Polypeptide specifically combined with Brucella antigen and application thereof

Abstract

The invention belongs to the technical field of bacterial detection, and particularly relates to a polypeptide specifically combined with a Brucella antigen and application of the polypeptide. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 or SEQ ID NO: 2. The polypeptide has high specificity with the Brucella, and the Brucella is detected based on the polypeptide, so that the sensitivity is high, the detection range is wide, and the Brucella in the concentration interval of 10 < 3 > CFU / mL to 10 < 8 > CFU / mL can be accurately detected.

Description

Technical Field

[0001] The invention belongs to the technical field of bacterial detection, and in particular relates to a polypeptide specifically binding to Brucella antigen and application thereof. Background Art

[0002] Brucella is a Gram-negative bacterium that can cause brucellosis, which is mainly manifested by fever, sweating, fatigue, joint and muscle pain, etc., and severe cases can cause loss of ability to work. Therefore, the detection of Brucella is of great significance.

[0003] At present, the detection methods of Brucella mainly include traditional culture method, serological test and molecular biology method. Among them, although the traditional culture method can provide a basis for diagnosis, it is time-consuming and requires professional laboratory conditions; although the serological test is easy to operate, there are problems of cross-reaction and false positive; although molecular biological methods such as PCR have high sensitivity and specificity, they are expensive and have high technical requirements for operators.

[0004] And enzyme-linked immunosorbent assay (ELISA) method has simple operation, and the characteristics of fast detection speed and high accuracy have the possibility of overcoming the above-mentioned Brucella detection method defect.ELISA method is based on antigen and antibody combination to realize Brucella detection, wherein by antibody binding antigen is one of Brucella detection means.So, digging a kind of antibody that can be combined with Brucella or antibody substitute (peptide) is of great significance to Brucella.

[0005] Phage display technology is a technology that uses the surface of bacteriophage to display foreign peptides. It can display a large number of different peptide libraries on the surface of bacteriophage and screen out peptides (antibody substitutes) with high affinity and specificity through specific binding with target antigens. This technology has been widely used in vaccine development, drug design and biological detection. Summary of the invention

[0006] Based on this, the present invention screens out two polypeptides that can specifically identify Brucella through phage display peptides, and the specificity is strong; in addition, when Brucella is detected through an ELISA test method based on the polypeptide, the accuracy is high and the detection range is wide.

[0007] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0008] In one aspect, the present invention provides a polypeptide that specifically binds to a Brucella antigen, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0009] Another aspect of the present invention provides a reagent or kit for detecting Brucella, which comprises the polypeptide of the present invention.

[0010] Preferably, the above reagent or kit is an enzyme-linked immunosorbent assay reagent or kit.

[0011] More preferably, the above-mentioned ELISA is a double antibody sandwich ELISA.

[0012] Preferably, the above reagents or kits further include a blocking buffer, an enzyme-labeled secondary antibody, a color developing solution, a stop solution or a washing solution.

[0013] In another aspect, the present invention provides a use of the polypeptide of the present invention in the preparation of a product for detecting Brucella.

[0014] In another aspect, the present invention further provides a use of the polypeptide of the present invention in the preparation of a product for diagnosing Brucella infection.

[0015] Preferably, in the above two applications, the Brucella is bovine Brucella.

[0016] Preferably, in the above two applications, the product comprises a reagent or a kit.

[0017] Preferably, in the above two applications, the reagent or kit is an enzyme-linked immunosorbent assay reagent or kit.

[0018] The beneficial effects of the present invention include:

[0019] (1) The polypeptide specifically binding to Brucella antigen provided by the present invention can specifically recognize and specifically bind to Brucella antigen, does not cross-react with Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii or Escherichia coli, and has high specificity.

