CN109575138A - A kind of application of ochratoxin A antiidiotype nano antibody as ochratoxin A antigen substitute - Google Patents

Abstract

The invention belongs to molecular biology fields, and in particular to a kind of application of ochratoxin A antiidiotype nano antibody VHH2-24 as kind of ochratoxin A antigen substitute.Present invention obtains the amino acid sequences ochratoxin A antiidiotype nano antibody as shown in SEQ ID NO:1, by optimizing peridium concentration, ochratoxin A monoclonal antibody working concentration, closed reagent, pH, methanol concentration, the VHH-ELISA reaction based on ochratoxin A antiidiotype nano antibody Surrogate antigen is established；Sample is carried out as the VHH-ELISA that substitution envelope antigen is established using VHH 2-24 and adds recovery experiment, shows the pollution detection that can be applied to ochratoxin A based on ochratoxin A antiidiotype nano antibody VHH-ELISA method that this research is established.

Description

A kind of ochratoxin A antiidiotype nano antibody is replaced as ochratoxin A antigen For the application of object

Technical field

The invention belongs to molecular biology fields, and in particular to a kind of ochratoxin A antiidiotype nano antibody conduct The application of ochratoxin A antigen substitute.

Background technique

Ochratoxin A (ocbratoxinA, OTA) is a kind of toxic fungal secondary metabolite, mainly by aspergillus and Mould generates, and mainly pollutes the industrial crops such as grain, vegetables and coffee, grape, cocoa, many due to that can pollute species, and produces The continuous development of the global and its present crop products world commerce of malicious fungi causes ochratoxin A in the whole world Wide-scale distribution, and seriously endangered the health of animals and humans.It has stronger renal toxicity and hepatotoxicity wind agitation, and has Teratogenesis shape, carcinogenicity, immunotoxicity and embryotoxicity, many countries all set up limit standard, therefore establish at low cost and fast Fast effective in-situ check and test method is particularly important.

Currently, the method for ochratoxin A detection has thin layer chromatography, high performance liquid chromatography, immunoassay With electrochemical sensor etc..Wherein, immunoassay is combined based on antigen and antibody specific, has detection sensitivity high, special The advantages that property is good, pre-treatment is easy, analysis is quick, low in cost, batch sample suitable for food safety detection it is quick Screening.But this method is established, a large amount of OTA reference substances and organic solvent synthesis detection antigen need to be used, while also needing OTA reference substance Standard curve is established, there is potentially hazardous to the health of operator.In addition, the waste liquid in experimentation, may be used also It can cause the secondary pollution of surrounding enviroment.

1963, Oudin etc. proposed the concept of idiotope, and idiotope refers to the epitope in the area antibody V, including hypervariable region With skeleton area.1974, Jerne proposed " immune network theory " (Immune Network Theory), i.e., immune system is each A cell is mutually distinguishable, stimulates, restricts, and forms complicated dynamic equilibrium network structure.The core of this network is only on antibody Special type (Idiotype, Id), idiotype have antigenicity, can induce body and generate anti-idiotype (anti-idiotypic Antibody, Anti-Id or AId), referred to as anti-Id.Anti- Id can be divided into tetra- seed type of α, β, γ and ε, and Ab2 β identifies Ab antigen Binding site can simulate the three-dimensional conformation of original antigen, play the role of Surrogate antigen molecule with haptens competitive binding Ab1.

Prepare that anti-idiotype production technology is relatively easy, and easy to operate, expense is lower using Anti-TNF-α body technique, But the later-period purification of serum is relatively complicated, and single limits throughput.And monoclonal antibody method is used, the production cycle is long, and preparation process also compares More complex, required high expensive, cell confluency and positive rate are far below Ab1 class monoclonal antibody, but the method once success, gained Hybridoma cell strain can save steadily in the long term, realize that once and for all prepares a large amount of AId, and method is simple, antibody mass Height, property stable homogeneous.Phage antibody library technique prepare AId it is easy, quickly, it is economical, gained antibody molecule amount is small, immunogene Property low, genotype it is consistent with phenotype, create new method to prepare humanized antibody, but this method requires antibody library storage capacity very High, diversity is enriched, otherwise later period screening technique also properly will be difficult to obtain the AId of function admirable.

Summary of the invention

The present invention is in view of the shortcomings of the prior art, be designed to provide a kind of ochratoxin A antiidiotype nano antibody work For the application of ochratoxin A antigen substitute.

For achieving the above object, the technical solution adopted by the present invention are as follows:

A kind of application of ochratoxin A antiidiotype nano antibody as ochratoxin A antigen substitute, it is described reddish brown Aspertoxin A antiidiotype nano antibody is ochratoxin A antiidiotype nano antibody VHH2-24, and amino acid sequence is such as Shown in SEQ IDNO:1, the nucleotide sequence of the amino acid is encoded as shown in SEQ ID NO:2.

