CN117288947B - Cattle necrotic bacillus antibody indirect ELISA detection kit and application thereof - Google Patents

Abstract

The present invention belongs to the field of biological detection technology, and specifically relates to an indirect ELISA detection kit for bovine necrotic bacillus antibody and its application. The present invention uses bovine necrotic bacillus leukotoxin LTA4 protein as the coating antigen in the indirect ELISA detection kit, which can realize the rapid detection and screening of bovine necrotic bacillus infection, and has the characteristics of simple operation, rapid and sensitive, accurate results, good specificity and repeatability; at the same time, the indirect ELISA detection kit for bovine necrotic bacillus antibody has the characteristics of simple preparation method, low cost and suitability for large-scale production, and provides a rapid, simple and sensitive diagnostic kit for detecting bovine necrotic bacillus infection for large-scale breeding in animal husbandry, and provides technical support for the prevention and control of bovine necrotic bacillus infection.

Description

An indirect ELISA detection kit for bovine necrotic bacillus antibody and its application

Technical Field

The invention belongs to the technical field of biological detection, and specifically relates to an indirect ELISA detection kit for bovine necrotic bacillus antibody and application thereof.

Background Art

Fusobacterium necrophorum is a Gram-negative strict anaerobe. As a typical opportunistic pathogen, it is widely present in nature and can cause a variety of necrotizing suppurative diseases in humans and animals. Fusobacterium necrophorum is divided into F. necrophorum subsp. necrophorum (Fnn subspecies) and F. necrophorum subsp. funduliforne (Fnf subspecies) according to DNA homology. Among them, Fnn subspecies is the main pathogen of cattle liver abscess, foot rot and necrotic laryngitis. It also plays an important role in the occurrence of mastitis and endometritis in dairy cows, and has a universal trend in cattle infection. At present, the diagnosis of bovine Fusobacterium necrophorum infection in veterinary clinics is mainly based on bacterial isolation and identification and PCR diagnostic technology. Since Bovine Fusobacterium necrophorum is strictly anaerobic and has strict requirements on culture conditions, the presence of oxygen in sample transportation and culture during bacterial isolation and identification and PCR diagnostic technology in clinical practice often leads to long detection time and poor stability of test results. At the same time, PCR technology as a conventional diagnostic method has also shown poor results in epidemiological surveys of early latent infection of bovine necrotic Clostridium difficile and multi-sample testing. Therefore, it is necessary to expand research on other diagnostic detection methods for necrotic Clostridium difficile.

Compared with PCR detection methods, ELISA detection methods have advantages such as high throughput and strong specificity in the clinical detection process. It detects antibodies in serum in an indirect way. When used in epidemiological surveys of multi-sample detection, ELISA detection methods are more convenient. The ELISA detection process is based on the specific binding between antigens and antibodies, and the detection signal is visualized through enzyme reactions to obtain corresponding results. The ELISA detection method can use bacterial exotoxins, viral surface proteins and related antibodies as antigens. Its detection application range is wide and can be used for the detection of various clinical proteins, hormones, antibiotics and antibodies in samples. However, there is currently no effective antigen for indirect ELISA detection of necrotic Bacillus.

Summary of the invention

The purpose of the present invention is to provide an indirect ELISA detection kit for bovine necrosis bacillus antibody and its application. By using bovine necrosis bacillus leukotoxin LTA4 recombinant protein as coating antigen, a bovine necrosis bacillus antibody detection kit with simple operation, rapid sensitivity, accurate results and strong specificity is established.

The invention provides the use of LTA4 recombinant protein as a coating antigen in preparing an indirect ELISA detection kit for bovine necrotic bacillus antibody.

Preferably, the amino acid sequence of the LTA4 recombinant protein is as shown in SEQ ID NO.1.

The invention also provides an indirect ELISA detection kit for bovine necrotic bacillus antibody, comprising: an enzyme-labeled plate coated with LTA4 recombinant protein, a blocking solution, a washing solution, a diluent, a positive control, a negative control, an enzyme-labeled secondary antibody, a color developing solution and a stop solution.

Preferably, the coating concentration of the LTA4 recombinant protein is 0.03125-2 μg/mL.

Preferably, the sealing liquid comprises 5% by weight of skim milk.

Preferably, the washing solution includes PBST solution; and the diluent includes PBS solution.

Preferably, the enzyme-labeled secondary antibody comprises HRP-labeled rabbit anti-bovine IgG.

Preferably, the dilution of the enzyme-labeled secondary antibody is 1:3000.

Preferably, the color developing solution includes TMB color developing solution; and the stop solution includes H ₂ SO ₄ solution.

The present invention also provides a method for detecting bovine necrotic bacillus for non-diagnostic purposes, comprising the following steps:

1) Dilute the LTA4 recombinant protein and coat it on an ELISA plate;

2) blocking and washing the coated ELISA plate to obtain a blocked ELISA plate;

3) adding the diluted sample, negative control, and positive control to the closed ELISA plate, incubating and washing, respectively, to obtain a primary ELISA plate;

4) adding the enzyme-labeled secondary antibody to the primary enzyme-labeled plate strip, incubating and washing, to obtain an intermediate enzyme-labeled plate containing the secondary antibody;

5) adding the color developing solution to the intermediate ELISA plate containing the secondary antibody to develop color in the dark, thereby obtaining a color developing ELISA plate;

6) Adding the stop solution to the color development plate to stop the color development, measuring the absorbance values of the diluted sample, negative control and positive control at OD _450nm , and calculating the negative critical value and the positive critical value;

Wherein, N is the negative serum OD _450nm , and P is the positive serum OD _450nm ;

7) Determine the result according to the diluted sample, negative critical value and positive critical value obtained in step 6). If the sample OD _450nm ≥ the positive critical value, it is judged as positive; if the sample OD _450nm ≤ the negative critical value, it is judged as negative; if the sample OD _450nm is between the positive critical value and the negative critical value, it is a suspicious sample and is retested.

