CN103743913B - A method for the rapid identification of host proteins that interact with aflatoxin B1 - Google Patents

Abstract

The invention relates to a method for finding out the binding protein of aflatoxin _B1 by solid phase chromatography. The method can be used to quickly and effectively find out the binding protein of the toxin, and further promote the research on the poisoning mechanism of the toxin. More specifically, the method includes coupling aflatoxin B ₁ to a carrier protein, immobilizing the coupling complex on a PVDF membrane, incubating the coupling complex with the total protein, performing non-specific washing and specific washing. removed and identified by mass spectrometry. Through the in vitro binding verification, it was found that the accuracy of the interacting protein of the mycotoxin obtained by this method was relatively high.

Description

A method for the rapid identification of host proteins that interact with aflatoxin B1

technical field

The invention belongs to the field of protein engineering, and specifically relates to a method for screening a protein combined with aflatoxin _B1 through solid phase chromatography and an in vitro verification analysis of whether it has binding ability to _AFB1 .

Background technique

Mycotoxins are metabolites produced by fungi growing in food or feed, which are extremely harmful to humans and animals. Over the years, there have been a large number of reports about mycotoxins contaminating food and crops, leading to food poisoning. Among them, the common mycotoxins are: aflatoxin, chrysogreen penicillin, citrinin, T-2 toxin, deoxynivalenol wait. Among them, Aflatoxins (AFT) are secondary metabolites mainly produced by Aspergillus flavus ( Aspergillus parasiticus ), and the contaminated food is mainly grains and oils such as peanuts, corn, rice, wheat, and peanut oil. Aflatoxin was classified as a Class 1 carcinogen by the Cancer Research Institute of the World Health Organization in 1993. It is a highly toxic and highly toxic substance, and its toxic effect is mainly damage to the liver. In natural food, aflatoxin B ₁ (Aflatoxin B ₁ , AFB ₁ ) is the most common and most harmful. Animal experiments have shown that AFB ₁ has strong liver toxicity and carcinogenic effects.

For a long time, scientists have conducted a lot of research on the mechanism of AFB ₁ 's toxin production and the mechanism of its toxicity. However, due to the characteristics of the toxin as a small molecule, there are few studies on the mechanism of the toxin being absorbed by animal cells. The present invention designs a method for quickly identifying the protein that interacts with AFB _1. The present invention finds out the binding protein of aflatoxin through solid-phase affinity chromatography. Using this method, it can quickly and effectively find out the protein that interacts with AFB ₁ The binding protein of the toxin lays a foundation for promoting the study of the toxicity mechanism of the toxin. The method includes coupling AFB ₁ with a carrier protein, immobilizing the coupling complex of the toxin and the carrier protein on a PVDF membrane, incubating the coupling complex with the total protein of the host, and performing non-specific washing and specific washing. removed and identified by mass spectrometry. The interacting protein of AFB ₁ obtained by this method has high accuracy and can lay a foundation for the study of the mechanism of the toxin being absorbed by animal cells.

Contents of the invention

The object of the present invention is to provide a method for rapidly identifying a host protein interacting with aflatoxin B1, a method for screening a protein combined with aflatoxin _B1 by solid phase chromatography, and whether it has a combination with _AFB1 In vitro validation assays. After screening and verification of this method, it has been found that 40S ribosomal protein SA and estradiol β dehydrogenase 5 can bind to AFB ₁ and are the interacting proteins of AFB ₁ .

To achieve the above object, the present invention adopts the following technical solutions:

A method for rapidly identifying a host protein interacting with aflatoxin B ₁ is to firstly couple aflatoxin B ₁ with bovine serum albumin, and then screen the protein that interacts with aflatoxin B 1 by solid phase chromatography. ₁ The bound protein is identified by mass spectrometry, and then the binding of the protein to aflatoxin B ₁ is verified by in vitro binding experiments.