[0020] (2) Based on the polypeptide specifically binding to Brucella antigen provided by the present invention, Brucella is detected with high sensitivity and wide detection line, and can accurately detect 10 3 CFU / mL-10 8 Brucella in CFU / mL concentration range. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The recovery rate of phage screening in the cycle of "adsorption-washing-elution-amplification";

[0022] Figure 2 The absorbance of 30 phage monoclones;

[0023] Figure 3 The specificity of different phage monoclones to Brucella is shown;

[0024] Figure 4 To verify the specificity of Brucella antibody (secondary antibody) for ELISA;

[0025] Figure 5 To verify the specificity of Brucella antibodies (secondary antibodies) at different concentrations by ELISA;

[0026] Figure 6 The detection range of phage display peptide No. 9 for Brucella detection;

[0027] Figure 7 This is the detection range of Brucella using phage-displayed peptide No. 25. DETAILED DESCRIPTION

[0028] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0029] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0030] An embodiment of the present invention provides a polypeptide that specifically binds to a Brucella antigen, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0031] It should be noted that, in the present invention, a polypeptide with high specificity to Brucella antigens is screened out by screening polypeptides through phage display peptides, and the polypeptide does not cross-react with Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii or Escherichia coli; and Brucella is detected by a double antibody sandwich method, with a wide detection range and high sensitivity; the sequence of the polypeptide is shown in SEQ ID NO: 1 (the phage display peptide No. 9 described below) or SEQ ID NO: 2 (the phage display peptide No. 25 described below).

[0032] The embodiment of the present invention also provides a reagent or a kit for detecting Brucella, which comprises the polypeptide of the present invention.

[0033] It should be noted that, based on the strong specificity between the polypeptide and the Brucella antigen in the present invention, Brucella can be detected through the specific binding of the polypeptide and the Brucella antigen.

[0034] In some specific examples, the above reagent or kit is an enzyme-linked immunosorbent assay reagent or kit.

[0035] It should be noted that the polypeptide of the present invention can be added with other auxiliary detection reagents to prepare a reagent or kit for detecting Brucella, and the other auxiliary detection reagents can be a reagent or kit based on enzyme-linked immunosorbent assay.

[0036] In some specific examples, the ELISA is a double antibody sandwich ELISA.

[0037] It should be noted that the above-mentioned ELISA may include direct ELISA, indirect ELISA or double antibody sandwich ELISA, among which the double antibody sandwich ELISA test method, i.e. double antibody sandwich ELISA, may be preferred due to its high sensitivity.

[0038] In some specific examples, the above reagents or kits further include a blocking buffer, an enzyme-labeled secondary antibody, a color developing solution, a stop solution or a washing solution.

[0039] It should be noted that the reagents or kits for the enzyme-linked immunosorbent assay in the present invention may also include a blocking buffer, an enzyme-labeled secondary antibody, a color developing solution, a stop solution or a washing solution; the above reagents are all well-known in the art and can be obtained commercially or homemade.

[0040] The embodiment of the present invention also provides an application of the polypeptide of the present invention in the preparation of a product for detecting Brucella.

[0041] The embodiment of the present invention also provides an application of the polypeptide of the present invention in the preparation of a product for diagnosing Brucella infection.

[0042] It should be noted that, based on the specificity of the polypeptide of the present invention to Brucella, it can be used in the preparation of detection products or diagnostic products for detecting Brucella or Brucella infection diseases. The detection method is accurate and has a wide detection range.

[0043] In some specific examples, in any of the above applications, Brucella is bovine Brucella.

[0044] It should be noted that the sources of Brucella include more than 60 kinds of livestock (such as cattle, pigs and sheep), poultry and wild animals. The polypeptides in the present invention have better specificity for bovine Brucella (Brucella abortus or Brucella bovis).

[0045] In some specific examples, in any of the above applications, the product includes a reagent or a kit.

[0046] In some specific examples, in any of the above applications, the reagent or kit is an enzyme-linked immunosorbent assay reagent or kit.

[0047] It should be noted that, in the application of the above polypeptide in the preparation of detection products or diagnostic products for detecting Brucella or Brucella infection diseases, the form of the reagent or kit is consistent with the above; it may include direct ELISA, indirect ELISA or double antibody sandwich ELISA, preferably the double antibody sandwich ELISA test method, i.e. double antibody sandwich enzyme-linked immunosorbent assay; in addition, the reagent or kit may also include a blocking buffer, an enzyme-labeled secondary antibody, a color developing solution, a stop solution or a washing solution; the above reagents are all well-known reagents in the art and can be obtained commercially or homemade.

