CN105316328B - Coding can specifically bind the nucleotides of DON polypeptide - Google Patents

Abstract

The invention belongs to biological technical field, and the nucleotides of peptide molecule of deoxynivalenol (Deoxynivalneol, DON) can be specifically bound by being related to coding, and sequence is TGT GCT CAT CAT GGT ATT CCG TAG TGT.Described peptide molecule can substitute traditional DON antibody and applied in DON immune analysis system, and with the tolerance of more preferable soda acid and heat endurance, the acquisition of the peptide molecule is without immunologic process, preparation is simple, cost is low, the cycle is short, the peptide molecule both can largely be expanded by way of biological culture, can also largely it be prepared by the method for chemical synthesis or genetic engineering, new path is provided for the preparation of DON antibody, there is preferable application value.

Description

Coding can specifically bind the nucleotides of DON polypeptide

Technical field

The invention belongs to biological technical field, and in particular to can specifically bind deoxynivalenol The peptide molecule of (Deoxynivalneol, DON) and its application.

Background technology

Deoxynivalenol (Deoxynivalneol, DON), also known as vomitoxin (Vomitoxin), category are single Spore aliphatic compound is held, is gained the name because it can cause the vomiting of pig, also there is certain harm, European Union's criteria for classification to human body For three-level carcinogenic substance.DON is mainly produced by sickle-like bacteria, is widely present in the crops such as barley, wheat, corn, oat and rice And its in product.DON has cytotoxicity, embryotoxicity and immunotoxicity etc., can cause apocleisis, vomiting, diarrhoea, fever and life Grow the health of the symptom such as slow, the serious threat mankind and animal.Due to DON high pollution rate and high toxicity, to DON in food Detect significant.

Detection DON more commonly used at present method mainly includes：High performance liquid chromatography (High performance Liquid chromatography, HPLC), gas chromatography (Gaschromatography, GC), TLC (Thin Layer chromatography, TLC), tablets by HPLC-MS (High performance liquid Chromatography-mass sepectrum, HPLC-MS), microbiological method and immunodetection etc., wherein immunology Analysis method is with its high sensitivity, simple to operate and the advantages that can realize scale selection, in DON field of fast detection It is widely used.

The core of immunological analysis method is specific recognition and combination between Ag-Ab, therefore prepares specificity Just turn into the premise and core of development immunological analysis method with reference to the antibody of antigen.The research of antibody develops rapidly in recent years, From traditional polyclonal, monoclonal antibody, it is developed to single-chain antibody, phage displaying antibody, single domain heavy chain antibody, anti- Idiotype antibody etc. is the genetic engineering antibody field of representative.Prepared compared to conventional antibodies and be subjected to animal immune, cell The complicated processes such as fusion, positive colony screening, the advantages that genetic engineering antibody is facilitated with its orientable transformation, elutriation, turn into mesh The focus of preceding research.In addition, numerous scholars in recent years including China have begun to be conceived to not with immune globulin The white novel antibodies research that structure is formed for main body, such as the aptamer based on SELEX technologies, polymerize based on molecular engram The artificial antibody of body technique and antibody analog based on Lipocalin protein families etc. achieve gratifying progress, are The replacement Journal of Sex Research of conventional antibodies provides sturdy theory and methodology basis.Make a general survey of the development trend of antibody, it can be seen that Novel antibodies have molecular weight it is small, it is simple in construction, can self evolve, prepare the characteristics of quick and development trend.For example, monoclonal Antibody, single-chain antibody, the molecular weight of single domain heavy chain antibody gradually decrease, and respectively 150,30-50,15-20kDa, with Lipocalin is that the antibody analog of skelemin is 18-20kDa, and aptamer is only then a bit of nucleotide sequence.

