CN107167591A - A kind of aflatoxin single domain heavy chain antibody affine in immunity sorbing material - Google Patents

A kind of aflatoxin single domain heavy chain antibody affine in immunity sorbing material Download PDF

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CN107167591A
CN107167591A CN201710266348.9A CN201710266348A CN107167591A CN 107167591 A CN107167591 A CN 107167591A CN 201710266348 A CN201710266348 A CN 201710266348A CN 107167591 A CN107167591 A CN 107167591A
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aflatoxin
heavy chain
chain antibody
aspergillus flavus
sorbing material
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涂追
许杨
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Nanchang University
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Nanchang University
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Abstract

The invention belongs to biological technical field, it is related to a kind of affine in immunity sorbing material based on single domain heavy chain antibody, especially for the affine in immunity sorbing material of aflatoxin.Including carrier, the aglucon on carrier is mounted in, it is characterised in that the material is using the single domain heavy chain antibody of specific recognition aflatoxin as aglucon, and the single domain heavy chain antibody of the specific recognition aflatoxin has SEQ ID NO.:Amino acid sequence shown in 13 grades.The antibody has acid and alkali-resistance, high temperature resistant and the characteristic such as is readily produced, and has important practical value for inexpensive, the reusable immunological detection method of aflatoxin.

Description

A kind of aflatoxin single domain heavy chain antibody affine in immunity sorbing material
Technical field
It is special the present invention relates to a kind of affine in immunity sorbing material based on single domain heavy chain antibody (also known as nano antibody technology) It is not the affine in immunity sorbing material for aflatoxin.
Technical background
Aflatoxin is that a class is main to be produced by the mycetogenetic hypertoxic cometabolism such as Aspergillus flavus, aspergillus parasiticus bacterium Thing.Their chemical constitution is similar, is two furocoumarin derivatives, mainly including aflatoxin B1(AFB1)、B2 (AFB2)、G1(AFG1)、G2(AFG1) and M1(AFM1) etc..In the food and feed of natural contamination, AFB1Pollution it is the most normal See, its toxicity and carcinogenicity are also most strong, it is I class carcinogenic substance to be delimited by the Agency for Research on Cancer of the World Health Organization.Countries in the world And area specifies AFB in strict aflatoxin limit standard, such as European Union's infant food1The μ g/ of allowance≤0.10 kg。
Existing Determination Methods of Aflatoxins mainly has instrumental method, immunoassay and TLC.Its Instrumental analytic approach has the advantages that sensitivity high, accuracy and reproducible, but for the requirement of analysis sample pre-treatments compared with It is high to remove the impurity in sample to reduce the interference to subsequent detection, it is necessary to try one's best.Traditional pre-treating method have liquid-phase extraction, SPE etc., processing procedure is cumbersome and specific not high.Affinity chromatography technology is based on specific between aglucon and test substance Identification, can by single stepping realize determinand in COMPLEX MIXED sample is isolated and purified.The aspergillus flavus poison used at present Plain affinity purification medium, as aglucon, is existed fast intolerant to organic reagent, activity typically using polyclonal antibody or monoclonal antibody The problem of prompt drop is low.Accordingly, it would be desirable to which developing the good new aglucon of active high, stability replaces conventional antibodies.Single domain heavy chain antibody It is made up of the variable region of alpaca heavy chain antibody, also known as nano antibody, with acid and alkali-resistance, high temperature resistant and the spy such as is readily produced Property, there is obvious advantage compared to conventional antibodies.
The content of the invention
It is an object of the invention to provide a kind of for the affine in immunity sorbing material of aflatoxin and its application.
To achieve the above object, the present invention is adopted the following technical scheme that：
For a kind of affine in immunity sorbing material (aflatoxin single domain heavy chain antibody affine in immunity suction of aflatoxin Enclosure material) include carrier and aglucon, the aglucon is single domain heavy chain antibody, with SEQ ID NO.:1、SEQ ID NO.:3、 SEQ ID NO.:5、SEQ ID NO.:7、SEQ ID NO.:9、SEQ ID NO.:11、SEQ ID NO.:Amino shown in 13 Acid sequence.The aglucon can specific recognition aflatoxin.
The aglucon can also be to pass through random or site-directed mutagenesis technique on the basis of the single domain heavy chain antibody of foregoing sequences Carry out the antibody that can be specifically bound with aflatoxin of house of correction acquisition.
The carrier is magnetic bead, agarose gel microsphere, silica gel microball or porous material.