During the re-test, if the sample OD _450nm > negative critical value, it is judged as positive, and if the sample OD _450nm ≤ negative critical value, it is judged as negative.

Beneficial effects:

The present invention provides an application of LTA4 recombinant protein as a coating antigen in the preparation of an indirect ELISA detection kit for bovine necrosis bacillus antibody. The present invention uses the bovine necrosis bacillus leukotoxin LTA4 protein as a coating antigen, can realize rapid detection and screening of bovine necrosis bacillus infection, and has the characteristics of simple operation, rapid sensitivity, accurate results, good specificity and repeatability, etc.

On this basis, the present invention also provides an indirect ELISA detection kit for bovine necrosis bacillus antibody, including: an enzyme label plate coated with LTA4 recombinant protein, a blocking solution, a washing solution, a diluent, a positive control, a negative control, an enzyme-labeled secondary antibody, a color developing solution and a stop solution. The experimental results show that the indirect ELISA detection kit for bovine necrosis bacillus antibody established by the present invention has no cross reaction to paratuberculosis bacillus, Brucella, etc., indicating that the kit has good sensitivity, specificity and repeatability, and has good accuracy; at the same time, the indirect ELISA detection kit for bovine necrosis bacillus antibody has the characteristics of simple preparation method, low cost and suitability for large-scale production, providing a quick, simple and sensitive diagnostic kit for detecting bovine necrosis bacillus infection for large-scale breeding in animal husbandry, and providing technical support for the prevention and control of bovine necrosis bacillus infection.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

FIG1 is an SDS-PAGE diagram of the expression of the recombinant protein of the bovine necrotic bacillus leukotoxin LTA4 in Example 1;

FIG2 is an SDS-PAGE image of the purified bovine necrotic bacillus leukotoxin LTA4 recombinant protein in Example 1;

FIG. 3 is a Western blot identification diagram of the prokaryotic expression of the recombinant protein LTA4 of the bovine necrotic bacillus leukotoxin in Example 1.

DETAILED DESCRIPTION

The invention provides the use of LTA4 recombinant protein as a coating antigen in preparing an indirect ELISA detection kit for bovine necrotic bacillus antibody.

In the present invention, the amino acid sequence of the LTA4 recombinant protein is preferably as shown in SEQ ID NO.1, specifically: SDAVIANYAGTVSGVARAAIGASTSVNEITGSTKAYVKDSTVIAKEET DDYITTQGQVDKVVDKVFKNLNINEDLSQKRKISNKKGFVTNSSATHTLKSLLANAAGSGQAGVAGTVNINKVYGETEALVENSILNAKHYSVKSGDYTNSIGVVGSVGVGGNVGVGASSDTNIIKRNTKTRVGKTTMSDEGFGEEAEITADSKQGISSFGVGVAAAGVGAGVAGTVSVNQFAGKTEVDVEEAKILVKKAEITAKRYSSVAIGNAAVGVAAKGAGIGAAVAVTKDESNTRARVKNSKIMTRNK. The nucleotide sequence encoding the LTA4 recombinant protein is preferably as shown in SEQ ID NO.2, specifically: 5'-TCTGATG CGGTAATTGCTAATTATGCAGGAACAGTGTCTGGAGTGGCCCGTGCAGCAATAGGAGCCTCAACCAGTGTGAATGAAATTACAGGATCTACAAAAGCATATGTAAAAGATTCTACAGTGATTGCTAAAGAAGAAACAGATGATTATTACTACTCAAGGGCAAGTAGATAAAGTGGTAGATAAAGTATTCAAAAATCTTAATATTAACGAAGACTTATCACAAAAAAGAAAAATAAGTAATAAAAAAGGATTTGTTACCAATAGTTCAGCT ACTCATACTTTAAAATCTTTATTGGCAAATGCCGCTGGTTCAGGACAAGCCGGAGTGGCAGGAACTGTTAATATCAACAAGGTTTATGGAGAAACAGAAGCTCTTGTAGAAAATTCTATATTAAATGCAAAACATTATTCTGTAAAATCAGGAGATTACACGAATTCAATCGGAGTAG TAGGTTCTGTTGGTGTTGGTGGAAATGTAGGAGTAGGAGCTTCTTCTGATACCAATATTATAAAAAGAAATACCAAGACAAGAGTTGGAAAAACTACAATGTCTGATGAAGGTTTCGGAGAAGAAGCTGAAATTACAGCAGATTCTAAGCAAGGAATTTCCTCTTTTGGAGTCGGAGTCGCAGCAGCCGGGGTAGGAGCCGGAGTGGCAGGAACCGTTTCCGTAAATCAATTTGCAGGAAAGACGGAAGTAGATGTG GAAGAAGCAAAGATTTTGGTAAAAAAAGCTGAGATTACAGCAAAACGTTATAGTTCTGTTGCAATTGGAAATGCCGCAGTCGGAGTGGCTGCAAAAGGAGCTGGAATTGGAGCAGCAGTGGCAGTTACCAAAGATGAATCAAACACGAGAGCAAGAGTGAAAAATTCTAAAATTATGACTCGAAACAAG-3’.

The method for preparing the LTA4 recombinant protein of the present invention preferably comprises: transferring the recombinant expression vector containing the LTA4 protein encoding gene into Escherichia coli for culturing to obtain a culture solution;

Inducing expression in the culture solution, and performing solid-liquid separation on the culture solution after the induced expression to obtain a precipitate;

The precipitate is resuspended and then ultrasonically disrupted, and solid-liquid separation is performed again to obtain a supernatant containing the LTA4 recombinant protein.