The method for screening the protein combined with aflatoxin B ₁ by solid phase chromatography is to display the bovine serum albumin-aflatoxin B ₁ coupling complex on the PVDF membrane activated by methanol, 3-5 Incubate overnight at ℃; after blocking the PVDF membrane incubated with bovine serum albumin-aflatoxin B ₁ with 2% bovine serum albumin solution, place the membrane in mouse liver total protein solution and incubate overnight at 3-5℃ In order to fully combine, at the same time, a PVDF membrane that only displayed bovine serum albumin molecules was placed in the mouse liver total protein solution as a negative control; For heterogeneous elution, shake at 3-5°C for 10 minutes, wash four times, perform specific elution with 2M NaCl, shake at 3-5°C for 30 minutes, and analyze the interacting proteins in the eluate by SDS-PAGE after concentration. After silver staining, differential bands were taken for identification by mass spectrometry.

The in vitro binding experiment is used to verify the binding of the protein to aflatoxin B ₁ , and after the aflatoxin B ₁ interaction protein identified by mass spectrometry is gene cloned, induced to express, and purified, the corresponding protein is verified by ELISA. Aflatoxin B ₁ binding;

The above is verified by an in vitro binding experiment, and the specific steps are: coating the bovine serum albumin-aflatoxin B ₁ coupling complex in the well, overnight at 3-5°C, and 2wt.% bovine serum albumin solution in the After blocking at 37°C for 2 h, add purified aflatoxin _B1- interacting protein and incubate at 37°C for 2 h, then add primary antibody and secondary antibody successively, and finally add TMB chromogenic solution and H ₂ SO ₄ stop solution successively, set at the same time The negative wells coated with bovine serum albumin were used as a control, and it was verified by this method that 40S ribosomal protein SA and estradiol β dehydrogenase 5 had binding properties to aflatoxin _B1 .

The primary antibody is an anti-protein His-tagged antibody, incubated at 37°C for 1 h; the secondary antibody is HRP-labeled goat anti-mouse IgG anti-His antibody, incubated at 37°C for 1 h.

The invention has the advantages that: the mycotoxin is a metabolite produced by fungi growing in food or feed, which is extremely harmful to humans and animals. Scientists have been conducting a lot of research on the toxicity mechanism of mycotoxins, especially AFB ₁ , but because the toxin is a small molecule (it is difficult to identify the host protein that interacts with it), the mechanism of toxin absorption by animal cells, And little is known about the proteins that interact with it in the cell after it is taken up by the cell. The invention provides a feasible, fast and simple method for screening proteins interacting with mycotoxins (taking aflatoxin as an example), and lays the foundation for further understanding of the toxin-causing mechanism of mycotoxins.

Description of drawings

Fig. 1 Structural molecular formula of AFB ₁ .

Fig. 2 The coupling route between AFB ₁ and BSA.

Fig. 3 The ultraviolet scanning images of AFB1, BSA and the coupling product; 1 is bovine serum albumin; 2 is aflatoxin B ₁ ; 3 is bovine serum albumin-aflatoxin B ₁ coupling complex.

Figure 4 SDS-PAGE analysis of AFB1 binding protein; where M is Marker; 1 is bovine serum albumin-aflatoxin B ₁ coupling complex; 2 is bovine serum albumin; 3 is bovine serum albumin-aflatoxin B ₁ coupling Linked complex; 4 is bovine serum albumin; 1 and 2 lanes are PBST washing groups, 3 and 4 are PBS washing groups.

In vitro expression and purification of AFB ₁ -binding proteins identified in FIG. 5 . Among them, Figure A: the expression and purification of Rpsa protein. M: Marker; 1: IPTG-induced PET28a strain; 2: IPTG-induced PET28a-rpsa recombinant strain; 3: purified Rpsa protein; B: expression and purification of Akr1c6 protein. M: Marker; 1: IPTG-induced PET28a strain; 2: IPTG-induced PET28a-akr1c6 recombinant strain; 3: purified Akr1c6 protein; C: expression and purification of Cyb5a protein. M: Marker; 1: IPTG-induced PET28a bacterial culture; 2: IPTG-induced PET28a-cyb5a recombinant bacterial culture; 3: purified Cyb5a protein.