The preparation method of the above-mentioned affine sorbing material of Aspergillus flavus toxin immuno, it is characterized in that：
When the carrier is magnetic bead, preparation method is：1mg carboxyl magnetic beads are taken in centrifuge tube, 500~1000 μ l is added and lives Change buffer solution (10mM, NaH2PO4, pH6.0), vortex mixed is uniform, and magnetic frame reclaims magnetic bead, then washs 2 with activation buffer Time.It is separately added into after 1~5mg carbodiimides (EDC) and n-hydroxysuccinimide (NHS), vortex mixed, stands 30min.With Coupling buffer (10mM, Na2HPO4, pH 7.4) and wash magnetic bead 3 times, aspergillus flavus resisting toxin single domain heavy chain antibody is added, room temperature is anti- 2~6h is answered, the immunomagnetic beads of covalent coupling aspergillus flavus resisting toxin single domain heavy chain antibody is obtained；
When the carrier is agarose gel microsphere, preparation method is：The dry glue that CNBr is activated is washed with 0.1M HCl 10~15 times, 5~10min is balanced every time.With coupling buffer (10mM, Na2HPO4, pH 7.4) wash 5~15 times, add anti- Aflatoxin single domain heavy chain antibody, reacts at room temperature 2~10h, obtains covalent coupling aspergillus flavus resisting toxin single domain heavy chain antibody Affine in immunity sorbing material；
When the carrier is silica gel microball, preparation method is：By silica gel microball pure water and phosphate buffer (PBS, 10mM, pH 6.0) alternately washing 5~10 times, with PBS suspension silica gel microball, add aspergillus flavus resisting toxin single domain heavy chain Antibody, is mixed, and adds 1~10mg/ml of final concentration carbodiimide (EDC), and rapid to mix, 4 DEG C of 12~24h of stirring reaction are obtained The affine in immunity sorbing material of aspergillus flavus resisting toxin single domain heavy chain antibody to covalent coupling.
Above-mentioned affine in immunity sorbing material (carrier is agarose gel microsphere and silica gel microball) is filled to chromatographic column, i.e., Aspergillus flavus toxin immuno affinity column is obtained, method is：According to chromatography column capacity, appropriate above-mentioned affine in immunity sorbing material is taken In after chromatographic column, PBS (10mM, the pH 7.4) washings for adding 5~10 times of bed volumes, 4 DEG C, 20% ethanol solution is stored in.
The immunomagnetic beads of covalent coupling aspergillus flavus resisting toxin single domain heavy chain antibody need not fill post, magnet point after directly adsorbing From.
The invention further relates to be mounted with the affinity column of the affine sorbing material of the Aspergillus flavus toxin immuno.
The application of the above-mentioned affine sorbing material of Aspergillus flavus toxin immuno, with the affine sorbing material of the Aspergillus flavus toxin immuno Sample extracting solution is purified, enrichment aflatoxin (AFB1、AFB2、AFG1Or AFG2).Aspergillus flavus toxin immuno is affine to be inhaled Application of the enclosure material in aflatoxin, purification aflatoxin contamination material is removed.This processing is not controlled with medical diagnosis on disease For the purpose for the treatment of.When the carrier of immune absorption material is agarose gel microsphere and silica gel microball, method is：It is clear with pure water first Immune absorption material is washed, sample extracting solution is added, is then eluted with pure water, the aspergillus flavus poison of again with methanol elution specific adsorption Element, the eluent of collection, the sample extracting solution after as purifying and be enriched with, available for subsequent analysis detection.When carrier is magnetic bead When, method is：Immunomagnetic beads is added in pure water, magnetic frame reclaims magnetic bead, then repeated washing 3~5 times adds immunomagnetic beads Enter in sample extracting solution, mix, magnetic frame reclaims magnetic bead, after pure water is cleaned 1~3 time, the Huang of methanol elution specific adsorption is bent Mould toxin, the eluent of collection, the sample extracting solution after as purifying and be enriched with, available for subsequent analysis detection.
The present invention is single domain heavy chain antibody for the affine in immunity sorbing material aglucon of aflatoxin, with SEQ ID NO.:Amino acid sequence shown in 1, the aglucon can specific recognition aflatoxin.The single domain heavy chain antibody is readily available, can To produce aglucon as single domain heavy chain antibody by biological method mass propgation, it is to avoid the cumbersome production method such as artificial antibody, Greatly reduce production cost.Production process need not contact aflatoxin, it is to avoid to producers' actual bodily harm.With resistance to Soda acid, high temperature resistant and the characteristic such as it is readily produced, these characteristics are for the inexpensive, reusable immune of aflatoxin Learning detection method has important practical value.
Embodiment
Below by the preparation and application of the affine sorbing material of Aspergillus flavus toxin immuno, the present invention will be further described, These specific embodiments are not construed in any way as limiting the application of the present invention.
Embodiment 1：
The structure of aspergillus flavus resisting toxin single domain heavy chain antibody (i.e. for the single domain heavy chain antibody of aflatoxin) non-immune libraries Build
By aflatoxin B1With keyhole limpet hemocyanin (keyhole limpet hemocyanin, KLH) covalent coupling, Obtain aflatoxin artificial antigen AFB1- KLH, takes 300 μ g AFB1After-KLH is emulsified with Freund's complete adjuvant, to alpaca (Lama pacos) carries out subcutaneous multi-point injection and is immunized.Booster immunization uses 150 μ g AFB1- KLH and incomplete Freund's adjuvant breast Change, be spaced 2 weeks and carry out, venous blood sampling after being immunized 7 days every time determines serum titer using indirect elisa method, selects serum titer Highest sample separates lymphocyte, extracts RNA.