The present invention preferably transfers the recombinant expression vector containing the LTA4 protein encoding gene into Escherichia coli for culture to obtain a culture solution. The recombinant expression vector containing the LTA4 protein encoding gene of the present invention preferably includes pGEX-6p-1-LTA4, wherein the initial vector in the recombinant expression vector is preferably pGEX-6p-1, and the LTA4 protein encoding gene is inserted between BamHI and XhoI. The source of the pGEX-6p-1 is not particularly limited and can be purchased from conventional commercial channels; the nucleotide sequence of the upstream primer LTA4U used to amplify the LTA4 protein encoding gene is shown in SEQ ID NO.3, specifically 5'-AGGAggatccTCTGATGCGGTAAT-3', wherein ggatcc is a BamHI restriction site; the nucleotide sequence of the downstream primer LTA4L is shown in SEQ ID NO.4, specifically 5'-AATctcgagCTTGTTTCGAGTCAT-3', wherein ctcgag is an XhoI restriction site. In the present invention, the system for double enzyme digestion of pGEX-6p-1 is 40 μL, including 1 μL BamHI, 1 μL XhoI, 2 μL pGEX-6p-1 plasmid, 4 μL 10×Kbuffer and 32 μL ddH ₂ O; the reaction conditions for double enzyme digestion are preferably 37° C. water bath for 3 hours.

The E. coli of the present invention preferably includes E. coli competent cells BL21 (DE3), i.e., E. coli BL21 (DE3). The culture method of the present invention is preferably shaking culture, the rotation speed of the shaking culture is preferably 190 to 220 rpm, more preferably 190 rpm, the temperature is preferably 37°C, and the shaking culture ends when the OD _600nm of the cultured bacterial solution is 0.6 to 0.8.

After obtaining the culture solution, the present invention preferably mixes the culture solution with an isopropylthiogalactoside inducer (IPTG inducer) and then performs induction culture to obtain a culture solution after induced expression. The final concentration of the isopropylthiogalactoside inducer in the culture solution is preferably 0.1-2 mmol/mL, more preferably 1 mmol/mL; the induction culture time is preferably 10-20 hours, more preferably 16 hours, and the temperature is preferably 16-37°C, more preferably 16°C.

After obtaining the culture fluid after induced expression, the present invention preferably performs solid-liquid separation on the culture fluid after induced expression to obtain a precipitate. The solid-liquid separation method of the present invention is preferably centrifugation, the centrifugation speed is preferably 8000 rpm, the temperature is preferably room temperature, and the time is preferably 10 minutes.

After obtaining the precipitate, the present invention preferably mixes the precipitate with a PBS solution and resuspends it, and then ultrasonically crushes the obtained resuspended liquid until it is clarified. The PBS solution of the present invention is preferably a precooled PBS solution; the power of the ultrasonic crushing of the present invention is preferably 40 to 60 Hz, more preferably 50 Hz; and intermittent ultrasonication is preferably used, with ultrasonication for 5 seconds and intermittent ultrasonication for 10 seconds as one cycle.

After the ultrasonic crushing, the present invention preferably performs solid-liquid separation on the obtained ultrasonic crushing mixture to obtain a supernatant. The solid-liquid separation is preferably centrifugal, and the centrifugal speed is preferably 8000 rpm, the temperature is preferably 4°C, and the time is preferably 10 minutes.

After obtaining the supernatant, the present invention preferably purifies the supernatant to obtain the LTA4 recombinant protein. The present invention preferably uses Tiandirenhe GlutathioneBeads (SA008100) for the purification. In the specific implementation process, the purification can be performed by referring to the instruction manual.

The invention uses the bovine necrosis bacillus leukotoxin LTA4 protein as the coating antigen. The LTA4 protein has genetic uniqueness, can realize the rapid detection and screening of bovine necrosis bacillus infection, and has the characteristics of simple operation, rapidity and sensitivity, accurate results, good specificity and repeatability, etc.

Based on this, the present invention also provides an indirect ELISA detection kit for bovine necrotic bacillus antibody, comprising: an enzyme-labeled plate coated with LTA4 recombinant protein, a blocking solution, a washing solution, a diluent, a positive control, a negative control, an enzyme-labeled secondary antibody, a color developing solution and a stop solution.

In the present invention, the amino acid sequence of the LTA4 recombinant protein is preferably as shown in SEQ ID NO.1. The coating concentration of the LTA4 recombinant protein on the ELISA plate is preferably 0.03125-2 μg/mL, more preferably 0.125 μg/mL. The ELISA plate of the present invention preferably includes a 96-well ELISA plate, the specification is preferably 8 wells × 12 strips, and can be disassembled by itself. The 96-well ELISA strips in the embodiment of the present invention are preferably purchased from Costar.

The washing liquid of the present invention preferably includes a PBST solution; the PBST solution is preferably prepared by mixing a PBS solution and Tween-20, and the pH of the PBS solution is preferably 7.4; the volume ratio of the PBS solution to Tween-20 is preferably 1:2000. The PBST solutions used in the present invention are all prepared in the above manner. The present invention preferably concentrates the washing liquid into a 25-fold solution as a stock solution of the washing liquid.

The sealing liquid of the present invention is preferably 5% by weight skim milk.

In the present invention, the diluent preferably includes a PBS solution, the concentration of the PBS solution is preferably a PBS solution, and the concentration of the PBS solution is preferably 0.01 mol/L. In the present invention, the diluent is preferably concentrated into a 25-fold solution as a stock solution of the serum diluent.