People are keen to research and development peptide mimics (peptido-mimetics) in recent years, and the first purpose is by with life The complicated molecule of thing activity is progressively modified, substituted, transforming, being finally compressed to the small molecule for only including functional unit, referred to as peptide The rational design of analogies.Therefore, peptide molecule can turn into potential novel antibody molecules.With phage-displayed polypeptides storehouse skill Art develops rapidly, by the elutriation of phage display peptide library, can quickly, simply obtain and specific target body molecular specificity knot The peptide molecule of conjunction.Phage display peptide library technology is mainly characterized by effectively filtering out and target target body specific bond Phage-displayed polypeptides, the technology are exploring the interphase interaction binding site of acceptor and part, are seeking high-affinity biology work Property ligand molecular, explore agnoprotein matter space structure epitope, the development etc. of new generation vaccine is widely used, but above-mentioned The application of phage-displayed polypeptides technology is confined to carry out affine elutriation by target body molecule of macro-molecular protein mostly.Due to egg White matter surface has the epitope of multiple copies, therefore is very easy to obtain the peptide molecule for corresponding to combine therewith.Current polypeptide Molecule is applied in immune analysis as the mimic epitope (antigen substitute) of antigen mostly, and using peptide molecule as small point The report that the substitute of sub- material antibody is applied to immune analysis is less, the reason is that small-molecule substance volume is too small, and Amino that surface is not enriched, carboxylic group, it is difficult to combined with peptide molecule, so as to cause elutriation difficult.

The present invention is by phage-displayed polypeptides storehouse technology, by the way of affine elutriation, from phage-displayed polypeptides storehouse In quickly screen the peptide molecule that can be specifically bound with DON, the peptide molecule has and traditional DON is polyclonal, Dan Ke The similar immunology detection characteristic of grand antibody molecule, DON immune analysis can be applied to as the substitute of traditional DON antibody System.This method avoid the animal immune prepared required for traditional monoclonal antibody molecule, cell fusion, monoclonal screening Etc. flow, have the characteristics that without immunologic process, screening is convenient, the cycle is short, it is low to prepare cost.

The content of the invention

The present invention with DON holoantigens (the conjugate DON-Ovalbumin of DON and oralbumin) for target molecule, by target Molecule is attached on ELISA Plate carrier, input phage random displaying ring seven peptide storehouse, is carried out the affine elutriation of liquid phase, is obtained one kind DON peptide molecule can be specifically bound, its amino acid sequence is：(during structure ring seven peptide storehouse, it is opened up AHHGIPQ The heptapeptide sequence head and the tail shown also respectively have a C residue, with circlewise structure, if including head and the tail cysteine residues are also included in, Then its amino acid sequence is CAHHGIPQC)

In aforementioned polypeptides molecular structure, capitalization English letter represents known natural L-form amino acid residue or its D- type respectively One kind of isomers, i.e. A represent alanine residue, and H represents histidine residue, and P represents proline residue, and it is residual that G represents glycine Base, I represent isoleucine residues, and Q represents glutamine residue, and C represents cysteine residues.

The invention further relates to the nucleotides of encoding such polypeptides molecule amino acid sequence (CAHHGIPQC), its sequence is:

TGT GCT CAT CAT GGT ATT CCG TAG TGT。

The present invention refers to that peptide molecule can be entered by way of Phage amplification, chemical synthesis or genetic engineering recombination expression Row is a large amount of to be prepared.Phage amplification refers to that the bacteriophage for having peptide molecule will be shown, by way of biology expands, amount reproduction Production displaying has the bacteriophage particles of peptide molecule.Chemical synthesis refers to the amino acid sequence according to the mimic epitope announced, and leads to The mode for crossing chemically synthesized polypeptide carries out Peptide systhesis.The mode of genetic engineering recombination expression refers to the base of coded polypeptide molecule Cause, by being cloned into expression vector, a large amount of preparations of peptide molecule are carried out in the form of polypeptide-fusion protein.

The invention further relates to application of the peptide molecule in the analysis of non-disease diagnostic purpose immunology detection.Immunology The type of the detection non-disease diagnostic purpose immunology based on Ag-Ab specific reaction such as including MBP enzyme linked immuno-adsorbent assay Analyze detection type.

The beneficial effects of the invention are as follows：The alternative traditional DON antibody molecules of peptide molecule mentioned by the present invention, as DON binding molecule directly applies to immunology detection analysis, and the peptide molecule has good structural stability, acid and alkali-resistance, resistance to height Temperature, prepare the features such as simple, quick.

