathogenic escherichia coli antibodies.

Preferably, the Eae-Stx2 fusion protein is used as a coating antigen, and an indirect ELISA method is established to detect the pathogenic escherichia coli antibody.

The invention also provides a kit for detecting pathogenic escherichia coli antibodies based on an indirect ELISA method, which comprises an ELISA plate, a sealing liquid, a washing liquid, a diluent, an ELISA reagent, a substrate chromogenic liquid, positive standard serum and negative standard serum, wherein the ELISA plate is coated with the Eae-Stx2 fusion protein.

Preferably, the Eae-Stx2 fusion protein is coated at an optimal concentration of 1 μg/mL, provided that the coating is performed at 37℃for 1h and then at 4℃overnight.

Preferably, the blocking solution is gelatin with the mass concentration of 5%, the washing solution is PBST solution, the dilution solution contains aqueous solutions with the 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-labeled reagent is staphylococcus protein A marked by horseradish peroxidase, the substrate chromogenic solution is 3,3', 5' -tetramethyl benzidine solution, the positive standard serum is Eae-stx2 antibody positive serum, and the negative standard serum is pathogenic escherichia coli antibody negative serum.

Preferably, the optimal dilution ratio of the enzyme-labeled reagent is 1:20000, the optimal reaction time of the substrate color development liquid is 12min, and the optimal dilution ratio of the positive standard serum and the negative standard serum is 1:100.

Preferably, when the kit is used for detecting serum of a sample to be detected, the optimal incubation time of the serum of the sample to be detected is 30min.

Preferably, whether the pathogenic E.coli antibodies are contained is judged by measuring the OD _450nm value of the serum of the sample to be tested, wherein the judging standard is that the serum sample to be tested does not contain the pathogenic E.coli antibodies when the OD _450nm values are all less than 0.362, and the pathogenic E.coli antibodies are contained in the serum sample to be tested when the OD _450nm values are all more than or equal to 0.362.

The above-mentioned serum samples to be tested include, but are not limited to, serum derived from pigs, cattle and human carrying/infecting pathogenic E.coli.

The invention discloses the following technical effects:

The invention constructs fusion protein Eae-Stx2 by connecting Eae protein and Stx2 protein through flexible linker, and establishes an indirect ELISA detection method of pathogenic E.coli antibody by using Eae-Stx2 fusion protein as coating antigen. The E.coli Eae-Stx2 fusion protein prepared by the invention has good antigenicity, and the indirect ELISA antibody detection method is established based on the antigenicity, has the characteristics of high sensitivity, strong characteristics and good repeatability, is suitable for detecting the conditions of carrying/infecting pathogenic E.coli antibodies of pigs, cattle and human beings, and can be used for defining the infection background and providing basic data for prevention and control of zoonosis.

The indirect ELISA kit and the method for detecting the pathogenic escherichia coli antibody are convenient for large-scale sample detection and epidemiological monitoring, and have wide popularization and application prospects and values.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the inducible expression of recombinant protein Eae-Stx2, M, molecular weight protein Marker, 1, BL21 (pCold I-Eae-Stx 2) before induction, 2, whole-cell lysate after induction, 3, BL21 (pCold I-Eae-Stx 2) whole-cell lysate supernatant, 4, BL21 (pCold I-Eae-Stx 2) whole-cell lysate centrifugation pellet;

FIG. 2 shows the Western blot identification results of recombinant protein Eae-Stx2, M, molecular weight protein Marker, 1, B21 (pCold I-Eae-Stx 2) before induction, and 2, whole-cell lysis protein after induction.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The test methods for specific experimental conditions are not specified in the examples below, and are generally carried out under conventional conditions, for example, by molecular cloning in Sambrook et al, conditions described in the laboratory Manual (New York: cold Spring Harber Laboratory Press, 1989), or under the conditions recommended by the manufacturer.

Example 1 selection of fragments of interest to be expressed

Sequence diversity and immunogenicity analyses were performed on pathogenic E.coli compactin and volunteer toxin 2, respectively, using DNASTAR.lasergene.v7.1 software, using computer software to analyze their conserved fragments, designing primers to amplify the fragments of interest, and using flexible linker ([ G ₃ ] S4) to ligate Eae and Stx2 and codon optimize, with addition of cleavage sites NdeI (CATATG) and XbaI (TCTAGA) at the N-and C-termini, respectively. The amino acid sequence is shown as SEQ ID No.1, and the optimized gene sequence is shown as SEQ ID No. 2.