In vitro binding verification of _AFB1- binding proteins identified in Figure 6 to _AFB1 . Coat the cross-linked product BSA-AFB1 in the enzyme-labeled well, then add the purified protein, primary antibody (anti-protein antibody), secondary antibody, chromogenic solution, stop solution, measure the OD value, and judge the yellow color by the OD value. Binding of Aspergillus toxin B1 to proteins.

Detailed ways

1. Preparation of polyacrylamide gel

(1) 2 mol/L Tris-HCl (pH8.8): Weigh 24.2 g Tris base and add appropriate amount of ultrapure water to dissolve, adjust the pH value to 8.8 with hydrochloric acid, add ultrapure water to make up to 100 mL.

(2) 1 mol/L Tris-HCl (pH6.8): Weigh 12.1 g Tris base, add appropriate amount of ultrapure water to dissolve, adjust the pH value to 6.8 with hydrochloric acid, add ultrapure water to make up to 100mL.

(3) 30% acrylamide storage solution: Weigh 29.2 g of acrylamide and 0.8 g of N',N'-methylenebisacrylamide, add ultrapure water to dissolve to 100 mL, and filter it with filter paper after it is completely dissolved. Store at 4°C protected from light.

(4) 10% SDS: Weigh 10 g SDS (electrophoresis grade), add ultrapure water to dissolve to 100 mL, and store at room temperature.

(5) 10% ammonium persulfate: Weigh 100 mg of ammonium persulfate, add ultrapure water to dissolve to 1 mL.

(6) 4× Separating Gel Buffer: Measure 75 mL 2 mol/L Tris-HCl (pH8.8), 4 mL 10% SDS, add 21 mL ddH2O, and store at 4 ℃ for several months.

(7) 4× Stacking Gel Buffer: Take 50 mL 1 mol/L Tris-HCl (pH6.8), 4 mL 10% SDS, add 46 mL ddH2O, it can be stored at 4 ℃ for several months.

(8) Electrode buffer: Weigh 3 g Tris base, 14.4 g glycine, 1 g SDS, add appropriate amount of ultrapure water to dissolve, adjust the pH value to 8.3 with hydrochloric acid, add ultrapure water to make up to 1000 mL. It can also be made into a 10× stock solution, which can be stored for a long time at room temperature.

(9) 5× sample buffer: weigh 10 mg bromophenol blue, add 0.6 mL 1 mol/L Tris-HCl (pH6.8), 2.5 mL glycerol, 2 mL 10 % SDS, 0.5 mL β-mercapto ethanol and 4.4 mL ddHO, can be stored at 4°C for several weeks, or at -20°C for several months.

(10) Coomassie Brilliant Blue Staining Solution: Weigh 1 g Coomassie Brilliant Blue R-250, 200 mL methanol, 50 mL glacial acetic acid, add distilled water to dissolve to 500 mL.

(11) Coomassie brilliant blue decolorization solution: measure 450 mL methanol, 100 mL glacial acetic acid, add distilled water to 1000

mL.

2. Names and methods of gene cloning and protein purification reagents

(1) 1.0% agarose gel: Weigh 0.2 g agarose into 20 mL0.5×TBE, heat to dissolve, pour it into the gel plate, insert a comb,

(2) 5×TBE: Weigh 54.0 g Tris base, 27.5 g boric acid, add 800 mL, add 20 mL 0.5 mol/L EDTA (pH8.0), until fully dissolved, dilute to 1 L with water, store at room temperature .

(3) LB medium: Weigh 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl, add 800 mL of deionized water, adjust the pH value to 7.0, and set the volume to 1 L. After high temperature and high pressure sterilization, store at 4°C .

(4) Ampicillin (Amp) (100 mg/mL): Dissolve 1 g of ampicillin sodium salt in a sufficient amount of water, finally dilute to 10 mL, filter and sterilize, aliquot into small portions and store at -20°C.

(5) Kanamycin (Kan) (50 mg/mL): Dissolve 0.5 g of Kanamycin in a sufficient amount of water, and finally dilute to 10 mL, filter and sterilize, aliquot into small portions and store at -20°C storage.