[0048] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0049] In the following example, the preparation of TSA medium includes: 10g of tryptone soy broth medium, 15g of agar powder, ultrapure water added to 1L, and autoclaved at 121℃ for 25min; the preparation method of blocking buffer includes: PBS with 1% BSA by mass, 10mg / ml BSA, filtered sterilization, and stored at 4℃; TBST buffer is 1×PBS+0.5% Tween-20 by volume; diluent is 1×PBS+0.1% Tween-20 by volume, 1mg / ml BSA; the preparation method of elution includes: 1.5g of glycine (0.2M glycine-HCl, 1mg / ml BSA) and 100mg of BSA, dissolved in 98ml ultrapure water, and HCl is used to adjust the pH to 2. 2 (about 1 ml of HCl), dilute to 100 ml, filter and sterilize, and store at room temperature; the preparation method of the neutralization solution includes: 1M Tris-HCl (pH 9), that is, 12.114g of Tris is dissolved in water, the pH is adjusted to 9.0, and the volume is adjusted to 100 ml; the preparation method of the LB solid culture medium includes: 10g of tryptone, 5g of yeast extract, 5g of NaCl, 15g of agar powder, add 1L of ultrapure water, and sterilize at 121°C for 25min; the preparation method of the coating buffer includes: 0.05M carbonate buffer at pH 9.6: weigh 1.5g of Na2CO3 and 2.9g of NaHCO3, add 800mL of ultrapure water to dissolve, adjust the pH to 9.6, and then dilute to 1L, and sterilize at 121°C for 25min.

[0050] 1. Screening of phage peptides that specifically bind to Brucella antigens

[0051] (I) Brucella culture and dilution

[0052] (1) Brucella culture

[0053] A disposable inoculation loop was used to dip the frozen stock solution of Brucella (Brucella abortus (Bovine Brucella) vaccine strain 104M, provided by Beijing Institute of Biology) on TSA medium for four-zone streaking, and cultured at 37°C for 72h; a single colony of Brucella was picked and placed in a conical flask containing 20mL of TSB medium, and cultured at 37°C and 220rpm for 32h to activate a live bacterial solution; 200μL of the live bacterial solution was drawn into a conical flask containing 20mL of TSB medium, placed in a constant temperature culture oscillator, and cultured at 37°C and 220rpm for 8h to perform secondary activation to obtain a second live bacterial solution; the second live bacterial solution was centrifuged at 5000rpm for 30min, the supernatant was discarded, the precipitate was collected, resuspended with PBS (0.01mol / L) and centrifuged again, washed 3 times, and collected in a centrifuge tube after being blown evenly.

[0054] (2) Dilution of Brucella bacterial solution

[0055] The spectrophotometer (U-3900, HITACHI, Japan) was used for detection. PBS was used as a blank control and the wavelength was zeroed at 600 nm. The two-live bacterial solution was evenly resuspended and PBS (0.01 mol / L) was added in small amounts several times to adjust the OD600 value to 1. At this time, the bacterial solution concentration was 1.45×10 10 CFU / mL; diluted with PBS (0.01 mol / L) to the required concentration when used.

[0056] (II) Screening of phage peptides that specifically bind to Brucella

[0057] The screening of phage peptides that specifically bind to Brucella was performed in 7 rounds of screening according to the cycle of "adsorption-washing-elution-amplification". The specific operation is as follows:

[0058] (1) Seven rounds of phage bioscreening

[0059] 1) Add Brucella solution (diluted with PBS, 50ul / well, 10 8 CFU / ml), 37℃ overnight until dry; take out the 96-well plate, add 300ul blocking buffer, incubate at room temperature for 1h; wash 6 times with TBST buffer, remove the residual liquid each time; take 50ul phage library solution (NEB, Ph.D TM .-12Phage Display Peptide LibraryKit v2, E8210S) (diluted to 2×10 11 pfu / ml); add to the blocked 96-well plate and incubate at room temperature for 1 hour; pour off the liquid TBST buffer and wash 6 times, removing the residual liquid each time; then add 50 μL of elution solution to each well, gently shake (110 rpm / min) for 10 minutes, and add 7.5 ul of neutralization solution to each well to obtain phage elution solution;