SEQ ID No.1:

SGDNTRLGIGGEYWRDYFKSSVNGYFRMSGWHESYNKKDYDERPANGFDIRFNGYLPSYPALGAKLMYEQYYGDNVALFNSDKLQSNPGAATVGVNYTPIPLVTMGIDYRHGTGNENDLLYSMQLRYQFDKPWSQQIEPQYVNELRTLSGSRYDLVQRNNNIILEYKKQDILSLNIPHDINGTEHSTQKIQLIVKSKYGLDGSGSGSGSSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVYTMTPGDVDLTLNWGRISNVLPEYRGEDGVRVGRISFNNISAILGTVAVILNCHHQGARSVRAVNEDSQPECQITGDRPVI.

SEQ ID No.2:

tcaggtgataatacaaggctaggaatagggggcgaatattggcgtgattacttcaaaagctctgtgaacggctatttccgcatgtccggctggcatgaaagctacaataagaaggactacgacgaacggccagctaatggcttcgacatccgtttcaatggttacctcccgagctatccggctttgggtgcgaaactgatgtatgagcaatactacggcgacaatgtagcgttgtttaacagcgataagctgcagagcaatccgggtgcggcaaccgttggtgttaactataccccgattccgctggtaaccatgggaattgattaccgccatggtacaggtaacgagaatgatctgctttactccatgcagctgcgttaccagtttgacaagccgtggagccaacagattgaaccgcaatatgttaacgagttgcgcaccctgtccggtagccgttatgacctggttcagc gtaataacaacatcatcctggagtacaaaaagcaagatatcctctcgctgaacattccgcatgatattaacggtactgagcacagcacccagaaaattcaactgattgttaaatccaaatacggcctggatggtagtggttctggctctggctcctcgggcaacaccatgacccgtgatgccagcagagccgtcttgcgcttcgtgacggttactgcggaagcgttgcgctttcgtcaaatccaacgtgaattccgtcaggcgctgtctgaaaccgctccggtttataccatgacgccgggcgacgtggatctgaccctgaattggggtcgtatttctaacgtgctgccggagtatcgtggtgaggacggcgtgcgcgtcggccgtatttcatttaacaacatcagcgcaatcttaggtacggtggcagtgatcttgaactgccaccaccagggtgccagaagcgttcgtgcggtgaacgaggacagccagccggaatgtcagatcaccggtgaccgccccgttatc.

EXAMPLE 2 prokaryotic expression and purification

Connecting the optimized target gene to pColdI expression vectors to construct recombinant expression plasmids pColdI-eae-stx2, transferring the recombinant plasmids into BL21 (DE 3) competent cells, selecting single bacterial colonies with correct bacterial liquid PCR and sequencing for amplification culture, adding IPTG with final concentration of 0.2-1 mM when bacterial liquid OD _600nm is about 0.6-1.0, overnight expressing at 16 ℃, collecting thalli, performing ultrasonic disruption, purifying proteins by adopting Sunrise Anti-HIS AFFINITY RESIN nickel columns, and identifying by SDS-PAGE and Westernblot.

The detection result shows that the recombinant fusion protein Eae-Stx2 is expressed in a soluble way and is consistent with the expected size (figure 1), and can specifically react with pathogenic escherichia coli positive serum (figure 2).

Example 3 optimization of ELISA reaction conditions

1. Determination of optimal coating concentration of recombinant protein antigen and dilution concentration of serum to be detected

The optimal coating concentration and serum dilution of the recombinant fusion protein Eae-Stx2 antigen are determined by using a chessboard method, the recombinant protein is diluted in a double ratio by using a carbonate buffer solution, the coating concentration is diluted to 2 mug/mL, 1 mug/mL, 0.5 mug/mL, 0.25 mug/mL, 0.125 mug/mL and 100 mug/hole, 4 ℃ overnight, the coated ELISA plate is sucked out of the coating solution by using a plate washer, PBST is washed 3 times, 5% gelatin sealing solution is added, 300 mug/hole and 37 ℃ wet box are incubated for 2 hours, after sealing, the recombinant protein is washed 3 times with the PBST, positive serum and negative serum of escherichia coli are respectively diluted in a double ratio by using PBS (PBS) to form a square matrix, 100 mug/hole, 37 ℃ wet box is incubated for 1h, SPA-HRP is added after PBST is washed 3 times, the working concentration is 1:20,000), 100 mug/hole is washed 100 mug, 37 ℃ wet box is incubated for 2 hours, the optimal coating concentration of the antigen is stopped, and the optimal antigen concentration is determined by using a 20 ℃ and a negative reader is then added to a positive serum dilution meter for 37 mug/hole, and a negative antigen dilution value is determined by comparing the optimal coating concentration with 39L and the antigen concentration with the positive antigen. When the OD value of positive serum is about 1.0, the negative value is low, and the antigen concentration with high P/N value and the working concentration of serum are the optimal antigen coating concentration and the optimal dilution concentration of serum to be detected. The results showed that the optimal antigen coating concentration was 1. Mu.g/mL and the optimal negative and positive serum dilution was 1:100.