(6) IPTG (isopropylthio-β-D-galactoside) (1 mol/L): Dissolve 238 mg of IPTG in 1 mL of water, sterilize by filtration, and store at -20°C.

(7) 0.1 mol/L CaCl2: Weigh 54 g CaCl2-6H2O, dilute the solution to 1 L with deionized water, filter and sterilize with a 0.22 μm filter, aliquot into 10 mL portions and store at -20°C.

(8) Buffer B, C, D, E (L-1): Weigh 13.8 g NaHPO4 H2O, 1.2 g Tris Base, 480.5 g urea, after dissolving, adjust the pH to 8.0, 6.3, 5.9, 4.5 respectively, use Adjust the pH before.

3. The name and preparation method of each reagent in the ELISA method

(1) 0.05 M carbonate coating buffer (pH 9.6): Weigh 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate and dissolve them in 800 mL of double distilled water, add 2 mol/L of sodium hydroxide Adjust its pH value to 9.6, and finally set the volume to 1000 mL.

(2) 1× Phosphate Buffer Saline (PBS): Weigh 8.0 g of NaCl, 0.2 g of KCl, 3.58 g of Na2HPO4·12H2O, 0.27 g of KH2PO4, dissolve them in 1 L of water, adjust the pH to 7.2-7.4, and autoclave at 121°C Bacteria for 20 min.

(3) 5% PBSM blocking solution: Add 4 g of skimmed milk powder to 100 mL of 1×PBS (pH 7.4).

(4) PBST eluent: 1×PBS containing Tween-20 at a concentration of 0.05%. Add 0.5 mL of Tween-20 to 1000 mL of 1×PBS (pH 7.4).

(5) Substrate chromogenic solution A: Weigh 27.2 g of sodium acetate and 3.2 g of citric acid into a 1000 mL beaker, add 0.6 mL of 30% hydrogen peroxide, and finally adjust the volume to 1000 mL.

(6) Substrate chromogenic solution B: Weigh 0.4 g sodium edetate, 1.9 g citric acid and 0.4 g tetramethylbenzidine (TMB) into a 1000 mL beaker, add 100 mL glycerol, and finally set Capacity up to 1000 mL.

(7) Stop solution (2 mol/L sulfuric acid): slowly drop 111 mL of concentrated sulfuric acid into 889 mL of double distilled water and mix well.

The present invention will be described in detail below in conjunction with specific examples. The following examples are to further illustrate the present invention, but should not be regarded as limiting the present invention.

Example 1 Rapid Identification of Host (Mice) Proteins Interacting with Aflatoxin B ₁

1) Coupling of mycotoxin and carrier protein

Aflatoxin B ₁ (Aflatoxin B ₁ referred to as AFB ₁ ) was purchased from Simga, USA. According to the structural characteristics of AFB ₁ , the carbodiimide method was used for coupling. First, carry out the oximation reaction, take 2mg AFB ₁ and 4mg CMO dissolved in 400 l pyridine, shake and react in the dark at 25°C for 4 hours, freeze-dry the reaction product to obtain the oximation product of AFB ₁ ; take 0.2mg AFB ₁ oximation Dissolve the product in 100 μL of DMF-water (6:9 V/V ), add 2 mg of EDC and mix well in the dark, then add 0.5% C-BSA solution, react for 4 hours at 25°C in the dark at 100 r/min, and then add EDC 2mg continued to react for 20h. The resulting coupling product was put into a dialysis bag in 0.01mol/L PBS (pH7.4), and dialyzed at 4°C for 3 days, during which time the dialysate was replaced. And the ultraviolet scanning was carried out on AFB ₁ , C-BSA and the coupling product, and the changes of the absorption peaks of the coupling product were compared.

2) immobilize the coupled complex on PVDF membrane;

Cut out a 10cm*1cm polyvinylidene fluoride membrane (PVDF membrane), activate it in methanol solution for 5s, wash it with PBS solution, place it in BSA-AFB ₁ solution, and shake slowly overnight at 4°C to let the protein molecules Binds sufficiently to the membrane to display _AFB1 on the membrane surface via BSA.