It is established based on the ochratoxin A antiidiotype nano antibody as ochratoxin A antigen substitute ELISA immunoassay method, includes the following steps:

(1) Competitive assays ELISA reacts: will be coated in after ochratoxin A antiidiotype nano antibody VHH2-24 dilution ELISA Plate, 4 DEG C of coatings are overnight；Next day is closed with confining liquid, is incubated for 1h at 37 DEG C；Into enzyme mark hole be added methanol/ PBS7.4 buffer is added to enzyme mark hole after diluting ochratoxin A monoclonal antibody and competitor with 7.4 buffer of PBS In, 37 DEG C of incubation 1h；The sheep anti-mouse antibody of horseradish peroxidase-labeled, 37 DEG C of incubation 1h are added；Add developing solution, 37 DEG C After reacting 15min, OD is measured₄₅₀, obtain Percentage bound B/B₀Value；

(2) standard curve is established: be at war with suppression using the ochratoxin A standard items of various concentration as the competitor ELISA reaction processed, detection obtain different Percentage bound B/B₀Value, using ochratoxin A log concentration value as abscissa, with corresponding Percentage bound B/B₀Value is used as ordinate, establishes Competitive assays ELISA examination criteria curve；

(3) it ochratoxin A concentration mensuration: is at war with inhibition using the sample containing ochratoxin A as competitor ELISA reaction, detection obtain Percentage bound B/B₀Value substitutes into the standard curve that step (2) are established, and conversion obtains ochratoxin The concentration of A.

According to the above scheme, 0.06 μ of μ g/mL~0.13 of peridium concentration of the ochratoxin A antiidiotype nano antibody g/mL。

According to the above scheme, the confining liquid is 3%BSA, 1.5%OVA or 3% skimmed milk power.

According to the above scheme, the pH value of the methanol/PBS buffer solution is 7.0~7.4.

According to the above scheme, methanol concentration is 5%~10% in the methanol/PBS buffer solution.

Beneficial effects of the present invention are as follows: the present invention is by positive bacteriophage ochratoxin A antiidiotype nano antibody VHH2-24 genetic transformation is to expression bacterial strain, and through inducing expression, purifying, identification, the protein form nanometer for obtaining function admirable is anti- Body；By optimizing peridium concentration, ochratoxin A monoclonal antibody working concentration, closed reagent, pH, methanol concentration, base is established In the VHH-ELISA of nano antibody Surrogate antigen；The present invention selects the samples such as corn, wheat, rice measurement ochratoxin A anti- The TIANZHU XINGNAO Capsul of idiotype nano antibody VELISA, for the rate of recovery between 80%~114.8%, accuracy is good, it was demonstrated that reddish brown Aspertoxin antiidiotype nano antibody Surrogate antigen VHH-ELISA method result is accurate, reliable, and being that one kind is effective, feasible exempts from Epidemic disease analysis method.

Detailed description of the invention

Fig. 1 is for judging the multifarious test result of phage display nano antibody non-immune libraries.

Fig. 2 is that phage-ELISA identifies positive colony.

Fig. 3 is VHH2-24 and anti-ochratoxin A, aflatoxin B₁(abbreviation AFB₁), zearalenone (referred to as ZEN), the combination situation of vomitoxin (abbreviation DON) monoclonal antibody.

Fig. 4 is that chessboard method optimizes VHH 2-24 peridium concentration and ochratoxin A monoclonal antibody working concentration.

Fig. 5 is influence of the closed reagent to VELISA.

Fig. 6 is influence of the phosphate buffer pH value to VELISA.

Fig. 7 is influence of the salt ionic concentration to VELISA.

Fig. 8 is influence of the methanol concentration to VELISA.

Fig. 9 is the VELISA based on VHH 2-24 to aflatoxin B₁, zearalenone, the intersection of vomitoxin it is anti- Answer situation.

Figure 10 is for VHH 2-24 to the competition curve of OTA in different sample substrates.

Specific embodiment

For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but it is of the invention Content is not limited solely to the following examples.

In following embodiment, it is CCTCC NO.C201329 that the ochratoxin A monoclonal antibody, which is by deposit number, Hybridoma cell strain 1H2 secrete and generate, hybridoma cell strain 1H2 is preserved in Chinese Typical Representative culture on March 7th, 2013 Collection (CCTCC), and patent " hybridoma cell strain 1H2, its generate anti-ochratoxin A monoclonal antibody and It is applied ", application number: disclosed in 201310115921.8.

The building of 1 phage display nano antibody non-immune libraries of embodiment

1. alpaca is immune

200 μ g of ochratoxin A monoclonal antibody (being dissolved in PBS7.4) is taken, is emulsified with isometric incomplete Freund's adjuvant Afterwards, subcutaneous multi-point injection is carried out to the male alpaca (Alpaca) of three one full year of life, is hereafter immunized once, is immunized 8 times altogether every two weeks, Four exempt from after start blood sampling detection.At the 4th time it is immune after 7~10 days, with EDTA vacuum blood collection tube jugular vein blood collection 10mL, simultaneously Gentle inversion heparin tube avoids blood clotting, then handles blood with the filter in LeukoLOCK kit, then successively uses 3mLPBS buffer and 3mL RNAlater buffer rinse filter, then required leucocyte is just trapped in filter, finally will Filter is sealed in -80 DEG C, in case proposing RNA use.