Brief description of the drawings

DON indirect competitive ELISA standard curves of the Fig. 1 based on peptide molecule.

Embodiment

Embodiment 1. and the affine elutriation and its identification of DON specific binding polypeptide molecules

1) affine elutriation and the specific method of DON specific binding polypeptide molecules are：Take 0.5mg DON-OVA holoantigens (coupling ratio 15:1) ELISA Plate hole (100 μ L/ holes), is coated in, 37 DEG C are incubated 2 hours.With PBST (10mM PBS pH 7.4 Include 0.1%Tween-20 (v/v)) after washing 10 times, add 37 DEG C of 350 μ l confining liquids (5% gelatin solution) and be incubated 2 hours. Washed 5 times with PBST, 100 μ l phage peptide libraries (phage display ring seven peptide storehouse, purchased from NEB companies, with PBS 10 is added per hole Dilution bacteriophage stoste again, about 2.0 × 10¹¹Pfu), 37 DEG C of oscillating reactions 2 hours.Uncombined bacteriophage is discarded, is washed with PBST Wash 15 times, the DON antibody for being dissolved in PBS with 100ng with reference to upper bacteriophage is at war with elution.10 μ l wash-out bacteriophages are taken to survey drop Degree, remaining, which is used to infecting 20mL, grows to the E.coli ER2738 bacterial strains of logarithm early stage and is expanded.Use PEG/ within second day NaCl deposition and purification bacteriophages, and determine the titre of bacteriophage after amplification.

Then the 2nd wheel and the 3rd wheel elutriation are carried out successively, and panning step is substantially the same with the first round, the phagocytosis added every time The scale of construction is 2 × 10¹¹Pfu, difference are：The phage-displayed polypeptides of 2nd, 3 wheel screenings and the plate for being coated with DON-OVA Bar incubation time is respectively 60min, 30min, and the concentration of competitive elution liquid (DON antibody) is respectively 75ng/mL, 25ng/mL.

2) identification of positive phage clones：Random picking 70 in the flat board of phage titre is determined after third round elutriation Individual bacteriophage spot, carries out the amplification of bacteriophage, and positive phage clones are carried out using Immunofluorescent antibody detection method Identification, specific method are：First, with 10mM PBS (pH 7.4) dilute DON-OVA antigens, 2 μ g/mL be coated with 96 hole elisa Plates, 4 DEG C be incubated overnight.After second day is washed 3 times with PBST (10mM PBS, 0.05%Tween-20 (v/v)), with containing 3% degreasing The PBS of milk powder is closed, and 37 DEG C are incubated 1 hour；Put into 100 μ l bacteriophages spots amplification liquid (1.0 × 10¹²Pfu), bitten with original As negative control, 37 DEG C are incubated 1 hour in thalline peptide storehouse；Add 1：The HRP of 5000 times of dilutions marks anti-M13 bacteriophages secondary antibody 100 μ l, 37 DEG C are incubated 1 hour；100 μ l tmb substrate liquid, lucifuge colour developing 5min are added, ELIASA reads the absorption at 450nm Value.Choose OD₄₅₀Phage clone of 2 times more than negative control is positive colony.

3) identification of DON peptide molecules is specifically bound：Carried out and DON specificity using the method for indirect competitive ELISA With reference to the identification of phage-displayed polypeptides, specific method is：DON-OVA, 2 μ g/mL coatings are diluted with 10mM PBS (pH 7.4) The μ L of ELISA Plate 100,4 DEG C of overnight incubations；After second day is washed 3 times with PBST (10mM PBS, 0.05%Tween-20 (v/v)), Closed with the PBS containing 3% skimmed milk power, 37 DEG C are incubated 1 hour；Put into 50 μ l and be accredited as the positive through indirect ELISA Phage clone (1.0 × 10¹¹Pfu) and 50 μ l DON standard items (concentration range 0-1000ng/mL), 37 DEG C are incubated 1 hour； Add 1：The μ l of anti-M13 bacteriophages secondary antibody 100 of 5000 dilution HRP marks, 37 DEG C are incubated 1 hour；Add 100 μ l tmb substrates Liquid, lucifuge colour developing 5min, reads OD₄₅₀.If the peptide molecule of phage display can specifically bind DON, DON is added The OD values in the hole of standard items will be less than control wells (not adding DON standard items).