The enzyme-labeled secondary antibody of the present invention preferably includes HRP-labeled rabbit anti-bovine IgG, and the dilution concentration of the enzyme-labeled secondary antibody is preferably 1:3000. The color developing solution of the present invention preferably includes TMB substrate color developing solution, and more preferably a ready-to-use TMB substrate color developing solution. In the present invention, if the TMB substrate color developing solution is not used immediately, it is preferred to store the TMB substrate color developing solution at 4°C; when the TMB substrate color developing solution is used after being stored at 4°C, it is preferred to use the TMB substrate color developing solution stored at 4°C after warming up to room temperature.

The stop solution of the present invention preferably includes H ₂ SO ₄ solution, and the concentration of the H ₂ SO ₄ solution is preferably 2 mol/L.

The present invention also provides a method for detecting bovine necrotic bacillus for non-diagnostic purposes, but the method for detecting bovine necrotic bacillus for diagnostic purposes also falls within the protection scope of the present invention, and specifically comprises the following steps:

1) Dilute the LTA4 recombinant protein and coat it on an ELISA plate;

2) blocking and washing the coated ELISA plate to obtain a blocked ELISA plate;

3) adding the diluted sample, negative control, and positive control to the closed ELISA plate, incubating and washing, respectively, to obtain a primary ELISA plate;

4) adding the enzyme-labeled secondary antibody to the primary enzyme-labeled plate strip, incubating and washing, to obtain an intermediate enzyme-labeled plate containing the secondary antibody;

5) adding the color developing solution to the intermediate ELISA plate containing the secondary antibody to develop color in the dark, thereby obtaining a color developing ELISA plate;

6) Add the stop solution to the color development ELISA plate to stop the color development, and measure the absorbance value at OD _450nm .

Calculate negative and positive cutoff values;

Wherein, N is the negative serum OD _450nm , and P is the positive serum OD _450nm ;

7) Result judgment standard: if the sample OD _450nm ≥ positive critical value, it is judged as positive; if the sample OD _450nm ≤ negative critical value, it is judged as negative; if the sample OD _450nm is between the positive critical value and the negative critical value, it is a suspicious sample and is re-tested.

The present invention dilutes the LTA4 recombinant protein and coats it on an ELISA plate. Specifically, the LTA4 recombinant protein is mixed and diluted with a coating solution and then coated to obtain a coated ELISA plate. The coating solution of the present invention preferably includes a carbonate buffer; the concentration of the coating solution is preferably 0.01 mol/L, and the pH value is preferably 9.6; the coating time is preferably 12 to 24 hours, more preferably 12 hours; the temperature is preferably 4 to 37°C, more preferably 4°C. The concentration of the LTA4 recombinant protein after dilution of the present invention is preferably 0.03125 to 2 μg/mL, more preferably 0.125 μg/mL; the dosage is preferably 100 μL/well.

After obtaining the coated ELISA plate, the present invention blocks and washes the coated ELISA plate to obtain a blocked ELISA plate. The blocking solution used for the blocking in the present invention is preferably skim milk with a mass percentage of 5%; the blocking is preferably a constant temperature blocking, and the temperature is preferably 37°C; the time is preferably 30min to 180min, more preferably 180min; the dosage is preferably 100 to 300μL/well, more preferably 200μL/well. The washing solution of the present invention is preferably a PBST solution, and the characteristics and preparation method of the PBST solution have been defined in the above technical scheme and will not be repeated; the number of times the washing solution is preferably 1 to 3 times, more preferably 3 times, and the time for each washing is preferably 3 to 5min, more preferably 5min.

After obtaining the closed ELISA plate, the diluted sample, negative control, and positive control are added to the closed ELISA plate for incubation and washing, respectively, to obtain a primary ELISA plate. The diluted sample of the present invention preferably includes a serum diluent, and the serum diluent is preferably obtained by mixing the serum to be tested with the diluent. The volume ratio of the serum to be tested and the diluent is preferably 1:25:1-800, and more preferably 1:100; the amount of the serum diluent is preferably 100-200 μL/well, and more preferably 100 μL/well; the present invention does not specifically limit the mixing method, and the conventional mixing method in the art can be used. The negative control of the present invention is preferably a negative serum control, and the positive control is preferably a positive serum control; the dilution multiple and the amount of the negative serum control and the positive serum control are preferably the same as those of the serum diluent. The present invention preferably also includes adding a blank control to the closed ELISA plate, and the blank control is preferably a PBST solution.

In this step, the incubation temperature is preferably 25-37°C, more preferably 37°C; the incubation time is preferably 1-2h, more preferably 1h; the washing solution used for washing is preferably PBST solution; the number of washes is preferably 1-3 times, more preferably 3 times; for each wash, the amount of washing solution is preferably 200-300μL/well, more preferably 300μL/well; the washing time is preferably 3-5min, more preferably 5min.

After obtaining the primary ELISA plate, the present invention adds the enzyme-labeled secondary antibody to the primary ELISA plate strip for incubation and washing to obtain an intermediate ELISA plate containing the secondary antibody. The present invention preferably dilutes the HRP-labeled rabbit anti-bovine IgG and then adds it to the primary ELISA plate for incubation and washing. In the present invention, the diluent used for the dilution is preferably a PBS solution; during the dilution, the volume ratio of the HRP-labeled rabbit anti-bovine IgG and the PBS solution is preferably 1:200 to 1:64000, and more preferably 1:3000. In the present invention, the amount of the diluted HRP-labeled rabbit anti-bovine IgG obtained after the dilution is preferably 100 to 200 μL/well, and more preferably 100 μL/well. In this step, the incubation temperature is preferably 25 to 37°C, and more preferably 37°C; the time is preferably 1h. In the present invention, the washing solution used for the washing is preferably PBST solution; the number of washing times is preferably 1 to 3 times, more preferably 3 times; during each washing, the amount of washing solution used is preferably 200 to 300 μL/well, more preferably 300 μL/well; the washing time is preferably 3 to 5 minutes, more preferably 5 minutes.