2. Total RNAs extraction

Alpaca is separated according to the operation manual of Life Technology company LeukoLOCK totalRNA extracts kit Total serum IgE in blood, concrete operations are as follows:

(1) it cracks/combines in liquid to 2.5mL and 70 μ LpH adjusting buffer is added, it is ready-to-use；(2) by the filter of sealing It places and restores to room temperature, open filter cap, washed away RNAlater buffer remaining in filter with 2mL syringe；(3) it uses 2.5mL syringe draws 2.5mL and now matches cracking/combination buffer to rinse filter primary, efflux with 15mLRNAse-free from Heart pipe is collected；(4) ddH for being free of nuclease is added₂O 2.5mL is vortexed and mixes, and 25 μ L Proteinase Ks is added, by centrifuge tube in room Warm 250rpm shakes 5min；(5) taking-up is stored in 4 DEG C of RNA combination magnetic bead, is vortexed after mixing and draws 50 μ L to above-mentioned centrifuge tube In, vortex centrifugal pipe, then isopropanol 2.5mL is added into centrifuge tube, room temperature shakes 5min；(6) by above-mentioned centrifuge tube in 3200rpm is centrifuged 3min, and supernatant is carefully sucked out and discards, is careful not to be drawn onto the magnetic bead of precipitating；(7) 600 μ L washing lotion I are added, instead The magnetic bead of multiple piping and druming precipitating makes it be uniformly dispersed, transfer suspension to 1.5mL centrifuge tube, then rinses 15mL centrifugation with 600 μ L washing lotion I Pipe is primary, and is transferred in same 1.5mL centrifuge tube；(8) 1.5mL centrifuge tube is centrifuged 30s in 16000g, carefully discarded Clearly；(9) 750 μ L washing lotions 2/3 are added, the concussion 30s that is acutely vortexed makes the magnetic bead of aggregate and precipitate scatter, and is centrifuged 30s in 16000g Magnetic bead is collected, is carefully discarded supernatant；(10) centrifuge tube is opened wide into lid and stands 2min in super-clean bench, it is residual in washing lotion 2/3 of volatilizing During which the alcohol stayed now mixes liquid with TURBODNase master, 4 μ LTURBO DNase (20U/ μ L) is taken to be added to 296 μ L It in LeukoLOCK DNase buffer, is transferred in above-mentioned centrifuge tube after mixing, blows and beats the magnetic of precipitating repeatedly with liquid-transfering gun Pearl is allowed to be uniformly dispersed, and 1000rpm shakes 10min at room temperature, and midway gently overturns mixing several times；(11) Xiang Shangshu centrifuge tube In sequentially add cracking/combine liquid (not plus pH adjust buffer) and each 300 μ L of isopropanol, crawl centrifugation 2s after mixing, then It is incubated for 3min at room temperature；(12) in 16000g be centrifuged 30s, abandon supernatant, add 750 μ L washing lotions 2/3, be acutely vortexed 30s, 16000g from Heart 30s abandons supernatant, 750 μ L washing lotions 2/3 is added again, and be acutely vortexed 30s, and 16000g is centrifuged 1min, abandons dry supernatant as far as possible, Room temperature opening stands 3min, vapors away remaining washing lotion, pays attention to place too long in order to avoid magnetic bead is over-drying；(13) it is added 60 μ L of eluent, be vortexed concussion 30s, and after 16000g is centrifuged 2min, the RNA solution eluted is transferred to another without nuclease 1.5mL centrifuge tube in, leave and take 1 μ LRNA solution nanodrop and survey concentration, then to take 3 μ L or so RNA solution to carry out agarose solidifying Gel electrophoresis analysis, remaining RNA solution are all inverted to cDNA immediately, prevent to degrade.

The synthesis of 3.cDNA

The first chain of cDNA is synthesized according to reverse transcription reagent box specification, steps are as follows:

(1) two 200 μ L is taken to be free of the PCR pipe of nuclease, it is each that 3 μ L 10mM dNTPmix, 3 μ L, 50 μM of oligo are added (dT) 20,24 μ LRNA is soft to mix；

(2) 65 DEG C of heating 5min, make template denaturation, and secondary structure is opened；

(3) it is immediately placed on cooled on ice at least 1min；

(4) premixed liquid now with synthesis cDNA:

(5) respectively add 30 μ L premixed liquids in Xiang Liangzhi PCR pipe, mixed with micropipettor；

(6) heat reaction mixture: 50 DEG C of annealing reaction 50min, then 85 DEG C of heating 5min make reaction terminating；

(7) 3 μ LRNAseH are respectively added into reaction mixture and mix, in 37 DEG C of heat treatment 20min, decompose incomplete The RNA of reaction；

The synthesis of (8) first chain cDNA is completed, and is saved backup after packing in -20 DEG C.

4. the amplification of antibody heavy chain variable region VHH gene

The respective degenerate primer pair of the variable region IgG2 and IgG3 VHH gene is taken, carries out polymerase respectively by template of cDNA Chain reaction (PCR) amplification, primer pair F, R2 are used for the clone of IgG3 hypotype for cloning IgG2 hypotype, primer pair F, R1, instead Answer system as follows:

PCR program are as follows:

Wherein the corresponding upstream primer of amplification IgG2, IgG3 is respectively primer R2, R1, and primer sequence is as shown in table 1.