[0060] 2) Use a disposable inoculation loop to dip the frozen stock of Escherichia coli (E. coli K12 ER2738) into four zones on the LB solid medium containing tetracycline, and invert it to incubate it overnight at 37°C; pick a single colony of E. coli and put it into a conical bottle containing 20ml LB liquid medium (self-prepared: 10g tryptone, 5g yeast extract, 5g NaCl, add 1L ultrapure water, and sterilize it at 121°C for 25min), and place it in a 37°C constant temperature incubator at 220rpm for 30min to reach OD 6000.01–0.05; take 5 μL of the phage eluate in step 1) to measure the titer and calculate the recovery rate (recovery rate = phage titer of the eluate / titer of the phage added for screening), add the remaining phage eluate to 20 mL of Escherichia coli culture in the early logarithmic growth stage, and culture at 37°C and 220 rpm for 4.5 h to obtain a mixed culture;

[0061] 3) Centrifuge the mixed bacterial solution at 4°C, 5000g for 10 min, transfer the supernatant to a new centrifuge tube, add 1 / 5 volume of 20% PEG / 2.5M sodium chloride (e.g., add 5ml 20% PEG / 2.5M sodium chloride to 20ml supernatant), vortex the mixed solution and place it at 4°C overnight; the next day, take the mixed solution and centrifuge it at 4°C, 12000g for 15min, after which phage precipitation can be seen, discard the supernatant, add 1ml TBS buffer to the phage precipitation and resuspend it, centrifuge it at maximum speed for 1min, remove residual cells and precipitation, add 200ul 20% PEG / 2.5M NaCl to the supernatant, and place it on ice for 60min; then centrifuge it at 4°C, 20000g for 10min, discard the supernatant, resuspend the phage precipitation in 200μl TBS buffer to obtain the amplified phage solution, completing the first round of screening; take 5ul phage amplification solution for titer determination, and the rest for the next round of screening;

[0062] 4) The 96-well plate was re-coated with Brucella solution, and the first round of phage amplification solution was diluted to 10 11 pfu / ml for the second round of screening, and so on for the third to seventh rounds until the recovery rate is basically stable.

[0063] (2) Eliminate nonspecific interference of bacteriophage (BSA, negative bacteria)

[0064] 1) Add 50ul BSA / well (10mg / mL) to the 96-well plate for phage screening and incubate at 37℃ overnight until dry; remove the 96-well plate, add 300ul blocking buffer, and incubate at room temperature for 1h; wash 6 times with TBST buffer, and remove all residual liquid each time; take 50ul of the seventh round of phage amplification solution (diluted to 10 10 pfu / ml); add to the blocked 96-well plate and incubate at room temperature for 1 hour; aspirate the supernatant, take 5ul for titer determination, and amplify the rest using steps 2) and 3) of the seven rounds of phage biological screening in (1) above to obtain the eighth round of phage amplification solution;

[0065] 2) Add 1 ml of negative bacterial mixed solution (Staphylococcus aureus solution, Staphylococcus epidermidis solution, Acinetobacter baumannii solution and Escherichia coli solution, 10% of each solution) into the centrifuge tube. 8CFU / ml); then add 10ul of the eighth round of phage amplification solution (the concentration of the eighth round of phage amplification solution is 10 10 pfu / ml) at 37°C for 1 h; centrifuge at 6010 g for 15 min; take 5 ul of supernatant for titer determination, and use (1) steps 2) and 3) of seven rounds of phage bioscreening for the remaining supernatant. In the last titer determination, 30 well-separated positive plaques are randomly picked from the plate with less than 100 plaques.

[0066] The results are as follows Figure 1 As shown, the phage screening recovery rate was basically stable in the 6th and 7th rounds of screening.

[0067] (3) Phage monoclonal amplification

[0068] According to step (1), Escherichia coli (E. coli K12 ER2738) was cultured to the early logarithmic growth stage, 30 well-separated blue plaques were picked up by the tip of a disposable pipette and placed in culture tubes containing 20 ml of E. coli culture solution, and cultured at 37°C with shaking for 45 hours for amplification; the subsequent steps were carried out according to the phage elution solution amplification step to obtain a monoclonal phage amplification solution.