After obtaining the intermediate ELISA plate containing the secondary antibody, the present invention adds a color developing solution to the intermediate ELISA plate containing the secondary antibody to develop color in the dark, thereby obtaining a color ELISA plate. In the present invention, the amount of the color developing solution is preferably 100 to 200 μL/well, more preferably 100 μL/well; the time for developing color in the dark is preferably 5 to 20 minutes, more preferably 15 minutes.

After obtaining the colorimetric ELISA plate, the present invention adds a stop solution to the colorimetric ELISA plate to stop the color development, and respectively measures the absorbance values at OD _450nm of the diluted sample, the negative control, and the positive control to calculate the negative critical value and the positive critical value. The amount of the stop solution of the present invention is preferably 100 to 200 μL/well, more preferably 100 μL/well.

In the present invention, Wherein, N is the negative serum OD _450nm , and P is the positive serum OD _450nm .

After obtaining the absorbance value, negative critical value and positive critical value of the diluted sample at OD _450nm , the present invention determines the result as follows: if the sample OD _450nm ≥ the positive critical value, it is judged as positive; if the sample OD _450nm ≤ the negative critical value, it is judged as negative; when the sample OD _450nm is between the positive critical value and the negative critical value, it is a suspicious sample and is re-tested.

When the retest is performed, if the OD _450nm of the diluted sample is greater than the negative critical value, it is judged as positive, and if the OD _450nm of the diluted sample is less than or equal to the negative critical value, it is judged as negative.

In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

Example 1

Preparation of recombinant protein of bovine necrotic bacillus leukotoxin LTA4

The pGEX-6p-1-LTA4 positive plasmid (LTA4 protein encoding gene inserted between BamHI and XhoI) was transformed into E. coli BL21 (DE3) competent cells, and after shaking culture at 190 rpm and 37°C, a bacterial solution was obtained, in which the positive plasmid was extracted using the Tiangen kit;

When the concentration of the bacterial solution measured by the spectrophotometer at OD _600nm is 0.6-0.8, IPTG is added to induce expression, and the amount of IPTG added is 1 mmol/mL at a final concentration, and the induction culture conditions are 16°C for 16 hours. The induced expression bacterial solution is centrifuged and the precipitate is ultrasonically broken to obtain the LTA4 protein expressed in the supernatant (pGEX-6P-1-LTA4 recombinant protein after induction);

The induced LTA4 recombinant protein, the induced pGEX-6p-1 empty vector, the uninduced LTA4 recombinant protein and the pGEX-6p-1 empty vector were ultrasonically disrupted and the supernatant and precipitate were separated.

The specific operation is as follows: take 100 mL of induced bacterial liquid, centrifuge at 8000 rpm for 10 min at room temperature, and discard the supernatant; add 20 mL of pre-cooled PBS solution to the centrifuged precipitate and mix well, perform ultrasonic disruption under ice-water mixture conditions (0°C) until clarified, and set the ultrasonic program to 50 Hz, ultrasonic 5 s, and rest for 10 s; centrifuge the ultrasonic product at 8000 rpm for 20 min at 4°C to separate the supernatant and precipitate, resuspend the precipitate with 200 μL of PBS, and obtain the induced pGEX-6P-1-LTA4 recombinant protein ultrasonic disruption supernatant after ultrasonication;

The induced pGEX-6p-1 empty vector, the uninduced LTA4 recombinant protein and the pGEX-6p-1 empty vector were ultrasonically disrupted and the supernatants were separated according to the above steps to obtain the induced pGEX-6p-1 empty vector ultrasonic disruption supernatant, the uninduced pGEX-6P-1-LTA4 recombinant protein ultrasonic disruption supernatant and the uninduced pGEX-6P-1 empty vector ultrasonic disruption supernatant, respectively.

The supernatant and precipitate of ultrafine crushing were analyzed by SDS-PAGE, and the induced expression results are shown in Figure 1, wherein M represents protein Marker, 1 represents the supernatant of ultrasonic crushing of pGEX-6P-1-LTA4 recombinant protein after induction, 2 represents the precipitate of ultrasonic crushing of pGEX-6P-1-LTA4 recombinant protein after induction; 3 represents the supernatant of ultrasonic crushing of pGEX-6P-1 empty vector after induction; 4 represents the precipitate of ultrasonic crushing of pGEX-6P-1 empty vector after induction; 5 represents the supernatant of ultrasonic crushing of uninduced pGEX-6P-1-LTA4 recombinant protein; 6 represents the precipitate of ultrasonic crushing of uninduced pGEX-6P-1-LTA4 recombinant protein.

It can be concluded from Figure 1 that the recombinant protein of bovine necrosis bacillus leukotoxin LTA4 was successfully expressed in the supernatant at 57 kDa, the induced pGEX-6p-1 empty vector was expressed at 25 kDa, and the uninduced recombinant protein of bovine necrosis bacillus leukotoxin LTA4 was not expressed, which was consistent with the expected results.