TABLE 1 determination of optimal coating concentration of antigen and optimal dilution of serum (checkerboard method)

2. Determination of recombinant antigen coating conditions

The ELISA plates were coated at the optimum antigen concentration, 100. Mu.L/well, and divided into 4 groups. Group I, 37 ℃ coating 2h, group II, 37 ℃ coating 3h, group III, 37 ℃ coating 1h plus 4 ℃ overnight, group IV, 4 ℃ overnight. The coated ELISA plate is washed, the interior of the wet box is sealed for 2 hours at 37 ℃, and the optimal serum dilution is added into each hole after sealing, and the wet box is acted for 1 hour at 37 ℃. After washing, 1:20,000 dilution of HRP-SPA was added per well, 100. Mu.L/well, and the wells were allowed to function for 1h at 37 ℃. After washing, 100. Mu.L/well of substrate was added, color development was performed at 37℃for 5min, the reaction was stopped by adding 2M H ₂SO₄, OD ₄₅₀ values of the microplate reader were recorded, and antigen coating times of negative positive serum OD ₄₅₀ values and negative values low and P/N values high were compared to determine the optimal coating conditions. The results show that group III (37 ℃ coating 1h plus 4 ℃ overnight) has the highest P/N value.

TABLE 2 optimal incubation conditions for antigens

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

3. Determination of the sealing liquid

The ELISA plates were coated with the optimal antigen concentration and optimal antigen coating conditions, and after washing, they were divided into 4 groups. Group I, 5% nonfat milk powder, group II, 5% calf serum, group III, 5% BSA (bovine serum albumin), group IV, 5% gelatin. The closing time in the wet box was 2.0h, and 3 parallel wells were made for each group. After blocking, washing, adding the optimal concentration of negative and positive serum, 100 uL/hole, and performing the action for 1h in a 37 ℃ wet box, after washing, adding 1:20,000 diluted HRP-SPA,100 uL/hole, and performing the action for 1h in a 37 ℃ wet box. After washing, 100. Mu.L/well of substrate was added, color developed for 5min at 37℃and the reaction was stopped by adding 2M H ₂SO₄, and the OD ₄₅₀ value of the microplate reader was recorded to determine the optimal type of blocking solution. The results showed that group IV (5% gelatin) had the highest P/N value.

TABLE 3 determination of optimal blocking solution

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

4. Determination of dilution factor of enzyme-labeled secondary antibody

The ELISA plate is coated with the optimal antigen concentration and the optimal antigen coating condition, and after washing, 5% gelatin blocking solution is added, and the interior of the wet box is blocked for 2 hours at 37 ℃. After closing, washing the mixture, adding the optimal concentration of negative and positive serum, and allowing the mixture to act in a 37 ℃ wet box for 1h. HRP-SPA was diluted 1:10,000, 1:20,000, 1:25,000 and 1:30,000, 3 replicates per dilution, and the duration of action was 60min. After washing, 100. Mu.L/well of substrate was added, developed for 5min at 37℃and the reaction was stopped by adding 2M H ₂SO₄, and the OD ₄₅₀ value of the microplate reader was recorded to determine the optimal secondary antibody dilution and time of action. The results show that the P/N value is highest when the secondary antibody dilution is 1:20,000.