The sequencing of peptide molecule encoding gene and its determination of amino acid sequence of embodiment 2. and DON specific bindings

1. the bacteriophage for there are specific binding DON peptide molecules by indirect competitive ELISA identification displaying is expanded, Extract the DNA sequencing template of bacteriophage.Simplified process is as follows：Phage amplification is carried out, after first step centrifugation, 800 μ l are contained Bacteriophage supernatant is transferred to a new centrifuge tube.Add 200 μ l PEG/NaCl precipitating phages.Precipitation is resuspended in 100 μ l after centrifugation Iodide buffer solution (10mM Tris-HCl (pH 8.0), 1mM EDTA, 4M NaI), 250 μ l absolute ethyl alcohols precipitation DNA is added, Precipitation (DNA sequencing template) is washed after centrifugation with 70% ethanol again.Precipitation is finally resuspended in 20 μ l aqua sterilisas, takes 2 μ l to carry out fine jade Sepharose electrophoretic analysis；5 μ L bacteriophages templates are taken to carry out DNA sequencing, its -96gIII sequencing primer is：5’-^HOCCC TCA TAG TTA GCG TAA CG-3’.DNA sequencing nucleotides sequence is classified as:TGT GCT CAT CAT GGT ATT CCG TAG TGT The amino acid sequence of peptide molecule (cysteine containing head and the tail) can be obtained according to DNA sequencing result and password sublist： CAHHGIPQC。

Embodiment 3. specifically binds application of the DON peptide molecules in ELISA

(1) sample extraction

5g testing samples are weighed, add 25ml PBS solutions, 200rpm vibrates 5 minutes；By extract solution with No. whatman1 After filter paper is filtered, 1ml filtrates are taken to add 1ml PBS (phosphate buffer, pH=7.2)

After mixing, as sample extracting solution is stand-by.

(2) it is coated with and closes

The DON-OVA (2 μ g/ml) diluted with 10mM PBS (pH 7.4), coated elisa plate, 4 DEG C of overnight incubations.Second day After being washed 3 times with PBST (10mM PBS, 0.05%Tween-20 (v/v)), closed with the PBS containing 3% skimmed milk power, It is stand-by with PBST board-washings 6 times after 37 DEG C are incubated 1 hour.

(3) foundation of standard curve

The lath handled well through step (2) is taken out, putting into 50 μ l displayings respectively per hole there are specific binding DON peptide molecules Bacteriophage (1.0 × 10¹²Pfu) and a series of various concentrations 50 μ L DON standard items (0-10,000ng/ml), 37 DEG C incubation 20min.Add 1:The anti-M13 bacteriophages secondary antibody of 5000 dilution HRP marks, 37 DEG C are incubated 1 hour.Then developed the color with tmb substrate, Read OD₄₅₀.Using DON log concentrations as abscissa, Percentage bound (adds the OD in DON hole₄₅₀/ do not add DON hole OD₄₅₀× 100%) it is ordinate, establishes indirect competition standard curve.

(4) detection of sample

The lath handled well through step (2) is taken out, putting into 50 μ l displayings respectively per hole there are specific binding DON peptide molecules Bacteriophage (1.0 × 10¹²Pfu) and testing sample extract solution, 37 DEG C are incubated 1 hour.Add 1:5000 dilution HRP marks resist M13 bacteriophage secondary antibodies, 37 DEG C are incubated 1 hour.Then developed the color with tmb substrate, read OD₄₅₀, calculations incorporated rate, and according to standard Curve, retrodict out the content of DON in sample.