The bovine necrosis bacillus leukotoxin LTA4 recombinant protein expressed in the supernatant was purified by column (the column was Tiandirenhe GlutathioneBeads (SA008100), and the operation was performed according to the instructions) to obtain the purified bovine necrosis bacillus leukotoxin LTA4 recombinant protein. The purified bovine necrosis bacillus leukotoxin LTA4 recombinant protein was analyzed by SDS-PAGE. The results are shown in Figure 2, wherein M represents protein Marker, and lanes 1, 2 and 3 are the pGEX-6P-1-LTA4 recombinant protein after the first elution purification, the pGEX-6P-1-LTA4 recombinant protein after the second elution purification, and the pGEX-6P-1-LTA4 recombinant protein after the third elution purification, respectively.

It can be concluded from FIG2 that the recombinant protein of bovine necrotic bacillus leukotoxin LTA4 has a single band at 57 kDa, and the protein concentration is 1.31 mg/mL, indicating that the recombinant protein of bovine necrotic bacillus leukotoxin LTA4 was successfully purified.

Using Biyuntian GST TagMousemAb (AF2891) as the primary antibody, the induced bovine necrotic bacillus leukotoxin LTA4 recombinant protein, the induced pGEX-6p-1 empty vector and the uninduced bovine necrotic bacillus leukotoxin LTA4 recombinant protein were ultrasonically fragmented and the supernatant and precipitate were separated (the steps are the same as above), and Western blot identification was performed. The results are shown in Figure 3, wherein M represents protein Marker, 1 represents the supernatant of ultrasonic fragmentation of induced pGEX-6P-1-LTA4 recombinant protein, 2 represents the precipitate of ultrasonic fragmentation of induced pGEX-6P-1-LTA4 recombinant protein, 3 represents the supernatant of ultrasonic fragmentation of induced pGEX-6P-1 empty vector, 4 represents the precipitate of ultrasonic fragmentation of induced pGEX-6P-1 empty vector, 5 represents the supernatant of ultrasonic fragmentation of uninduced pGEX-6P-1-LTA4 recombinant protein, and 6 represents the precipitate of ultrasonic fragmentation of uninduced pGEX-6P-1-LTA4 recombinant protein.

It can be concluded from Figure 3 that in the supernatant of ultrasonic fragmentation of the bovine necrotic bacillus leukotoxin LTA4 protein expressed in the supernatant obtained after IPTG induction, there is a reaction band of about 57 kDa in size, in the supernatant and precipitate of ultrasonic fragmentation of the pGEX-6p-1 empty vector after IPTG induction, there is a reaction band of 25 kDa in size, and the uninduced bovine necrotic bacillus leukotoxin LTA4 recombinant protein has no reaction band. The results show that the bovine necrotic bacillus leukotoxin LTA4 recombinant protein was successfully expressed and has reactogenicity.

Example 2

An indirect ELISA detection kit for bovine necrotic bacillus antibody, comprising the following components: a positive serum control, a negative serum control, a TMB substrate colorimetric solution, a 96-well enzyme-labeled plate, a serum diluent, an enzyme-labeled secondary antibody, a blocking solution, a washing solution, and a stop solution;

Washing solution: 1 pack of commercial PBS powder is prepared into 2000 mL of deionized water. 1 mL of Tween-20 (0.05%) is added into 2000 mL of PBS and concentrated 25 times as the storage solution.

TMB substrate colorimetric solution (commercial Solebol colorimetric solution PR1200).

Stop solution (2 mol/L H ₂ SO ₄ ) distilled water 178.3 mL, add concentrated sulfuric acid (98%) 21.7 mL dropwise.

Preparation method of ELISA plate: Use 0.05M carbonate buffer (pH 9.6) as coating solution, dilute the purified bovine necrosis bacillus leukotoxin LTA4 recombinant protein in Example 1, and add 100 μL/well to the ELISA plate to ensure that the concentration of bovine necrosis bacillus leukotoxin LTA4 recombinant protein is 0.125 μg/mL. Coat overnight at 4°C, discard the coating solution the next day, and wash 3 times. The amount of washing solution used for each wash is 300 μL/well, the washing time is 5 minutes, pat dry, add 5% skim milk blocking solution at 200 μL/well, stand at 37°C for 2 hours, wash 3 times, the washing method is the same as before, and pat dry. After drying at room temperature until there are no water droplets, put it into a packaging bag, add a desiccant, and store it in a vacuum.

Example 3

The basic operation steps of the indirect ELISA kit in Example 2 are as follows:

The purified bovine necrosis bacillus leukotoxin LTA4 recombinant protein was diluted to 2 μg/mL with 0.01 mol/L carbonate buffer (pH 9.6) as antigen coating solution, and then added to a 96-well ELISA plate, 100 μL per well, to ensure that the concentration of bovine necrosis bacillus leukotoxin LTA4 recombinant protein in each well was 0.125 μg/mL, and coated at 4°C for 12 h;

The 96-well ELISA plate coated overnight was blocked by adding 5% skim milk at 200 μL/well and kept at 37°C for 2 h, followed by conventional washing with PBST;

The serum to be tested was added at the optimal dilution ratio of 1:100 (volume ratio) (i.e., 1 mL of serum to be tested was mixed with 100 mL of PBST buffer), and the negative serum control, positive serum control, and blank control (PBST buffer) were added to a 96-well ELISA plate at 100 μL/well. After incubation at 37°C for 1 hour, conventional PBST washing was performed;

According to the dilution multiple of HRP-labeled rabbit anti-bovine IgG 1:3000 (i.e., 1 μL HRP-labeled rabbit anti-bovine IgG was mixed with 16000 μL PBS solution), 100 μL/well was added to a 96-well ELISA plate, incubated at 37°C for 1 h, and washed with PBST as usual;

Take out the TMB substrate colorimetric solution from the 4°C refrigerator and return it to room temperature. Add 100 μL/well of the colorimetric solution and color develop for 15 min at room temperature in the dark.