The amplification of table 1VHH antibody gene and sequencing primer sequence table

5. the building in phage display nano antibody library

Enzyme is carried out to carrier pComb3X and VHH respectively using SfiI, connects the VHH segment after digestion with T4 ligase With carrier pComb3X, take 3 μ L connection products into 25 μ L E.coli ER2738 competent cells, light mixed rear be all sucked out turns It moves in the electric revolving cup (internal diameter 1mm) of pre-cooling, is quickly placed in electroporation and carries out electrotransformation；After electric shock, 1mL37 is added immediately DEG C preheating SOC culture medium into electric revolving cup, gently inhaled with liquid-transfering gun and play mixing, be transferred to and shake in tube, in 37 DEG C of shaking tables 250rpm concussion recovery culture 1h；It repeats electricity to have turned 10 times, 3 μ L connection product, collects the bacterium solution of 10 conversions and take 1 μ L every time It is coated on LB-Amp plate after diluting 10 times with sterile water, in 37 DEG C of incubator overnight incubations for estimating storage capacity.It will be complete Portion's transformed bacteria is gone in 200mLSB culture medium, and carbenicillin is added to 50 μ g/mL, tetracycline to 20 μ g/mL；250rpm, 37 DEG C culture is to OD₆₀₀It is 0.6；1mL helper phage (1 × 10 is added¹³Pfu/mL), 37 DEG C of standings infect 30min；250rpm, 37 DEG C culture 2h, be added kanamycins to concentration be 70 μ g/mL, continue overnight incubation；Bacterium solution is centrifuged by next day, 4 DEG C, 10000rpm is centrifuged 15min；It takes supernatant into sterile centrifugation tube, adds 1/4 volume PEG/NaCl solution, ice bath 2h, then be centrifuged, abandon Supernatant (contains 1 × protease inhibitors, 0.02%NaN with 10mL re-suspension liquid₃, 0.5%BSA PBS buffer solution) be resuspended precipitating； Phage solution is filtered with 0.22 μm of filter, removes remaining bacterium etc.；Gained phage solution is phage display nanometer Antibody library is dispensed, carries out mark, is stored in -70 DEG C.

6. the identification of phage display nano antibody non-immune libraries

From 20 monoclonals are chosen on plate respectively into 1mLSB culture medium, 37 DEG C are cultivated to bacterium solution OD₆₀₀About 0.8, it takes out Bacterium solution send company to be sequenced using gback as primer, analyzes cloned sequence, determines the diversity level in built library.Sequencing result is such as Shown in Fig. 1, the amino acid sequence of the Insert Fragment of selected 20 clones is different, shows that constructed phage display nanometer is anti- Body library diversity is good, can be used for subsequent screening operation.

The elutriation and identification of 2 ochratoxin A antiidiotype nano antibody of embodiment

(1) elutriation of ochratoxin A antiidiotype nano antibody

Using ochratoxin A monoclonal antibody as target, by the reddish brown song for reducing primary concentration of envelope, competitive elution by wheel Mould toxin A standard concentration, is used alternatingly closed reagent, carries out affine enrichment elutriation, obtains and is directed to ochratoxin A monoclonal The antibody of antibody, i.e. antiantibody, also referred to as anti-idiotype, panning step are as follows:

(1) be coated with: 6 holes are wrapped in coating ochratoxin A monoclonal antibody, 20 μ g/mL, 100 holes μ L/, with coating buffer Dilution；Adsorption hole is removed, 3%BSA/PBS is coated with, 6 holes are wrapped in 100 holes μ L/；4 spend better than the 37 DEG C incubations of night coated effect 2h；

(2) it closes: 3%PBSTM, 300 holes μ L/, after 37 DEG C of incubations 1h, hand washing plate 3 times；

(3) library to envelope antigen hole: 100 holes μ L/, 37 DEG C of reaction 1h, then room temperature shaker is added to react 1h, then with band filter core Pipette tips hand-wash this 6 holes, and board-washing 10 times；

(4) it elutes: preparing 100ng/mL ochratoxin A standard items, room temperature shaker reacts 1h；

(5) it goes to adsorb: eluent being transferred to adsorption hole, 100 holes μ L/ react at room temperature 30min；

(6) collect 600 μ L eluates, referred to as " 1st output ", first round elutriation is completed；1st output need to be by expanding Increase the elutriation that could be used for the second wheel；

(7) " 1st output " titre is measured, 10 μ L " 1st output ", gradient dilution to 10 are taken³、10⁴、10⁵, this 3 Dilution respectively takes 10 μ L to infect 90 μ L ER2738 (OD 0.8), 37 DEG C of standing 30min, is coated with LB-Amp (ammonia benzyl) plate, and 37 It DEG C is incubated overnight, the monoclonal number on secondary number of days plate, estimates titre.

In subsequent elutriation, coated antibody concentration is gradually reduced, and ochratoxin A concentration gradually decreases in eluent, Three-wheel elutriation is carried out, panning process is shown in Table 2, and elutriation enrichment the results are shown in Table 3.