3) Incubate the coupling complex with the total protein, and the coupling complex and the total protein combine with each other;

According to the operation steps of the Cell Membrane Protein and Cytoplasmic Protein Extraction Kit of Beyontien Company, the total protein of mouse liver was extracted, the protein concentration was measured, and the extracted total protein was diluted to 2mg/ml with PBS solution. After the PVDF membrane incubated with BSA-AFB ₁ was blocked with 2% BSA solution, the membrane was taken out and placed in the total protein solution of mouse liver, and incubated overnight at 4°C with slow shaking to allow protein molecules to bind to AFB ₁ or BSA. At the same time, a PVDF membrane coated only with BSA molecules was placed in the mouse liver total protein solution as a negative control.

4) Perform non-specific washing and specific elution to obtain proteins that interact with mycotoxins.

Take 4°C pre-cooled PBS or PBST buffer for non-specific washing of the incubated membrane, 40 mL each time, shake at 4°C for 10 min, and wash four times. Take 4°C pre-cooled PBS buffer solution containing 2M NaCl for specific elution of the washed membrane, shake at 4°C for 30 min, and collect the elution solution. The obtained elution solution was put into a dialysis bag in 0.01mol/L PBS (pH7.4), dialyzed at 4°C for 2 days, during which the dialysate was changed, and the dialyzed solution was concentrated with PEG20000. Then the experimental group and the control group were further analyzed by SDS-PAGE. After silver staining, there were 8 different bands, which were tapped for mass spectrometry identification. Specific mass spectrometry conditions are as follows:

LC conditions: High performance liquid chromatography: Thermo Scientific Accera System; Chromatographic column: BioBasic C18 Column (100 x 0.18 mm, particle size: 5 um); Sample volume: 10uL; Mobile phase: A: aqueous phase (0.1% formic acid) ; B: Hexanonitrile (0.1% formic acid); Gradient: 5%–35%B in 20 minutes, 35%–95%B in 2 minutes; Flow rate: 2.5uL/min;

MS conditions: mass spectrometer: LTQ-XL (Thermo Scientific); spray voltage: 3.5 kV; capillary temperature: 275°C; sheath gas flow rate: 15arb; precursor ion scanning range: 400-2000m/z; Isolation width: 2 Da.

Secondary mass spectrometry conditions: AGC Target 1e4, 1 microscans; collision energy: 35% CID.

Search: The database used for the search is the Mus musculus.fasta protein library downloaded from UNIPROT (http://www.uniprot.org/). The raw data obtained by mass spectrometry were analyzed with Proteome Discoverer 1.2 software for relative quantitative analysis.

5) Analysis of mass spectrometry results

Analyze the mass spectrometry results, delete the proteins that are inconsistent with the corresponding band sizes and the matching peptides are too small, and obtain 32 proteins; after literature analysis, three proteins that need further verification are finally determined: 40S ribosomal protein SA (Rpsa) and estradiol beta dehydrogenase 5 (Akr1c6), cytochrome b5 (Cyb5a).

Table 1 Mass spectrometry analysis of AFB ₁ binding protein bands

6) Expression and purification of AFB ₁ binding protein

Total mouse RNA was extracted and cDNA was obtained after reverse transcription. Design primers for rpsa, akr1c6 and cyb5a genes, use the obtained cDNA as a template to clone the corresponding genes, construct them on the vector PET28a, and then transfer them into E.coli. After verification, add IPTG to the bacteria to induce protein expression, and then use The nickel column was used to separate and purify the recombinant protein to obtain the corresponding protein.