RNA extraction is carried out with reference to TAKARA company RNAiso reagents specification.Using RNA as template, oligo dT are to draw Thing, with reference to the TAKARA companies reverse transcriptase specification synthesis chains of cDNA first.
Using PrimeSTAR high-fidelity DNA polymerases, the variable region encoding gene for obtaining heavy chain antibody through nest-type PRC (is adopted 1) primer is shown in Table.First round PCR expands cDNA with primer AlpVh-LD and CH2-R respectively, and reaction condition is, 98 DEG C, 10s, 55 DEG C, 20s, 72 DEG C, 1min, 20 circulations, 98 DEG C, 10s, 68 DEG C, 1min, 72 DEG C of extension 10min.
By first round PCR primer with 1.2% agarose gel electrophoresis, 600bp~750bp DNA fragmentation is reclaimed, as Second wheel PCR template, respectively with primer AlpVh-SfiI and AlpVHHR1-NotI, AlpVh-SfiI and AlpVHHR2- NotI, is expanded, and reaction condition is, 98 DEG C, 10s, 50 DEG C, 20s, 72 DEG C, 40s, 5 circulations, 98 DEG C, 10s, 68 DEG C, 40s, 30 circulations, 72 DEG C of extension 10min.Reclaim, quantify through DNA fragmentation QIAquick Gel Extraction Kit, saved backup in -20 DEG C.It will bite Bacterium grain pHEN1 and pcr amplification product use Sfi I, Not I double digestions respectively, reclaimed through Ago-Gel, it is quantitative after, rubbed with 1: 3 You compare, and at 16 DEG C, connect overnight.
The library construction of table 1 and identification primer used
Note：Underscore represents restriction endonuclease recognition sequence
Connection product is dissolved in 10 μ L sterilized waters after ethanol precipitation, and Electroporation Transformation e. coli tg1 is carried out in ten times. Take 10 μ L electric shock, culture after bacterium solution doubling dilution, be coated with ampicillin 2 × YT culture plates, 37 DEG C, be inverted culture 12~ 16h, bacterium colony PCR is carried out using primer M13-R and pHEN-R, calculates storage capacity；Remainder is all coated on 24cm × 24cm ammonia Parasiticin 2 × YT culture plates, are inverted 12~16h of culture by 37 DEG C.With 10mL, 2 × YT culture mediums scrape the lawn on culture plate After washing, the glycerine of final concentration 15~30% is added, is dispensed, -80 DEG C save backup.
According to the storage capacity result of calculating, be inoculated with the living cells of 10 times of storage capacities in 20mL 2 × YT (contain 2% glucose, 100 μ g/mL ampicillins), 30 DEG C, 220r/min is cultivated to OD600 up to 0.5, and helper phage is added by infection multiplicity 20: 1 Body, 37 DEG C, 220r/min, 60min.Culture is centrifuged, (contains 100 μ g/mL ampicillins and 50 μ g/ with 50mL 2 × YT ML kanamycins) precipitation is resuspended, 30 DEG C, after 220r/min incubated overnights, 3000g centrifuging and taking supernatants add 5 × PEG/NaCl molten Liquid, places 1h or 4 DEG C overnight on ice, and 12000rpm centrifugation 30min, resuspension is deposited in the phosphate buffer containing 10% glycerine (PBS, 0.01M, pH 7.4), that is, obtain aspergillus flavus resisting toxin single domain heavy chain antibody non-immune libraries, takes 10 μ L to determine titre, remaining - 80 DEG C are sub-packed in save backup.
Embodiment 2：
The elutriation and identification of aspergillus flavus resisting toxin single domain heavy chain antibody
It is literary from the gained aspergillus flavus resisting toxin single domain heavy chain antibody non-immune libraries of embodiment 1 using the method for the affine elutriation of solid phase Elutriation is directed to the single domain heavy chain antibody of aflatoxin in storehouse.By AFB1With oralbumin (albumin, OVA) covalent coupling, Obtain artificial antigen AFB1-OVA.The artificial antigen AFB that 100 μ L PBS dilute is added per hole1- OVA, 4 DEG C, coating is stayed overnight, often The coating concentration for taking turns elutriation is respectively 100,75,50 μ g/mL；Coating buffer is suctioned out, PBS board-washings 3 times add 300 μ L 3% per hole BSA-PBS, closes 2h by 37 DEG C；PBS board-washings 6 times, add 100 μ L phage antibody libraries (containing about 2 × 1011CFU), 37 DEG C, incubate Educate 1.5h；Uncombined bacteriophage is suctioned out, with PBST (containing 0.5%Tween-20) board-washing 5 times (by wheel increase by 5 times), then PBS is used Board-washing 10 times (board-washing number of times is by wheel increase by 5 times)；With the elution absorption of 100 μ L eluents (glycine-HCI, pH2.2) in enzyme mark Bacteriophage in hole, eluate is neutralized with 50 μ L Tris-HCl (1mol/L, pH 8.0), takes 10 μ L to be used for titer determination, remaining It is used for next round elutriation after eluate amplification.Second wheel and third round elutriation use competitive elution, respectively with 50 and 25ng/mL's AFB1Solution is incubated 1h at 37 DEG C.