The elutriation in 2 bacteriophage nano antibody library of table

The every wheel elutriation bacteriophage of table 3 is enriched with result

(2) identification of positive phage clones

(1) from 30 monoclonals of random picking on the output titre plate of last wheel elutriation, on LB-Amp plate Bacterium is protected, while accessing 3mLSB culture medium；

30 μ L M13KO7 helper phages, 37 DEG C of standings are added until OD value is 0.8 in (2) 37 DEG C of shaking table culture 4-5h 30min；

(3) 37 DEG C of shaking table culture 1h add final concentration of 70 μ g/mL Kana, 37 DEG C of cultures；

(4) next day respectively takes 500vL bacterium solution to be centrifuged, 8000rpm, 3min, and supernatant dilutes 10 times and is used for Phage-ELISA；

(5) elisa plate is closed, 3%PBSTM, 300 holes μ L/, 37 DEG C of incubation 1h, is machine-washed plate 3 times；

(6) bacteriophage supernatant (10 times of dilution) of each clone is added in corresponding 3 holes, every 50 μ L of hole, Isosorbide-5-Nitrae, 7,10 Column plus 50 μ L standard items, 2,3,5,6,8,9,11,12 column plus 50 10% methanol of μ L/PBS buffer solution, microplate reader concussion after adding It mixes；

(7) 37 DEG C of standing 1h are hand-washed plate 10 times；

(8) secondary antibody, anti-M13/ horseradish peroxidase, 1:5000 dilution, 37 DEG C of incubation 1h；

(9) it develops the color, 37 DEG C of incubation 15min detect the light absorption value at 450nm.

Phage-ELISA is done to 30 clones, find they and ochratoxin A monoclonal antibody association reaction difference compared with Greatly.In Phage-ELISA measurement, with antibody response OD₄₅₀Value is higher, while OD after OTA is added₄₅₀Value is substantially reduced biting for phenomenon Thallus clone is determined as the positive, as a result as shown in Fig. 2, by four-wheel elutriation obtain altogether 9 positive colonies (be followed successively by 7,10,15, 7,21,23,26,27, No. 30 clones).According to above-mentioned ELISA as a result, positive colony is chosen from guarantor's bacterium plate, activation culture After send to Wuhan Jin Kairui biotech firm and carry out sequence analysis, sequencing primer gback carries out gene surveys to 9 positive colonies Sequence selects the ochratoxin A nano antibody VHH2-24, amino acid sequence such as SEQ ID of the highest phage display of OD value Shown in NO:1, nucleotide sequence is as shown in SEQ ID NO:2.

The expression and purification of 3 ochratoxin A antiidiotype nano antibody VHH2-24 of embodiment

(1) preparation of Top10F ' competent cell:

(1) LB- tetracyclin plates, 37 DEG C of overnight incubations are lined with the appropriate Top10F ' of picking；

(2) picking Top10F ' monoclonal is into 5mL LB culture medium, and 37 DEG C, 250rpm cultivates to OD₆₀₀It is 0.6~0.8；

(3) bacterium solution is gone into 4 DEG C of centrifugations in pre-cooling centrifuge tube (about 1.3mL/ pipe), 8000rpm, 8min；

(4) it is immediately placed in after being centrifuged on ice, abandons supernatant, the 0.1M CaCl of 1mL pre-cooling is added₂Thallus is resuspended in solution, inhales Beat ice bath 30min after mixing；

At (5) 4 DEG C, is put back into rapidly on ice after 8000rpm centrifugation 8min, thoroughly exhaust liquid, what precipitating was pre-chilled with 100 μ L 0.1MCaCl₂Solution is resuspended, as Top10F ' competent cell.

(2) extraction and conversion of positive phage clones plasmid:

(1) it from the glycerol stock of VHH2-24/ER2738, chooses and lines LB- carboxylic benzyl plate, 37 DEG C of overnight incubations in right amount；

(2) it chooses VHH2-24/ER2738 single bacterium to fall in 2mL SB culture medium, OD is arrived in 37 DEG C of cultures₆₀₀It is 0.8, extracts The plasmid of VHH2-24；

(3) on ice, add 1.5 μ LVHH 2-24 plasmids into 100 μ L Top10F ' competent cells of above-mentioned preparation, gently It mixes, places 30min on ice；

(4) 42 DEG C of thermal shock 90s put back to rapidly on ice, place 5min；

(5) in superclean bench, 700 μ L LB culture mediums, 37 DEG C, 200rpm recovery culture are added into each centrifuge tube 45min；

At (6) 4 DEG C, 12000rpm is centrifuged 4min, inhales and abandons part supernatant, leaves and takes about 150 μ L liquid, be coated with LB- after mixing Carboxylic benzyl plate, 37 DEG C are incubated overnight.

(3) inducing expression and purifying of nano antibody

On the plate of VHH 2-24/Top10F ', chooses single bacterium and fall on 3mLSB culture medium, 37 DEG C, 250rpm shaking table culture After overnight, 1mL bacterium is transferred into 200mLSB culture medium, continues culture to OD₆₀₀It is 0.6, is added 200 μ L 1M IPTG, 37 DEG C Overnight induction.4 DEG C, bacterium is received in 8000rpm, 15min centrifugation, and according to bacterial sediment quality, lysate B-PER is added by 20mL/g, Room temperature slowly shakes 10min after fully dispersed precipitating, then 12000rpm is centrifuged 20min, and supernatant is that the thick of nano antibody takes liquid. It is dialysed crude extract with 0.01M PBS7.4, after 0.22 μm of water phase filter membrane, in case ni-sepharose purification.