Embodiment 4 and a large amount of preparations of DON specific binding polypeptide molecules

1) in a manner of Phage amplification

It is inoculated with being added with the bacteriophage that DON is specifically bound to 20ml in ER 2738 culture, 37 degree of 220rpm Shaken cultivation 4.5h.Culture is transferred in another centrifuge tube, 4 DEG C of 10000rpm centrifuge 10min, by 80% turn of the top of supernatant Enter in a fresh tube, add the PEG/NaCl of 1/6 volume, 120min is stood at 4 DEG C.4 DEG C of 10000rpm centrifugations PEG/NaCL are quiet Put solution 15min.Supernatant is abandoned, residual supernatant is sucked after of short duration centrifugation.Add 1mL TBS to be resuspended, as bacteriophage is expanded Increase liquid.

2) prepared in a manner of polypeptide-fusion protein

A.PCR expands the external source encoding gene of peptide molecule

PCR reaction systems：(50μL)

PCR reaction conditions：

Using PCR primer QIAquick Gel Extraction Kit purified pcr product, trace dna quantitative instrument quantifies.Peptide molecule is (containing head and the tail half Cystine) coding gene sequence be：TGT GCT CAT CAT GGT ATT CCG TAG TGT,

B. the double digestion of external source encoding gene and expression vector

The external source code gene of ACC65I and Eag I enzymes and expression vector is respectively adopted, and (pMAl-pIII, NEB company, can Express MBP fusion proteins) carry out double digestion.

C. after digestion product connection and conversion

Plasmid pMal-PIII and purpose fragment are mixed with 1: 15 (mol ratio), 12h is connected in 20 DEG C of water-baths, takes 15 μ L Connection product is added in 100 μ L competent cell DH5 α, is fully mixed.After ice bath 35min, 42 DEG C of water-bath heat shock 60s, ice immediately 800 μ L LB liquid mediums are added after bath 3min, 37 DEG C, 200rpm culture 1h, 8000rpm centrifugation 5min, supernatant is sucked and leaves and takes About 300 μ L, it is coated in LB-A solids (Amp) culture medium, 37 DEG C are incubated overnight, and obtain being subcloned positive colony.

D. Cloning Transformation

The subclone that above-mentioned steps obtain is extracted into purpose plasmid with Tiangeng kit, takes 10 μ L thin to 100 μ L competence In born of the same parents TB1, fully mix.After ice bath 20min, 45 DEG C of water-bath heat shock 60s, the training of 800 μ L LB liquid is added after ice bath 5min immediately Nutrient solution, 37 DEG C, 200rpm culture 45min, 7000rpm centrifugation 6min, suck supernatant and leave and take about 300 μ L, be coated on LB-A solids (Amp) in culture medium, 37 DEG C are incubated overnight, and obtain positive colony.E. the expression of polypeptide-MBP fusion proteins

By the positive clone molecule of above-mentioned acquisition, a single bacterium colony is chosen from flat board and is inoculated in 5mL LB-A, in 0.5% sucrose, 37 DEG C, 220r/min, shaken cultivation is overnight, and overnight culture is inoculated in 50mL LB-A by 1% inoculum concentration (v/v), and 0.2% In sucrose culture medium, 3 bottles of inoculation respectively, 37 DEG C, 220r/min shaken cultivations, when culture bacterial concentration OD600 reaches 0.6 When, IPTG to final concentration of 0.3mmol/L is added into three bottles of cultures, 120r/min shaken cultivations, (IPEG is molten by inducer Liquid) in 4 DEG C, 5000g, centrifugation 15min collects bacterial sediment, abandons supernatant.Cell is resuspended in 400mL 30mM Tris-HCl, 25% sucrose, pH 8.0 (80mL/g wet cell weights), EDTA to 1mM is added, shakes 5-10min, 8000g, 4 DEG C of centrifugations at room temperature 15min, supernatant is abandoned, precipitation is resuspended in the 5mM MgSO4 of 400ml precoolings, shakes 15min, 8000g, 4 DEG C on ice, centrifuges 20min, retain supernatant, 8mL 1M Tris-HCl, pH 7.4 is added into supernatant, obtain polypeptide-MBP fusion proteins.