[0069] (4) Phage ELISA verification

[0070] Brucella solution (50ul / well, 108CFU / ml) was added to a 96-well plate, and a negative control, skim milk powder solution (50ul / well, 1×PBS+1% skim milk powder solution) was set up in each well, and the plate was incubated at 37°C overnight until dry. The 96-well plate was taken out, and 200ul of blocking solution (1×PBS+1% skim milk powder solution) was added to each well, and the plate was blocked at 37°C for 1h, the blocking solution was removed, and the plate was washed with TBST buffer (1×TBS+0.5% Tween-20) for 5 minutes. The plate was washed 5 times, and the residual liquid was poured out after each wash; 50ul of the 30 phage monoclonal amplification solutions obtained in the above step (3) were added to each well, and incubated at room temperature for 1h. The phage solution was poured out, and the plate was washed 5 times with TBST buffer (1×PBS+0.5% Tween-20), and the residual liquid was poured out after each wash; 100μL (1:5000) of HRP-labeled mouse anti-M13 phage monoclonal antibody (Sino Biological, 11973-MM05T-H), incubate at 37°C for 1 hour, wash the plate 5 times with TBST buffer; add 100uL TMB ELISA Substrate colorimetric solution to each well, place the 96-well plate in a dark place to avoid light for 10 minutes, add 100ul450nm TMB ELISA Substrate stop solution to each well to stop the reaction, detect the absorbance at 450nm, and the absorbance value of the experimental well / negative well is greater than 2.1, which is considered positive.

[0071] Test results such as Figure 2 As shown in the figure, the absorbance of 30 phage monoclones was different, and the 20 best phage monoclones with absorbance ratio greater than 2.1 were selected ( Figure 2 1 to 7, 9, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22, 23 and 25) were sent for sequencing.

[0072] (5) Phage sequencing

[0073] The above 20 phage monoclones were sent to a sequencing company for sequencing. The sequencing primer was 5'-CCCTCATAGTTAGCGTAACG-3'. The sequencing results were analyzed using biological software to find out the sequences inserted by the phage. There were 20 inserted sequences.

[0074] (6) ELISA to verify peptide specificity and screen peptides

[0075] Add 50 μL of Brucella solution (10 8 CFU / mL), and set 1% skim milk powder (1×PBS+1% skim milk powder solution, 50ul / well), negative bacteria (50ul / well, 10 8CFU / mL, Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii and Escherichia coli respectively) as negative control; incubate at 37°C overnight until dry. After incubation, operate the plate washer to wash the 96-well plate with TBST buffer, add 200ul blocking solution (1×PBS+1% skim milk powder solution) to each well, block at 37°C for 1h, pour off the blocking solution, wash 5 times with TBST buffer (1×TBS+0.5% Tween-20), and pour out the residual liquid each time; add 50μL of 20 different phage monoclonal clones to different bacterial solution wells, incubate at room temperature for 1h; wash 5 times with TBST buffer, add 100uL TMB ELISA Substrate colorimetric solution to each well, place the 96-well plate in a dark place to avoid light for 10min, and add 100ul450nm TMBELISA to each well. The reaction was stopped with substrate stop solution, and the absorbance was detected at 450 mm. The absorbance value of the experimental well / the absorbance value of the negative well was considered positive when it was greater than 2.1.

[0076] Test results such as Figure 3 As shown, Figure 3 In the figure, the ordinate is the absorbance ratio, wherein 1% milk is the absorbance of Brucella / the absorbance of milk, S.aureus is the absorbance of Brucella / the absorbance of Staphylococcus aureus, S.epidermidis is the absorbance of Brucella / the absorbance of Staphylococcus epidermidis, A.baumannii is the absorbance of Brucella / the absorbance of Acinetobacter baumannii, and E.coli is the absorbance of Brucella / the absorbance of Escherichia coli. The results showed that the absorbance of the experimental wells (3, 9, 19, 21 and 25) was 2.1 times greater than that of the negative control wells, indicating that these five phage-displayed peptides had no cross-reaction with BSA (the above-mentioned 1% skim milk powder) and had good specificity for Brucella; among them, the phage-displayed peptides No. 9 and 25 had the best specificity.