Using Ni-NTA HisBind Resin kits nano antibody crude extract, steps are as follows:

(1) the sterile ddH of column volume is successively chromatographed with 10 times₂O, 0.01mol/L PBS7.4 balances pillar；

(2) crude extract after filtration sterilization is added in chromatographic column, after mixing with filler, shakes 1h at room temperature, makes nanometer Antibody is sufficiently combined with filler；

(3) mixed liquor is fitted into column, balances pillar with the 0.01mol/L PBS7.4 of 10 times of column volumes；

(4) each 10mL of imidazoles-PBS7.4 of 20mM, 40mM, 300mM are prepared respectively by 1M imidazoles, crosses 0.22 μm of water phase filter Film, is used as eluent and carries out gradient elution, collects efflux with 2mL centrifuge tube；

(5) pillar is crossed with the imidazoles-PBS7.4 of the 20mM of 10mL, does not collect efflux；

(6) pillar is crossed with the imidazoles-PBS7.4 of the 40mM of 10mL, 3mL efflux before collecting

(7) pillar is crossed with the imidazoles-PBS7.4 of the 300mM of 10mL, collects whole effluxes；

(8) pillar is washed with the imidazoles-PBS7.4 of the 1M of 10 times of column volumes, does not collect efflux, makes non-specific binding Foreign protein is all eluted；

(9) successively with the PBS of 10 times of column volumes, the ddH of 10 times of column volumes₂O, 20% ethyl alcohol of 10 times of column volumes washes column, Finally saved with isometric 20% ethyl alcohol water seal column.

SDS-PAGE electrophoretic analysis is made to each pipe efflux, the efflux for having obvious nano antibody band is mixed, in PBS 4 DEG C of dialysed overnights in buffer, then with PEG8000 to antibody-solutions carry out be concentrated or using concentration tube be concentrated to get VHH2-24 nano antibody solution, and it is sub-packed in -20 DEG C of preservations.

The specificity analysis of 4 antiidiotype nano antibody VHH2-24 of embodiment

(1) the specificity analysis of antiidiotype nano antibody VHH2-24: being 0.2 μ with coating buffer difference compound concentration Ochratoxin A antigen, that is, OTA-BSA, aflatoxin B of g/mL₁Antigen, that is, AFB₁- BSA, zearalenone antigen are ZEN-BSA and vomitoxin antigen, that is, DON-BSA, 4 DEG C of overnight coated elisa plates；Next day, with 3% skimmed milk power/conventional phosphoric acid The closing of salt buffer conventional phosphate Tween buffer solution, 300 holes μ L/, 37 DEG C of incubation 1h, by anti-ochratoxin A, Huang Qu Mould toxin B₁, zearalenone and vomitoxin four kinds of monoclonal antibodies since 10 μ g/mL, with conventional phosphoric acid salt buffer Liquid carries out doubling dilution, 37 DEG C of incubation 1h；Add the goat-anti mouse monoclonal antibody of horseradish peroxidase-labeled, 37 DEG C of incubation 1h； Developing solution is added, terminate liquid is added in 37 DEG C of colour developing 15min, measures OD₄₅₀。

According to the above method be coated with, close after, by VHH2-24 with conventional phosphoric acid salt buffer successively twice dilution, press more than The concentration of optimization prepares anti-ochratoxin A, aflatoxin B₁, four kinds of monoclonals of zearalenone and vomitoxin it is anti- Body is separately added into 50 μ LVHH2-24 dilutions and the corresponding monoclonal antibody of 50 μ L into each hole, and microplate reader oscillation mixes, and 37 DEG C be incubated for 1h；Horseradish peroxidase-labeled sheep anti-mouse antibody, 37 DEG C of reaction 1h are added by 1:5000 dilution again；Same above method Colour developing measures OD₄₅₀Value.

Test results are shown in figure 3, and Fig. 3 shows that VHH2-24 can inhibit ochratoxin A monoclonal antibody and antigen The combination of OTA-BSA, with the continuous increase of VHH2-24 concentration, inhibiting effect is more and more obvious, and fights aflatoxin B₁, zearalenone and vomitoxin monoclonal antibody and corresponding antigens combination there is no inhibiting effect, show VHH2-24 have There is good selectivity, is only specifically bound with ochratoxin A monoclonal antibody variable region.