After three-wheel elutriation, the monoclonal of random picking is rescued using helper phage KM13, exhibition is respectively obtained Show the phage particle of antibody variable region, then bacteriophage is determined with indirect phage-ELISA and indirect competition phage-ELISA The binding activity and specificity of grain, experiment setting negative control and ground control, specific load procedure are shown in Table 2.
The indirect phage-ELISA of table 2 is loaded table
Send biotechnology service company to carry out sequencing ELISA positive colonies, obtain the DNA sequence dna of Insert Fragment, It encodes the single domain heavy chain antibody for aflatoxin, specific as follows：
G8(SEQ ID NO.:2):
CAGTTGCAGCTCGTGGAGTCAGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCA GCCTCTGGACGCACCGGCACAATCTATGGCATGGGCTGGTTCCGCGAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGC GACTCTTTGGTGGACTGTTGGTGCCCCATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCTAGAGACAACG ACAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCACGTATTACTGTGCATTAGATAAC CGCCGCAGTTATGTTGATTACCACTCCGTAAGTGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTC A
G4(SEQ ID NO.:4)：
CAGGTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTACGAGACTCTCCTGTGCA GCCTCTGGACGCACCGGCACAATCTATGGCATGGGCTGGTTCCGCGAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGC GACTATTTGGTGGACTGTTGGTGCCCCATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCTAGAGACAACG ACAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCATTTATTACTGTGCATTAGATAAC CGCCGCAGTTATGTTGATTACTACTCCGTAAGTGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTC A
D7：(SEQ ID NO.:6)：
CAGTTGCAGCTCGTGGAGTCCGGTGGAGGCTTGGTGCAGGTTGGGGGGTCTCTGAGACTCTCCTGTGCA GCCTCTGGACGCACCGGCACAATCTATGGCATGGGCTGGTTCCGCGAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGC AACTATTTGGTGGACTGTTGGTGCTCCATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCTAGAGACAACG CCAAGAACACGGTATATCTGCAAATGAATAGCCTGAAAGTTGAGGACACGGCCATTTATTACTGTGCATTAGATAAC CGCCGCAGTTATGTTAATTACTACTCCTCAAGTGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTC A
C6:(SEQ ID NO.:8)：
CAGGTGCAGCTCGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTACA GCCTCTGGACGCACCGGCACAATCTATGGCATGGGCTGGTTCCGCGAGGCTCCAGGGAAGGAGCGTGAGTTTGTTGC GACTATTTGGTGGACTGTTGGTGCCCCATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCTAGAGACAACG ACAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCATTTATTACTGTGCATTAGATAAT CGCCGCAGTTATGTTGATTACCACTCCGTAAGTGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTC A
H4:(SEQ ID NO.:10)：
CAGTTGCAGCTCGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTACA GCCTCTGGACGCACCGGCACAATCTATGGCATGGGCTGGTTCCGCGAGGCTCCAGGGAAGGAGCGTGAGTTTGTTGC GACTATTTGGTGGACTGTTGGTGCCCCATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCTAGAGACAACG ACAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCATTTATTACTGTGCATTAGATAAT CGCCGCAGTTATGTTGATTACCACTCCGTAAGTGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTC A
H8：(SEQ ID NO.:12)：
CAGGTGCAGCTCGTGGAGTCGGGGGGAGGAGCGGTGCAGGCTGGGGGCTCTTTGAGACTCTCCTGTGCA GCCTCTGGACGCACCGGCACAATCTATGGCATGGGCTGGTTCCGCGAGGCTCCAGGGAAGGAGCGTGAGTTTGTTGC GACTATTTGGTGGACTTTTGATGCCCCATACTATGCAGACTCCGTGAAGGGTCGATTCACCATCTCTAGAGACAACG ACAAGAACACGGTGTATCTACAAATGAACAACCTGAGCCCTGAGGACACGGCCATTTATTACTGTGCATTAGATAAT CGCCGCAGTTATGTTGATTACCGCTCCGTAAGTGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTC A
E12:(SEQ ID NO.:14)：
CAGTTGCAGCTCGTGGAGTCGGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTCACACTCTCCTGTGCA GCCTCTGGACGCACCTTCACAACGTATGGCATGGGCTGGTTCCGCGAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGC AACTATGTGGTGGACTGTTGGTGCCCCATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCACTAGAGACAGCG CCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGTTAGATACC CGCCGCAGTTATGTTGATTACCGCTCCTCAAGTGAGTATGATTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTC A
The ammonia of the corresponding single domain heavy chain antibody for aflatoxin can be obtained according to DNA sequencing result and password sublist Base acid sequence：
G8(SEQ ID NO.:1)：
QLQLVESGGGLVQAGDSLRLSCAASGRTGTIYGMGWFREAPGKEREFVATLWWTVGAPYYADSVKGRFT ISRDNDKNTVYLQMNSLKPEDTATYYCALDNRRSYVDYHSVSEYDYWGQGTQVTVSS