Table 1 Primer sequence list

7) In vitro binding verification of AFB ₁ binding protein to aflatoxin B ₁

The binding of _AFB1 to the identified _AFB1- binding proteins was analyzed by enzyme-linked immunosorbent assay (ELISA). Take the cross-linked product BSA-AFB ₁ , dilute it with 0.05M carbonate buffer (pH 9.6) to a concentration of 2 μg/mL, coat 100 μL/well on a microtiter plate, overnight at 4°C; wash the plate 3 times with PBS, Block with 2% BSA, 200 μL/well, incubate at 37°C for 2 h; wash the plate 3 times with PBS, take the purified protein Rpsa and Akr1c6, dilute with 2% BSA to a concentration of 10 μg/mL, add 100 μL/well to the enzyme-labeled well Medium; wash the plate 3 times with PBS, dilute the HIS antibody at 1:4000 with 2% BSA, add to the microplate, 100 μL/well, incubate at 37°C for 1 h; wash 3 times with PBST and PBS, wash with 2% BSA Dilute horseradish peroxide-labeled goat anti-mouse IgG at 1:6000, 100 μL/well, incubate at 37°C for 1 h; wash with PBST and PBS 3 times, add TMB for color development, 100 μL/well, incubate at 37°C 10 min; stop the reaction with 2 M H ₂ SO ₄ , 50 μL/well; measure the absorbance at 450 nm. At the same time, BSA, trx and GFP proteins were set as negative controls, and BSA-AFB ₁ cross-linked products, Rpsa protein, Akr1c6 protein, and Cyb5a protein were used as positive controls. The results showed that Rpsa protein and Akr1c6 protein had binding ability to AFB ₁ , but Cyb5a protein had no obvious binding ability to AFB ₁ .

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

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Claims (4)

1. A method for rapidly identifying a host protein interacting with aflatoxin B ₁ , characterized in that: first aflatoxin B ₁ is coupled with bovine serum albumin, and then screened by solid phase chromatography The protein that binds to aflatoxin B ₁ is identified by mass spectrometry, and the binding of the protein to aflatoxin B ₁ is verified by in vitro binding experiments; The method for screening the protein combined with aflatoxin B ₁ by solid phase chromatography is to display the bovine serum albumin-aflatoxin B ₁ coupling complex on the PVDF membrane activated by methanol, 3-5 Incubate overnight at ℃; after blocking the PVDF membrane incubated with bovine serum albumin-aflatoxin B ₁ with 2% bovine serum albumin solution, place the membrane in mouse liver total protein solution and incubate overnight at 3-5℃ In order to fully combine, at the same time, a PVDF membrane that only displayed bovine serum albumin molecules was placed in the mouse liver total protein solution as a negative control; Heterotropic elution, shake at 3-5°C for 10-15min, wash three to four times, perform specific elution with 2M NaCl, shake at 3-5°C for 25-30min, and concentrate the interacting proteins in the eluate SDS-PAGE analysis was carried out, and differential bands were taken after silver staining for identification by mass spectrometry. 2. a kind of rapid identification according to claim 1 and the method for the host protein of aflatoxin B ₁ interaction, it is characterized in that: described in vitro binding experiment verification protein and aflatoxin B ₁ binding, After gene cloning, induced expression, and purification of the aflatoxin B ₁ interacting protein identified by mass spectrometry, the binding of the corresponding protein to aflatoxin B ₁ was verified by ELISA. 3. a kind of rapid identification according to claim 2 and the method for the host protein of aflatoxin B ₁ interaction, it is characterized in that: described by in vitro binding experiment verification, concrete steps are: bovine serum albumin-flavonoid The aspergillus toxin B ₁ coupling complex was coated in the wells, overnight at 3-5°C, 2wt.% bovine serum albumin solution was blocked at 37°C for 2 h, and the purified aflatoxin B ₁ interaction protein was added Incubate at 37°C for 2 hours, then add primary antibody and secondary antibody successively, and finally add TMB color development and H ₂ SO ₄ stop solution successively, and set negative wells coated with bovine serum albumin as a control, and 40S ribosomes were verified by this method Protein SA and estradiol beta dehydrogenase 5 bind to aflatoxin _B1 . 4. A method for rapidly identifying a host protein that interacts with aflatoxin B ₁ according to claim 3, characterized in that: the primary antibody is an anti-protein His-tag antibody, incubated at 37°C for 1 h; The secondary antibody was HRP-labeled goat anti-mouse IgG anti-His antibody, and incubated at 37°C for 1h.