5 nano antibody VHH2-24 of embodiment is established as ELISA (VELISA) method of ochratoxin A Surrogate antigen

The operating process that nano antibody VHH2-24 is reacted as the Competitive assays ELISA of ochratoxin A Surrogate antigen Are as follows: (1) will substitution ochratoxin A antigen coat is in ELISA Plate after the dilution of ochratoxin A antiidiotype nano antibody, 4 DEG C Coating is overnight；Next day is closed with confining liquid, is incubated for 1h at 37 DEG C；(2) methanol/PBS 7.4 is added into enzyme mark hole to buffer Liquid is added in enzyme mark hole after diluting ochratoxin A monoclonal antibody and competitor with 7.4 buffer of PBS, and 37 DEG C incubate Educate 1h；(3) sheep anti-mouse antibody of horseradish peroxidase-labeled, 37 DEG C of incubation 1h are added；(4) developing solution, 37 DEG C of reactions are added After 15min, OD is measured₄₅₀。

(1) VELISA condition optimizing

(1) best VHH peridium concentration and ochratoxin A monoclonal antibody working concentration: VHH2-24 is successively diluted 100 ×, 200 ×, 400 ×, 800 ×, 1600 ×, 3000 × times, corresponding concentration is respectively 0.5,0.25,0.13,0.06, 0.03,0.015 μ g/mL, coated elisa plate, using chessboard method respectively to nano antibody VHH2-24 peridium concentration and Aspergillus ochraceus poison Plain A monoclonal antibody working concentration optimizes, and takes nano antibody concentration and ochratoxin A list of the OD450 value 1.0 or so Clonal antibody concentration is optimal working concentration, and ochratoxin A monoclonal antibody extension rate is respectively 100,200,400,800 Times, corresponding concentration is respectively 10,5,2.5,1.25 μ g/mL, carries out indirect ELISA analysis by this concentration, establishes standard curve (Fig. 4).Different according to each combination colour developing value, finally selected VHH2-24 working concentration is 0.13 μ g/mL, corresponding Aspergillus ochraceus poison Plain A MAb concentration is 2.5 μ g/mL.

(2) best closed reagent: in optimal VHH2-24 peridium concentration and ochratoxin A monoclonal antibody working concentration Under, inquire into the influence that different closed reagents react VELISA.3%BSA, 1.5%OVA and 3% skimmed milk power are used respectively, are established Standard curve (Fig. 5) determines that 3% skimmed milk power is best confining liquid.

(3) optimal pH: ochratoxin A monoclonal antibody is diluted with the phosphate buffer that pH is 5,6,7,8 respectively, is used Each buffer doubling dilution ochratoxin A standard items containing 10% methanol, VELISA standard curve such as Fig. 6 of foundation work as pH When=7, reaction sensitivity highest is raised and lowered pH and has an impact to reaction sensitivity.

(4) best salt ionic concentration: adapted ddH respectively₂Tetra- kinds of solution dilution Aspergillus ochraceus poison of O, PBS, 2 × PBS, 4 × PBS Plain A monoclonal antibody establishes VELISA detection with each buffer doubling dilution ochratoxin A standard items for containing 10% methanol The standard curve of ochratoxin A.As shown in fig. 7, detection sensitivity is most under pure water reaction system and 4 × PBS reaction system It is low, and PBS and 2 × PBS reaction buffer have no significant effect reaction system, therefore, select PBS as reaction buffer.

(5) most suitable methanol concentration: in the next best nano antibody peridium concentration of the above optimization, ochratoxin A Dan Ke Under the conditions of grand antibody working concentration, closed reagent and optimal phosphate buffer, continue to inquire into different methanol concentrations to VELISA The influence of reaction.It is respectively 5%, 10%, 20%, 40% conventional phosphoric acid salt buffer conventional phosphate tween with methanol concentration Buffer soln dilutes ochratoxin A and ochratoxin A monoclonal antibody, establishes suppression curve (Fig. 8), wherein methanol is dense When degree is 5% and 10%, the IC that is obtained by curve₅₀Very close to, but IC when methanol concentration 10%₅₀It is smaller, and it is bent to compete fitting The trend of line is relatively more preferable.

(6) cross reacting rate of VELISA: using VHH2-24 as Surrogate antigen, according to optimal conditions, AFB is chosen₁、ZEN、 Tri- kinds of mycotoxins of DON make competitor, measure the cross reacting rate of VELISA.As seen from Figure 9, the ELISA is to OTA sensitivity It is higher, the IC of VHH2-24₅₀Value reaches 0.015g/mL, and and AFB₁, ZEN, DON cross reaction is not present, then VELISA is special It is anisotropic good, it can pointedly detect the ochratoxin A in agricultural product.

The foundation of embodiment 6VHH-ELISA sample analysis method

(1) nano antibody VHH2-24 is in different sample substrates to the competition curve of OTA

Pre-treatment is carried out to three kinds of corn, rice, wheat blank samples, by three kinds of sample extracting solutions respectively with PBS dilution 4 Again, after 8 times and 20 times, with extracting solution doubling dilution ochratoxin A standard items, the VELISA competition of each matrix extracting solution is established Suppression curve, and be compared with the standard curve established under the reaction system without matrix, three kinds of samples are right as the result is shown VELISA leads to false positive results there are certain matrix effect.It finds simultaneously, when diluting 4 times of extracting solution with 4%BSA/PBS, Matrix effect can be reduced, approach matrix curve with when no matrix, the results are shown in Figure 10.