G4(SEQ ID NO.:3)：
QVQLVESGGGLVQAGGSTRLSCAASGRTGTIYGMGWFREAPGKEREFVATIWWTVGAPYYADSVKGRFT ISRDNDKNTVYLQMNSLKPEDTAIYYCALDNRRSYVDYYSVSEYDYWGQGTQVTVSS
D7：(SEQ ID NO.:5)：
QLQLVESGGGLVQVGGSLRLSCAASGRTGTIYGMGWFREAPGKEREFVATIWWTVGAPYYADSVKGRFT ISRDNAKNTVYLQMNSLKVEDTAIYYCALDNRRSYVNYYSSSEYDYWGQGTQVTVSS
C6:(SEQ ID NO.:7)：
QVQLVESGGGLVQAGGSLRLSCTASGRTGTIYGMGWFREAPGKEREFVATIWWTVGAPYYADSVKGRFT ISRDNDKNTVYLQMNSLKPEDTAIYYCALDNRRSYVDYHSVSEYDYWGQGTQVTVSS
H4:(SEQ ID NO.:9)：
QLQLVESGGGLVQAGGSLRLSCTASGRTGTIYGMGWFREAPGKEREFVATIWWTVGAPYYADSVKGRFT ISRDNDKNTVYLQMNSLKPEDTAIYYCALDNRRSYVDYHSVSEYDYWGQGTQVTVSS
H8:(SEQ ID NO.:11)：
QVQLVESGGGAVQAGGSLRLSCAASGRTGTIYGMGWFREAPGKEREFVATIWWTFDAPYYADSVKGRFT ISRDNDKNTVYLQMNNLSPEDTAIYYCALDNRRSYVDYRSVSEYDYWGQGTQVTVSS
E12:(SEQ ID NO.:13)：
QLQLVESGGGLVQPGGSLTLSCAASGRTFTTYGMGWFREAPGKEREFVATMWWTVGAPYYADSVKGRFT ITRDSAKNTVYLQMNSLKPEDTAVYYCALDTRRSYVDYRSSSEYDYWGQGTQVTVSS
Using indirect competition phage-ELISA methods to cross reaction of the positive colony from several different aflatoxin hypotypes Rate is measured, by AFB1、AFB2、AFG1、AFG2And AFM1Five kinds of standard items are diluted to 12 different working concentrations, same Under conditions of carry out indirect competition phage-ELISA measure, respectively draw competitive ELISA curve, calculate inhibiting rate be 50% when Standard concentration (IC50), according to formula：Cross reacting rate (%)=(AFB1IC50/ analog IC50) × 100%, the class It is AFB like thing2、AFG1、AFG2Or AFM1, obtain positive colony of the present invention (the single domain heavy chain antibody for being directed to aflatoxin) right In AFB150% inhibition concentration.As a result show, positive colony (the single domain heavy chain antibody for being directed to aflatoxin) of the present invention is right In AFB1With preferable specificity, to AFG1And AFG2Also there is certain binding ability.
Embodiment 3：
It is prepared by the scale of aspergillus flavus resisting toxin single domain heavy chain antibody
Encode anti-AFB1The acquisition of the DNA fragmentation of single domain heavy chain antibody：1. it is double using restriction enzyme SfiI/NotI The anti-AFB of digestion phasmid pHEN-1Single domain heavy chain antibody genes, agarose gel electrophoresis reclaims anti-AFB1Single domain heavy chain antibody base Cause；2. directly by anti-AFB1Single domain heavy chain antibody coded sequence send biotechnology service company to carry out chemical synthesis；3. design is special Expanded in specific primer, the cDNA storehouses originated by round pcr from alpaca (Lama pacos).
By obtained anti-AFB1Single domain heavy chain antibody genes fragment is cloned into expression vector pET25, is reflected through PCR and digestion It is fixed, build and complete anti-AFB1The colibacillus expression plasmid of single domain heavy chain antibody.
Expression plasmid is converted to e. coli bl21, picking single bacterium colony carries out induced expression.Single bacterium colony is accessed into 4mL In LBA (the μ g/mL ampicillin of Luria-Bertani broth with 100) fluid nutrient medium, 37 DEG C, 250r/min shakes Swing culture 12h；It is transferred to the inoculum concentration of 1% culture volume in 50mL LBA fluid nutrient mediums, 37 DEG C, 250r/min Shaken cultivation is to OD6000.5 (about needing 2.5~3h) is reached, final concentration 0.1mM IPTG, 30 DEG C, 200r/min induction trainings is added Support.
Induced cultures 8000r/min is centrifuged, and 20mL phosphate buffers (pH 7.4) are added in cell precipitation and are mixed, 8000r/min is centrifuged, and removes supernatant, retains cell precipitation；10mL same buffers are added in cell precipitation, mixes, surpasses on ice Sound wave clasmatosis is handled, and ultrasonication condition is 200W, crushes 2s, interval 3s, totally 240 circulations, broken to cell at 4 DEG C The 12000r/min that minces centrifuges 20min, takes supernatant to carry out affinitive layer purification and SDS-PAGE electrophoretic analysis, or add in supernatant Enter the glycerine of final concentration 30%, mix, be stored in -20 DEG C of refrigerator-freezers stand-by.