Stop solution was added at 50 μL/well to stop color development, and the microplate reader was set to read the value of OD _450nm .

Example 4

Screening of the best application conditions for the indirect ELISA kit in Example 2

(1) Screening of optimal antigen coating concentration and negative and positive serum working concentrations

The purified bovine necrosis bacillus leukotoxin LTA4 recombinant protein was diluted and coated at 2 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.25 μg/mL, 0.125 μg/mL, 0.0625 μg/mL, and 0.03125 μg/mL. At the same time, the negative and positive sera of bovine necrosis bacillus were diluted in multiples according to the gradient of 1:25, 1:50, 1:100, 1:200, 1:400, and 1:800, and the HRP-labeled rabbit anti-bovine IgG secondary antibody was diluted at 1:5000. The composition of the indirect ELISA kit was the same as that of Example 2, and the basic operation steps were the same as those of Example 3. The P/N value was calculated using the square matrix method. The P/N value, N is the negative serum OD _450nm , and P is the positive serum OD _450nm . The highest P/N value was selected as the optimal antigen coating concentration and the optimal negative and positive serum dilution multiple. The results are shown in Table 1.

Table 1 Optimal antigen coating concentration and optimal serum working concentration

From the data in Table 1, it can be seen that when the P/N value is the highest, the optimal antigen pack concentration is 0.125 μg/mL and the optimal serum working concentration is 1:100.

(2) Screening of the optimal enzyme-labeled antibody working concentration

The purified bovine necrosis bacillus leukotoxin LTA4 protein was coated at the optimal antigen coating concentration of 0.125 μg/mL, and the standard bovine necrosis bacillus negative and positive serum was diluted at the optimal serum dilution multiple of 1:100. The enzyme-labeled secondary antibody was diluted by 1:2000, 1:3000, 1:4000, 1:5000, 1:8000, 1:16000, 1:32000, and 1:64000 according to the working range of the instructions using the square matrix method. The composition of the indirect ELISA kit is the same as that of Example 2, and the basic operating steps are the same as those of Example 3. The P/N value was calculated using the square matrix method, and when the P/N value was the highest, the optimal monoclonal antibody and enzyme-labeled antibody working concentrations were calculated, and the results are shown in Table 2.

Table 2 Optimal enzyme-labeled antibody working concentration

From the data recorded in Table 2, it can be seen that when the P/N value is the highest, the optimal enzyme-labeled antibody working concentration is 1:3000.

(3) Screening of TMB substrate colorimetric solution conditions

TMB substrate colorimetric solution 100 μL/well, temperature was room temperature, 37°C, colorimetric time was 5 min, 10 min, 15 min, 20 min (see Table 3 for details), each treatment was repeated 3 times, and a blank control was set at the same time. The composition of the indirect ELISA kit was the same as in Example 2, and the basic operation steps were the same as in Example 3. The P/N value was calculated using the square matrix method, and the action conditions of the TMB substrate colorimetric solution were calculated. The results are shown in Table 3.

Table 3 Results of the color development solution conditions

The results are shown in Table 3. Under room temperature conditions, 15 minutes of color development is the best condition for the color developing solution.

Example 5

Example 2: Test on positive/negative threshold, specificity, sensitivity and repeatability of indirect ELISA kit

1. Determined by indirect ELISA test, 30 negative bovine serum samples with known background were tested to determine the negative and positive critical values, and blank serum was used as a control to obtain The negative and positive results are as follows: The calculated negative critical value is 0.413. The calculated positive critical value is 0.491. When the negative and positive judgment criteria are: the sample P/N value ≥ 0.491 is judged as positive, and the sample P/N value ≤ 0.413 is judged as negative. If the sample P/N is between 0.413 and 0.491, it is a suspicious sample and needs to be retested. If it is still between the two, the result is judged as positive.

2. The indirect ELISA detection method and related judgment criteria for detecting bovine necrosis bacillus antibodies developed by the present invention were used to detect bovine necrosis bacillus positive serum, bovine paratuberculosis positive serum and bovine Brucella positive serum respectively. The basic operation steps of the indirect ELISA kit were the same as those in Example 3. The P/N value of the bovine necrosis bacillus positive serum was >0.491, and the P/N value of the bovine paratuberculosis positive serum and the bovine Brucella positive serum was <0.413. The results are shown in Table 4. It shows that the indirect ELISA kit for detecting bovine necrosis bacillus antibodies developed by the present invention has good specificity.

Table 4 Specificity test results

3. The indirect ELISA detection method for bovine necrosis bacillus antibody established by the present invention was used to detect the dilution ratio of the positive serum of bovine necrosis bacillus. The positive serum was diluted from 100 times, and diluted 2 times to 1:6400, and detected by the established indirect ELISA. When the serum was diluted to 1:1600, the value of OD _450nm was 0.432, and the value of P/N was between the positive and negative critical values, and a re-test was required. The value of P/N in the re-test result was still between the positive and negative critical values, and the result was determined to be positive. Therefore, the sensitivity of the indirect ELISA detection method established in this experiment was 1:1600. See Table 5.

Table 5 Sensitivity test results

4. The indirect ELISA antibody detection kit for bovine necrosis bacillus established by the present invention was used to conduct intra-batch and inter-batch repeatability tests. The intra-batch repeatability test was to take the 96-well ELISA plate coated with the same batch and test 3 known negative sera and 3 positive sera. The inter-batch repeatability test was to take the detachable 96-well ELISA plate coated with different batches and test 3 known negative sera and 3 positive sera.

The basic operation steps of the indirect ELISA kit are the same as those in Example 3. The P/N value and average inhibition rate of each sample in the intra-batch repeatability test and the inter-batch repeatability test are calculated. Standard deviation SD and coefficient of variation CV%, Samples 1 to 3 are known positive samples, and samples 4 to 6 are known negative samples. The test results are shown in Tables 6 and 7.