TABLE 4 determination of optimal secondary antibody dilution

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

5. Determination of the optimal time of action of serum

The ELISA plates were coated with the optimal antigen concentration and optimal antigen coating conditions and divided into 4 groups. Washing the coated ELISA plate, sealing for 2h in a 37 ℃ wet box, adding the optimal serum dilution into each hole after sealing, and respectively acting for 20min, 40min, 60min and 90min in the 37 ℃ wet box. After washing, HRP-SPA diluted by 1:20,000 is reacted for 1h in a 37 ℃ wet box, fresh substrate solution is added into each hole after washing, color development is carried out for 5min at 37 ℃, 2M H ₂SO₄ is added to stop reaction, and OD ₄₅₀ value of an enzyme label instrument is recorded to determine proper acting time of serum to be detected. The results showed that serum was incubated for 30min with the highest P/N value.

6. Determination of substrate reaction time

The ELISA plates were coated with the optimal antigen concentration and optimal antigen coating conditions and divided into 4 groups. The coated ELISA plate is washed, the interior of the wet box is sealed for 2 hours at 37 ℃, and the optimal serum dilution is added into each hole after sealing, and the wet box is acted for 1 hour at 37 ℃. After washing, HRP-SPA diluted by 1:20,000 is acted for 1h in a 37 ℃ wet box, substrate solution is added into each hole after washing, the color development time under the condition of 37 ℃ is respectively 5min, 8min, 12min and 15min, 2M H ₂SO₄ is added to terminate the reaction, and the OD ₄₅₀ value of an enzyme labeling instrument is recorded to determine the optimal substrate color development time. The results showed that when incubated for 12min, the P/N reached a higher value, and the negative control was not higher than 0.3.

TABLE 6 optimal incubation time for serum

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

7. Determination of the critical value

The indirect ELISA antibody detection kit is used for respectively detecting 30 parts of positive serum and 30 parts of negative serum which are known to be standard, and the critical value is determined by ROC curve analysis. The results showed that when OD _450nm <0.362, sensitivity (100%) and specificity (98.6%) were high, this OD value was chosen as the threshold, positive when OD _450nm was not less than 0.362, and negative when OD _450nm was not less than 0.362.

8. Specific cross-test

Coating an ELISA plate with purified recombinant protein under the optimal coating condition, washing and sealing, respectively diluting positive serum of Sal, listeria Monocytogenes (LM), campylobacter Jejuni (CJ), streptococcus (SS 2), pasteurella (PAS) and non-pathogenic Escherichia coli K12 (K-12) by 1:100, setting pathogenic Escherichia coli positive serum control and negative serum control, performing 1h in a wet box at 100 mu L/hole and 37 ℃, adding HRP-SPA diluted by 1:20,000 and performing 1h in a wet box at 37 ℃, adding substrate for developing for 10min in each hole after washing, stopping the reaction by a stopping solution, recording OD ₄₅₀ value on an ELISA instrument, and judging results. The results show that after the Eae-stx2 fusion protein antigen reacts with positive serum of different pathogens, the OD _450nm values are less than 0.362, and the Eae-stx2 fusion protein antigen is not in nonspecific binding with the positive serum of the pathogens (Table 7), so that the established ELISA method has better specificity.

TABLE 7 specificity detection

9. Sensitivity to

Pathogenic E.coli O157:H2 positive control serum was diluted 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800 times with sample dilutions, and ELISA assays were performed on the positive serum at different dilutions, respectively. The results showed that the test results were still positive (OD _450nm > 0.362) at a serum dilution of 1:3200, indicating higher sensitivity of the method (Table 8).

TABLE 8 sensitivity detection

Example 4

Assembly and use methods of indirect ELISA kit for detecting pathogenic escherichia coli antibodies, result judgment and specificity, sensitivity and stability of kit

1. Kit assembly

According to the optimization of the detection conditions in example 3, an indirect ELISA kit for detecting pathogenic large intestine was assembled, which comprises the following components:

(1) The ELISA plate is coated with compactin protein, and the preparation method is as follows:

① Antigen coating 100. Mu.L of recombinant eae-stx2 protein with the concentration of 1.0. Mu.g/mL is added to each hole of the ELISA plate, and the ELISA plate is coated at 37 ℃ for 1h and 4 ℃ overnight.

② Washing, namely taking out and drying the liquid, and washing the liquid 3 times for 3 min/time by using PBST.

③ Blocking with 5% gelatin-PBST, 300. Mu.L/well, 37℃wet box in 2h, washing as above.

(2) 10 Xwashing solution pH 7.4 volume 100mL 10 Xwashing solution means 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 respectively.