By optimizing induced expression condition (such as Host Strains, expression vector, Fiber differentiation time, temperature and IPTG concentration Deng), destination protein (single domain heavy chain antibody) expression quantity can be further improved, is largely to prepare anti-AFB1Single domain heavy chain antibody is carried Approach is supplied.
A small amount of preparations of the Aspergillus flavus toxin immuno of embodiment 4 is affine magnetic bead
Using nanometer magnetic bead as carrier, it is coupled after aspergillus flavus resisting single domain heavy chain antibody, obtains Aspergillus flavus toxin immuno magnetic Pearl, specific preparation method is as follows：
The magnetic bead (purchased from the fortune nanosecond science and technology Co., Ltd of Wuxi hundred, carboxyl magnetic bead 300nm) of 1mg carboxyl modifieds is taken in centrifugation Guan Zhong, adds 500 μ l activation buffers (10mM, NaH2PO4, pH 6.0), vortex mixed is uniform, and magnetic frame reclaims magnetic bead, then uses Activation buffer is washed 2 times.It is separately added into after 2mg carbodiimides (EDC) and n-hydroxysuccinimide (NHS), vortex mixed, Stand 30min.With coupling buffer (10mM, Na2HPO4, pH 7.4) and wash magnetic bead 3 times, add and be dissolved in the anti-of coupling buffer Aflatoxin single domain heavy chain antibody 1mg, reacts at room temperature 3h, magnetic bead is washed with coupling buffer 3 times, adds 500 μ l and contains 1% (w/v) coupling buffer of bovine serum albumin(BSA) (BSA) or 1% (w/v) ovalbumin (OVA) closes unreacted active group Group, reacts at room temperature 30min.Magnetic bead is washed with coupling buffer 3 times, PBS solution (10mM, pH7.4,0.02%w/v, Na3N) weight 4 DEG C are stored in after outstanding.
The preparation of the Aspergillus flavus toxin immuno of embodiment 5 is affine sorbing material and affinity column
Using agarose microbeads as carrier, aspergillus flavus resisting single domain heavy chain antibody is coupled, specific preparation method is as follows：
The dry glue that CNBr is activated is washed 10 times with 0.1M HCl, and 5min is balanced every time.With coupling buffer (10mM, Na2HPO4, pH 7.4) wash 10 times, add aspergillus flavus resisting toxin single domain heavy chain antibody (every gram of agarose microbeads of 2mg/), room temperature 4h is reacted, the agarose gel microsphere covalent coupling for making aspergillus flavus resisting toxin single domain heavy chain antibody be activated with CNBr.It is slow with coupling Fliud flushing (10mM, Na2HPO4, pH 7.4) and after washing 2 times, confining liquid room temperature reaction 2h is added to close unreacted active group. With alternately washing 3 times of 5 phosphate buffers (10mM, pH 7.4) accumulated by colloid and acetate buffer solution (0.1M, pH 4.0), obtain The covalent coupling affine in immunity sorbing material of aspergillus flavus resisting toxin single domain heavy chain antibody.Take the above-mentioned affine in immunity absorption of 0.2ml Material adds 20% ethanol in after the chromatographic column that capacity is 1ml, PBS (10mM, the pH 7.4) washings of 5~10 times of bed volumes Solution, 4 DEG C of preservations.
The preparation of the Aspergillus flavus toxin immuno of embodiment 6 is affine sorbing material and affinity column
Using silica gel microball as carrier, aspergillus flavus resisting single domain heavy chain antibody is coupled, specific preparation method is as follows：
Alternately washing 5~10 times of 2g silica gel microballs pure water and phosphate buffer (PBS, 10mM, pH 6.0) are taken, 10ml is used PBS suspension silica gel microball, adds 5mg aspergillus flavus resisting toxin single domain heavy chain antibodies, mixes, and adds final concentration 5mg/ml's Carbodiimide (EDC), rapid to mix, 4 DEG C of 12~24h of stirring reaction obtain covalent coupling aspergillus flavus resisting toxin single domain heavy chain The affine in immunity sorbing material of antibody.The above-mentioned affine in immunity sorbing materials of 0.2ml are taken in the chromatographic column that capacity is 1ml, 5~10 times After PBS (10mM, the pH 6) washings of bed volume, add and contain 0.02% (w/v) Na3N PBS (10mM, pH 6), 4 DEG C of preservations.
The Aspergillus flavus toxin immuno-affinity column column capacity of embodiment 7, recovery of standard addition, reuse are determined
Affinity column prepared by embodiment 5 or embodiment 6 is washed with 5ml PBS (10mM, pH 7.4), adds 5ml Concentration is 100ng/ml AFB1Standard solution, 10ml pure water elution, is eluted with 1ml methanol, collects the efficient liquid of eluent AFB in phase chromatogram detection eluent1Content, calculate affinity column capacity be 650~800ng.