Embodiment 5 and the immunology detection stability experiment of DON specific binding polypeptide molecules

1) resistance to acids and bases is tested

PH=4.0 can will be respectively used with the DON peptide molecules specifically bound and DON monoclonal antibodies, 5.0,6.0, 7.4,8.0 buffer solution is diluted to working solution, carries out indirect competitive ELISA measure respectively, and specific method is：With 10mM PBS The DON-OVA (2 μ g/ml) of (pH 7.4) dilution, coated elisa plate, 4 DEG C of overnight incubations.Second day with PBST (10mM PBS, 0.05%Tween-20 (v/v)) washing 3 times after, closed with the PBS containing 3% skimmed milk power, 37 DEG C be incubated 1 hour after, It is stand-by with PBST board-washings 6 times.Take out the lath handled well, put into respectively per hole 50 μ l can specifically bind DON peptide molecule/ A series of 50 μ lDON standard items (0-10,000ng/ml) of DON monoclonal antibodies (20 μ g/ml) and various concentrations, 37 DEG C of incubations 20min.Add 1:The sheep anti-mouse igg secondary antibody of anti-M13 bacteriophages secondary antibody/HRP marks of 5000 dilution HRP marks, 37 DEG C are incubated 1 Hour.Then developed the color with tmb substrate, read OD₄₅₀.Using DON log concentrations as abscissa, Percentage bound (adds DON hole OD₄₅₀/ do not add DON hole OD₄₅₀× 100%) it is ordinate, establishes indirect competition standard curve.

Experimental result is shown：Indirect competitive ELISA using peptide molecule as DON recognition components, in pH=4.0,5.0, When 6.0,7.4,8.0, its IC₅₀Respectively 2100,2300,2290,2180ng/ml；Using DON monoclonal antibodies as recognition component Indirect competitive ELISA, there is not good blocking effect in pH=4.0,5.0,6.0, smoothed curve, the immunology of antibody is presented Analytical performance is significantly affected, shows the peptide molecule of DON antibody surrogate things and has more preferable soda acid patience.

2) high temperature resistance is tested

Temperature will can be respectively placed in as 37 DEG C, 45 DEG C with the DON peptide molecules specifically bound and DON monoclonal antibodies, 50 DEG C, 60 DEG C, 30min is incubated in 70 DEG C of water-bath, carries out indirect ELISA experiment after taking-up respectively, specific method is：Use 10mM The DON-OVA (2 μ g/ml) of PBS (pH 7.4) dilutions, coated elisa plate, 4 DEG C of overnight incubations.Second day with PBST (10mM PBS, 0.05%Tween-20 (v/v)) after washing 3 times, closed with the PBS containing 3% skimmed milk power, 37 DEG C be incubated it is 1 small Shi Hou, it is stand-by with PBST board-washings 6 times.The lath handled well is taken out, 50 μ l being incubated by different temperatures are put into respectively per hole DON peptide molecule/DON monoclonal antibodies (20 μ g/ml), 37 DEG C of incubation 20min can be specifically bound.Add 1:5000 dilutions The sheep anti-mouse igg secondary antibody of the anti-M13 bacteriophages secondary antibody of HRP marks/HRP marks, 37 DEG C are incubated 1 hour.Then shown with tmb substrate Color, read OD₄₅₀, using OD values at 37 DEG C as 100%, calculate OD under different temperatures₄₅₀The stability % of value.

Experimental result is shown:Indirect ELISA using peptide molecule as DON recognition components, temperature be 37 DEG C, 45 DEG C, At 50 DEG C, 60 DEG C, 70 DEG C, its stability is respectively 100%, 93%, 88%, 80%, 72%；Using DON monoclonal antibodies as The indirect ELISA of DON recognition components, when temperature is 37 DEG C, 45 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, its temperature degree is respectively 100%, 70%, 50%, 20%, 10%, show compared to traditional DON monoclonal antibodies, the polypeptide point of DON antibody surrogate things Son has more preferable high temperature resistance.

Claims (1)

1. coding can specifically bind the deoxynivalenol (nucleosides of the peptide molecule of (Deoxynivalneol, DON) Acid, its sequence are:TGT GCT CAT CAT GGT ATT CCG TAG TGT.