Table 6 Intra-batch repeatability test results

Table 7 Results of inter-batch repeatability test

It can be seen from the data recorded in Table 6 and Table 7 that the CV% values of the kit of the present invention are all less than 10%, indicating that the established indirect ELISA antibody detection kit for bovine necrotic bacillus has good repeatability.

5. The indirect ELISA kit and PCR identification method for detecting antibodies to bovine necrotic bacillus developed by the present invention were used, wherein the PCR identification method was performed with reference to the method in the following literature (Jiang Jiancheng, Zhang Siyao, He Xianjing, et al. Isolation and identification of necrotic bacillus from a cattle farm in Hebei Province [J]. Chinese Journal of Biological Products, v.32(07):770-773.), and 90 clinical bovine serum samples with known identification results (19 of which were positive and 71 were negative) were tested. The basic operating steps of the indirect ELISA kit were the same as those in Example 3, and its P/N value was calculated. The results are shown in Table 8.

Table 8 Kit compliance test results

As shown in Table 8, the PCR identification results included 20 positive sera and 70 negative sera, and the indirect ELISA results included 20 positive sera and 70 negative sera. Finally, the consistency rate of the indirect ELISA and PCR identification of Necrobacterium necrotum of the present invention was 95.2%.

Example 6

Indirect ELISA kits for testing clinical samples

The indirect ELISA kit for detecting antibodies to necrotic bacillus developed by the present invention was used to detect 300 clinical serum samples from a cattle farm in Heilongjiang Province. The basic operating steps of the indirect ELISA kit were the same as those of Example 3, and the test results are shown in Table 9.

Table 9 Clinical sample testing results

As shown in Table 9, the kit of the present invention can specifically detect Bacillus necroticus, and the positive rate is only 3.3%.

It can be concluded from the above examples that the present invention uses a prokaryotic expression system to prepare a recombinant protein of the bovine necrotic bacillus leukotoxin LTA4, and further uses the recombinant protein of the bovine necrotic bacillus leukotoxin LTA4 as a coated diagnostic antigen. At the same time, the present invention provides an indirect ELISA detection kit for bovine necrotic bacillus antibody prepared by the recombinant protein, which has the characteristics of accurate results, simple operation, good specificity, high sensitivity, strong repeatability, etc., and is mainly used for screening bovine necrotic bacillus infection.

Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims (7)

1. An indirect ELISA detection kit for a necrotic bovine bacillus antibody, which is characterized by comprising: an ELISA plate coated with LTA4 recombinant protein, a sealing solution, a washing solution, a diluent, a positive control, a negative control, an ELISA secondary antibody, a chromogenic solution and a stop solution; the amino acid sequence of the LTA4 recombinant protein is shown as SEQ ID NO. 1;

The coating concentration of the LTA4 recombinant protein is 0.03125-2 mug/mL.

2. The necrotic bovine antibody indirect ELISA detection kit according to claim 1, wherein the blocking solution comprises skimmed milk with a mass percentage of 5%.

3. The necrotic bovine antibody indirect ELISA detection kit according to claim 1, wherein the washing solution comprises PBST solution; the dilutions included PBS solutions.

4. The necrotic bovine antibody indirect ELISA detection kit according to claim 1, wherein the enzyme-labeled secondary antibody comprises HRP-labeled rabbit anti-bovine IgG.

5. The indirect ELISA detection kit of necrobacter bovis antibodies according to claim 4, characterized in that the dilution of the second enzyme-labeled antibody is 1:3000.

6. The necrotic bovine antibody indirect ELISA detection kit according to claim 1, wherein the color development liquid comprises TMB color development liquid; the termination solution comprises H ₂SO₄ solution.

7. A method for detecting necrotic bovine bacteria for non-diagnostic purposes, comprising the steps of:

1) Diluting the LTA4 recombinant protein, and coating the diluted LTA4 recombinant protein on an ELISA plate, wherein the amino acid sequence of the LTA4 recombinant protein is shown as SEQ ID NO. 1;

2) Sealing and washing the coated ELISA plate to obtain a sealed ELISA plate;

3) Respectively adding a diluted sample, a negative control and a positive control into the closed ELISA plate for incubation and washing to obtain a primary ELISA plate;

4) Adding the secondary enzyme-labeled antibody into the primary enzyme-labeled strip for incubation and washing to obtain a secondary enzyme-labeled plate containing the secondary antibody;

5) Adding the color developing solution into the secondary-antibody-containing medium-grade ELISA plate for light-shielding color development to obtain a color developing ELISA plate;

6) Adding a stop solution into the chromogenic ELISA plate for chromogenic termination, respectively measuring light absorption values at OD _450nm of a diluted sample, a negative control and a positive control, and calculating a negative critical value and a positive critical value;

The said Wherein, N is negative serum OD _450nm, and P is positive serum OD _450nm;

7) Judging the result according to the diluted sample, the negative critical value and the positive critical value obtained in the step 6), wherein the OD _450nm of the diluted sample is more than or equal to the positive critical value, the diluted sample is judged to be positive, the OD _450nm of the diluted sample is less than or equal to the negative critical value, the diluted sample is judged to be negative, and the diluted sample is a suspicious sample when the OD _450nm of the diluted sample is between the positive critical value and the negative critical value, and rechecking is carried out;

And in the rechecking process, the OD _450nm of the diluted sample is larger than a negative critical value, the diluted sample is judged to be positive, and the OD _450nm of the diluted sample is less than or equal to the negative critical value, and the diluted sample is judged to be negative.