Peanut, corn sample (being free of aflatoxin) that 5g is crushed are taken, 50ng, 100ng and 200ng is separately added into AFB1Or AFG1Standard items, sample, vortex oscillation 15min, fast qualitative filter paper mistake are extracted with 60% (v/v) methanol-water solution Filter, takes 5ml filtrates, plus PBS (10mM, pH 7.4) dilutions.
Affinity column prepared by embodiment 5 or embodiment 6 is washed with 5ml PBS (10mM, pH 7.4), adds 10ml Sample extraction dilution, 10ml pure water elution, is eluted with 1ml methanol, collects eluent high performance liquid chromatography detection AFB1Or AFG1Content, calculate the rate of recovery.As a result show the affinity column of preparation to AFB1Or AFG1Average recovery rate be respectively 85~ 101%, 80~97%.After reusing 10 times, affinity column is to AFB1Or AFG1Average recovery rate>80%.
SEQUENCE LISTING
<110>University Of Nanchang
<120>A kind of aflatoxin single domain heavy chain antibody affine in immunity sorbing material
<130> 2015
<160> 21
<170> PatentIn version 3.3
<210> 1
<211> 125
<212> PRT
<213>Artificial sequence
<400> 1
Gln Val Gln Leu Val Glu Asn Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Ile Tyr Ser Leu Val
20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ala Asp Ile Thr Arg Val Gly Asn Thr Asn His Ala Asn Ser Arg Glu
50 55 60
Asp Arg Phe Thr Ile Ser Thr Gly Val Pro Trp Asn Thr Val Ile Leu
65 70 75 80
Ser Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Arg Thr Leu Ser Arg Val Leu Gly Thr Lys Arg Asp Glu Tyr
100 105 110
Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 2
<211> 375
<212> DNA
<213>Artificial sequence
<400> 2
caggtgcagc tcgtggagta ggggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtacag cctctggaag catctatagc ctcgttgcca tgggctggta ccgccaggct 120
ccagggaagc agcgcgagtg ggtcgcagat attactcgtg ttggtaacac aaaccatgcg 180
aactccaggg aggaccgatt caccatctcg acaggtgtcc cctggaacac ggtgattctg 240
tctatgaaca gcctggaacc tgaggacacg gccgtctatt actgtgcagc acgtcggacg 300
ctctcacggg tacttggcac gaagagagat gagtataact actggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 3
<211> 126
<212> PRT
<213>Artificial sequence
<400> 3
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Thr Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Gly Thr Ile Tyr
20 25 30
Gly Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Trp Trp Thr Val Gly Ala Pro Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Leu Asp Asn Arg Arg Ser Tyr Val Asp Tyr Tyr Ser Val Ser Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 4
<211> 378
<212> DNA
<213>Artificial sequence
<400> 4
caggtgcagc tcgtggagtc tgggggagga ttggtgcagg ctgggggctc tacgagactc 60
tcctgtgcag cctctggacg caccggcaca atctatggca tgggctggtt ccgcgaggct 120
ccagggaagg agcgtgagtt tgtagcgact atttggtgga ctgttggtgc cccatactat 180
gcagactccg tgaagggccg attcaccatc tctagagaca acgacaagaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccattt attactgtgc attagataac 300
cgccgcagtt atgttgatta ctactccgta agtgagtatg actactgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 5
<211> 126
<212> PRT
<213>Artificial sequence
<400> 5
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Val Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Gly Thr Ile Tyr
20 25 30
Gly Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Trp Trp Thr Val Gly Ala Pro Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Val Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Leu Asp Asn Arg Arg Ser Tyr Val Asn Tyr Tyr Ser Ser Ser Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 6
<211> 378
<212> DNA
<213>Artificial sequence
<400> 6
cagttgcagc tcgtggagtc cggtggaggc ttggtgcagg ttggggggtc tctgagactc 60
tcctgtgcag cctctggacg caccggcaca atctatggca tgggctggtt ccgcgaggct 120
ccagggaagg agcgtgagtt tgtagcaact atttggtgga ctgttggtgc tccatactat 180
gcagactccg tgaagggccg attcaccatc tctagagaca acgccaagaa cacggtatat 240
ctgcaaatga atagcctgaa agttgaggac acggccattt attactgtgc attagataac 300
cgccgcagtt atgttaatta ctactcctca agtgagtatg actactgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 7
<211> 126
<212> PRT
<213>Artificial sequence
<400> 7
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Gly Thr Ile Tyr
20 25 30
Gly Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Trp Trp Thr Val Gly Ala Pro Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Leu Asp Asn Arg Arg Ser Tyr Val Asp Tyr His Ser Val Ser Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 8
<211> 378
<212> DNA
<213>Artificial sequence
<400> 8
caggtgcagc tcgtggagtc ggggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtacag cctctggacg caccggcaca atctatggca tgggctggtt ccgcgaggct 120
ccagggaagg agcgtgagtt tgttgcgact atttggtgga ctgttggtgc cccatactat 180
gcagactccg tgaagggccg attcaccatc tctagagaca acgacaagaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccattt attactgtgc attagataat 300