(2) application of the VHH-ELISA in actual sample detection

In order to evaluate the accuracy of VELISA detection architecture, using newly-established VELISA method carried out corn, rice, The addition recovery test of wheat samples.Respectively the corn of blank, rice, wheat samples are added with the reddish brown song of 10,20,50 μ g/kg Mould toxin A standard solution the results are shown in Table 4 with the OTA content in newly-established method VELISA method test sample extracting solution.OTA Addition recovery test show that sample TIANZHU XINGNAO Capsul shows that this research is established anti-based on nanometer between 80~114.8% Body VELISA method can be applied in the pollution monitoring of ochratoxin A.

Table 4 adds recovery test based on the sample of nano antibody VHH-EILSA

Note: each result of a is interior average value in triplicate on the same day

Test is the average value of test result in five days between b group

Obviously, above-described embodiment is only intended to clearly illustrate made example, and is not the limitation to embodiment.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or It changes.There is no necessity and possibility to exhaust all the enbodiments.And the obvious variation or change therefore amplified It moves within still in the protection scope of the invention.

Sequence table

110 > Inst. of Oil Crops, Chinese Academy of Agriculture of <

A kind of application of the ochratoxin A antiidiotype nano antibody of 120 > of < as ochratoxin A antigen substitute

160 > 2 of <

<210> 1

<211> 153

<212> PRT

<213>alpaca

<400> 1

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

5 10 15 20

Ser Cys Ala Ala Ser Gly Val Ile Phe Ser Leu Glu Thr Met Gly Trp Tyr Arg Gln Ala

25 30 35 40

Pro Gly Lys Gln Arg Glu Met Val Ala Ile Ile Ala Arg Asp Ser Lys Thr Asn Tyr Val

45 50 55 60

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Tyr Ala Lys Asp Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Asp Ser Trp

85 90 95 100

Val Gly Ala Trp Arg Asp Glu Tyr Leu Glu Val Trp Gly Gln Gly Thr Leu Val Thr Val

105 110 115 120

Ser Ser Ala His His Ser Glu Asp Pro His Gly Gln Ala Gly Gln His His His His His

125 130 135 140

His Gly Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser

145 150 153

<210> 2

<211> 367 bp

<212> DNA

<213>alpaca

<400> 2

caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggagt catcttcagt ctcgaaacca tgggctggta ccgccaggct 120

ccagggaagc agcgcgagat ggtcgcaatt attgctcgtg atagtaagac aaactatgta 180

gactccgtga agggccgatt caccatctcc agagactacg ccaaggatac ggtgtatctg 240

caaatgaaca gcctgagacc tgaggacacg gccgtctatt actgtcatgc agatagttgg 300

gttggtgcct ggcgcgatga gtatctcgaa gtttggggcc agggcaccct ggtcactgtc 360

tcctcag 367

Claims (6)

1. a kind of application of ochratoxin A antiidiotype nano antibody as ochratoxin A antigen substitute, the reddish brown song Mould toxin A antiidiotype nano antibody is ochratoxin A antiidiotype nano antibody VHH2-24, amino acid sequence such as SEQ Shown in ID NO:1, the nucleotide sequence of the amino acid is encoded as shown in SEQ ID NO:2.

2. applying according to claim 1, which is characterized in that made based on the ochratoxin A antiidiotype nano antibody For the ELISA immunoassay method that ochratoxin A antigen substitute is established, include the following steps:

(1) Competitive assays ELISA reacts: ELISA Plate will be coated in after the dilution of ochratoxin A antiidiotype nano antibody, 4 DEG C Coating is overnight；Next day is closed with confining liquid, is incubated for 1h at 37 DEG C；7.4 buffer of methanol/PBS is added into enzyme mark hole, It is added in enzyme mark hole after ochratoxin antibody and competitor are diluted with 7.4 buffer of PBS, 37 DEG C of incubation 1h；It is added 1: The sheep anti-mouse antibody of 5000 horseradish peroxidase-labeleds, 37 DEG C of incubation 1h；It adds developing solution, after 37 DEG C of reaction 15min, surveys Determine OD₄₅₀, obtain Percentage bound B/B₀Value；

(2) standard curve is established: be at war with inhibition using the ochratoxin A standard items of various concentration as the competitor ELISA reaction, detection obtain different Percentage bound B/B₀Value, using ochratoxin A standard concentration logarithm as abscissa, with Corresponding Percentage bound B/B₀Value is used as ordinate, establishes Competitive assays ELISA examination criteria curve；

(3) ochratoxin A concentration mensuration: the sample containing ochratoxin A is at war with as competitor and inhibits ELISA anti- It answers, detection obtains Percentage bound B/B₀Value substitutes into the standard curve that step (2) are established, and conversion obtains the dense of ochratoxin A Degree.

3. applying according to claim 2, which is characterized in that the coating of the ochratoxin A antiidiotype nano antibody 0.06 μ of μ g/mL~0.13 g/mL of concentration.

4. applying according to claim 2, which is characterized in that the confining liquid is 3%BSA, 1.5%OVA or 3% degreasing Milk powder.

5. applying according to claim 2, which is characterized in that the pH value of the methanol/PBS buffer solution is 7.0~7.4.

6. applying according to claim 2, which is characterized in that in the methanol/PBS buffer solution methanol concentration be 5%~ 10%.