(3) 10 Xserum dilution pH 7.4 volume 100mL 10 Xserum dilution 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 by mass concentration.

(4) Enzyme-labeled reagent, horseradish peroxidase-labeled staphylococcal protein A (Boster, inc.), was diluted 1:25000 with PBST, 25mL.

(5) The substrate color development solution is a single-component 3,3', 5' -tetramethyl benzidine solution (Nanjing Assistant research company) with a concentration of 100mL.

(6) Stop solution, 2M sulfuric acid solution, 50mL.

(7) Positive serum Eae-stx2 antibody positive serum, 0.5mL. Purified Eae-stx2 recombinant protein was obtained as in example 2, and was fully emulsified with an equal volume of ISA201 (complete freund's adjuvant was used as soon as no complete freund's adjuvant was used for booster immunization), followed by subcutaneous multipoint injection of rabbits (Jinling species rabbit farm, male, 2.0 kg) for immunization. Ear blood was taken before immunization to isolate serum as a pre-immunization serum control. The primary antigen amount was 500 μg/dose, boost was applied for 21 days, the ear vein was bled for 10 days after boost, and the antibody titer was determined by ELISA. After the antibody titer reaches a higher level (more than or equal to 1:12800), taking blood by heart, collecting serum as pathogenic escherichia coli positive serum, subpackaging the serum into sterile glass bottles in a quantity of 0.5mL, sealing and preserving at-20 ℃.

(8) Negative serum 0.5mL of pathogenic E.coli antibody negative serum. Pathogenic escherichia coli negative rabbits (Jinling species rabbit farm), heart blood collection, serum separation as pathogenic escherichia coli negative serum, in a 0.5mL bottle amount in sterile glass bottle, sealing, and preserving at-20 ℃.

2. The detection of pathogenic E.coli using the kit of the invention comprises the following steps:

Each kit contained 2 96 ELISA plates vacuum packed with tin foil, each plate containing a removable ELISA microwell strip.

(1) The kit is taken out from the refrigerator and placed at room temperature for 30min. The 10 Xwashing solution and the 10 Xserum dilution were diluted with distilled water into 1 Xwashing solution and serum dilution, respectively.

(2) And (3) sample adding, namely taking out the ELISA plate, fixing the ELISA plate on a frame, respectively setting a negative and positive serum hole, a sample hole to be detected and a blank control, and recording the positions of the holes. The sample to be detected, positive serum and negative serum are respectively diluted by 1:40 times by serum diluent, and are respectively added into the hole to be detected and the hole of the standard substance, and the concentration is 100 mu L/hole. The blank control was not added.

(3) Incubation in a 37 ℃ wet box for 1h.

(4) Washing the plate, namely discarding liquid, beating the water-absorbing paper, adding 250 mu L of washing liquid into each hole, standing for 3min, throwing away the washing liquid, and repeating the washing of the plate for 3 times.

(5) And adding the enzyme-labeled reagent, namely adding 100 mu L of the enzyme-labeled reagent into each hole.

(6) And (3) washing the plate in the same step (4).

(7) Color development, namely adding 100 mu L of substrate color development liquid into each hole, gently shaking for 30s, and reacting for 15min at room temperature in a dark place.

(8) Stop reaction was stopped by adding 50. Mu.L of stop solution per well.

(9) Measurement of the optical density absorbance (OD _450nm) of each well was measured on an automatic ELISA.

(10) And (5) judging results.

Example 5 clinical application

56 Pig serum samples are clinically collected by using a pathogenic escherichia coli indirect ELISA antibody kit. The results showed that the positive detection rate of the samples was 12.5% (Table 9).

TABLE 9 clinical sample test results

Number of samples	Positive and negative	Negative of
			56	12.5%(7/56)	87.5%(49/56)

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit 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 through a flexible linker, and 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 gene encoding 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, characterized in that the kit comprises an ELISA plate coated with the Eae-Stx2 fusion protein according to claim 1, 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.

6. The kit according to claim 5, characterized in that 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.

7. The kit as claimed in 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 developer 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 the 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 the OD _450nm value of the serum sample to be tested is measured to determine whether it contains pathogenic Escherichia coli antibodies, and the judgment standard is: when the OD _450nm values are all <0.362, the serum sample to be tested does not contain pathogenic Escherichia coli antibodies; when the OD _450nm values are all ≥0.362, the serum sample to be tested contains pathogenic Escherichia coli antibodies.