[0077] 2. Detection limit test of phage peptides specifically binding to Brucella antigens

[0078] (I) ELISA to verify the specificity of Brucella antibody (secondary antibody)

[0079] In a 96-well plate, except for the last row, add Brucella solution (10 8CFU / mL, 70μL / well), overnight at 4℃; operate the plate washer to wash the 96-well plate with PBST buffer (1×PBS+0.05% Tween-20) as the plate washing solution, add 300μL / well of blocking buffer (PBSTBA, 1×PBS+0.05% Tween-20+1% BSA), incubate at 37℃ for 2h, and then wash the 96-well plate with PBST; Brucella antibody (NOVUS, USA, NB100-73111, monoclonal antibody) was added with 0.01mol / LPBS at a ratio of 1:12 5 ratio dilution and mix well, add 100 μL PBS to each well except the first row, add the diluted antibody to the first row at 200 μL / well, add 100 μL / well to the detection wells in the last row, and dilute in 1:2 ratio from the first row, dilute in sequence to the second to last row, take 100 μL of liquid and discard, incubate at 37°C for 1h, and wash the plate with PBST; add 100 μL (1:10000) PBST-diluted goat anti-mouse IgG2a (HRP) to each well, incubate at 37°C for 40min, and wash the plate with PBST; add 100 μL TMB ELISA Substrate colorimetric solution to each well, place the 96-well plate in a dark place to avoid light for 10min, add 100 μL 450nm TMB ELISA Substrate stop solution to each well to stop the reaction, and detect the absorbance at 450nm.

[0080] Test results such as Figure 4 and 5 As shown, the absorbance of the Brucella group was significantly higher than that of the PBS control group, and the absorbance increased with the increase of antibody concentration, indicating that there was a concentration-dependent binding ability between the Brucella antibody (NOVUS, USA, NB100-73111, monoclonal antibody) and Brucella.

[0081] (II) Double antibody sandwich enzyme-linked immunosorbent assay for quantitative detection of Brucella

[0082] Dilute phage display peptide No. 9 to 5ug / ml with coating buffer, 100ul / well, and incubate at 4℃ overnight; remove the coating buffer, wash the plate 3 times with washing buffer (1×PBS+0.05% Tween-20), 5 minutes each time; add 100ul Brucella solution of different concentrations (10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL and 10 8CFU / mL), incubate at 37°C for 1 hour; wash the plate 3 times with washing buffer (1×PBS+0.05% Tween-20), 5 minutes each time; add 100ul horseradish peroxidase-labeled Brucella specific antibody (NOVUS, USA, NB100-73111, monoclonal antibody) solution diluted 1:1000 with dilution buffer, incubate at 37°C for 1h; wash the plate 3 times with washing buffer, 5 minutes each time; add 100ul TMB ELISA Substrate colorimetric solution to each well, place the 96-well plate in a dark place to avoid light for 10 minutes, add 100ul 450nm TMBELISA Substrate stop solution to each well to stop the reaction, detect the absorbance at 450nm and draw a standard curve.

[0083] The results of the phage display peptide No. 9 test are as follows Figure 6 The results showed that the linear range of phage display peptide No. 9 for detecting Brucella was 10 3 CFU / mL-10 8 CFU / mL, indicating that this method can accurately detect 10 3 CFU / mL-10 8 Brucella in CFU / mL concentration range.

[0084] The phage display peptide No. 25 was used to detect Brucella according to the above method. The test results are as follows Figure 7 The results showed that the linear range of phage display peptide No. 25 for detecting Brucella was 10 3 CFU / mL-10 8 CFU / mL, indicating that this method can accurately detect 10 3 CFU / mL-10 8 Brucella in CFU / mL concentration range.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A polypeptide that specifically binds to a Brucella antigen, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. A reagent or a kit for detecting Brucella, characterized in that: Comprising the polypeptide of claim 1.

3. The reagent or kit according to claim 2, characterized in that The reagent or kit is an enzyme-linked immunosorbent assay reagent or kit.

4. The reagent or kit according to claim 3, characterized in that The ELISA was a double antibody sandwich ELISA.

5. The reagent or kit according to claim 3 or 4, characterized in that The reagents or kit may also include a blocking buffer, an enzyme-labeled secondary antibody, a color developing solution, a stop solution or a washing solution.

6. Use of the polypeptide according to claim 1 in the preparation of a product for detecting Brucella.

7. Use of the polypeptide according to claim 1 in the preparation of a product for diagnosing Brucella infection.

8. The use according to claim 6 or 7, characterized in that: Brucella is bovine Brucella.

9. The use according to claim 6 or 7, characterized in that: Products include reagents or kits.

10. The use according to claim 9, characterized in that: The reagent or kit is an enzyme-linked immunosorbent assay reagent or kit.

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