cgccgcagtt atgttgatta ccactccgta agtgagtatg actactgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 9
<211> 126
<212> PRT
<213>Artificial sequence
<400> 9
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Gly Thr Ile Tyr
20 25 30
Gly Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Trp Trp Thr Val Gly Ala Pro Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Leu Asp Asn Arg Arg Ser Tyr Val Asp Tyr His Ser Val Ser Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 10
<211> 378
<212> DNA
<213>Artificial sequence
<400> 10
cagttgcagc tcgtggagtc ggggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtacag cctctggacg caccggcaca atctatggca tgggctggtt ccgcgaggct 120
ccagggaagg agcgtgagtt tgttgcgact atttggtgga ctgttggtgc cccatactat 180
gcagactccg tgaagggccg attcaccatc tctagagaca acgacaagaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccattt attactgtgc attagataat 300
cgccgcagtt atgttgatta ccactccgta agtgagtatg actactgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 11
<211> 126
<212> PRT
<213>Artificial sequence
<400> 11
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Gly Thr Ile Tyr
20 25 30
Gly Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Trp Trp Thr Phe Asp Ala Pro Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Ser Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Leu Asp Asn Arg Arg Ser Tyr Val Asp Tyr Arg Ser Val Ser Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 12
<211> 378
<212> DNA
<213>Artificial sequence
<400> 12
caggtgcagc tcgtggagtc ggggggagga gcggtgcagg ctgggggctc tttgagactc 60
tcctgtgcag cctctggacg caccggcaca atctatggca tgggctggtt ccgcgaggct 120
ccagggaagg agcgtgagtt tgttgcgact atttggtgga cttttgatgc cccatactat 180
gcagactccg tgaagggtcg attcaccatc tctagagaca acgacaagaa cacggtgtat 240
ctacaaatga acaacctgag ccctgaggac acggccattt attactgtgc attagataat 300
cgccgcagtt atgttgatta ccgctccgta agtgagtatg actactgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 13
<211> 126
<212> PRT
<213>Artificial sequence
<400> 13
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr Tyr
20 25 30
Gly Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Met Trp Trp Thr Val Gly Ala Pro Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Thr Arg Asp Ser Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Asp Thr Arg Arg Ser Tyr Val Asp Tyr Arg Ser Ser Ser Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 14
<211> 378
<212> DNA
<213>Artificial sequence
<400> 14
cagttgcagc tcgtggagtc ggggggaggc ttggtgcagc ctggggggtc tctcacactc 60
tcctgtgcag cctctggacg caccttcaca acgtatggca tgggctggtt ccgcgaggct 120
ccagggaagg agcgtgagtt tgtagcaact atgtggtgga ctgttggtgc cccatactat 180
gcagactccg tgaagggccg attcaccatc actagagaca gcgccaagaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc gttagatacc 300
cgccgcagtt atgttgatta ccgctcctca agtgagtatg attactgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 15
<211> 18
<212> DNA
<213>Artificial primer
<400> 15
cttggtggtc ctggctgc 18
<210> 16
<211> 49
<212> DNA
<213>Artificial primer
<220>
<221> misc_feature
<222> (44)..(44)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (47)..(47)
<223> n is a, c, g, or t
<400> 16
tcgcggccca gccggccatg gcccagktgc agctcgtgga gtcnggngg 49
<210> 17
<211> 33
<212> DNA
<213>Artificial primer
<400> 17
cgagtgcggc cgcggggtct tcgctgtggt gcg 33
<210> 18
<211> 34
<212> DNA
<213>Artificial primer
<400> 18
cgagtgcggc cgcttgtggt tttggtgtct tggg 34
<210> 19
<211> 23
<212> DNA
<213>Artificial primer
<400> 19
ggtacgtgct gttgaactgt tcc 23
<210> 20
<211> 24
<212> DNA
<213>Artificial primer
<400> 20
agcggataac aatttcacac agga 24
<210> 21
<211> 20
<212> DNA
<213>Artificial primer
<400> 21
gccccattca gatcctcttc 20

Claims (7)

1. a kind of affine sorbing material of Aspergillus flavus toxin immuno, including carrier, are mounted in the aglucon on carrier, it is characterised in that should Material is using the single domain heavy chain antibody of specific recognition aflatoxin as aglucon, the list of the specific recognition aflatoxin Domain heavy chain antibody has SEQ ID NO.:Amino acid sequence shown in 13.
2. the affine sorbing material of Aspergillus flavus toxin immuno according to claim 1, it is characterised in that the aglucon be SEQ ID NO.:On the basis of amino acid sequence shown in 13 by random or site-directed mutagenesis technique carry out house of correction acquisition can be with The antibody of aflatoxin specific binding.
3. the affine sorbing material of Aspergillus flavus toxin immuno according to claim 1, it is characterised in that the carrier is magnetic Pearl.
4. the affine sorbing material of Aspergillus flavus toxin immuno according to claim 1, it is characterised in that the carrier is agar Sugared gel or silica gel microball or porous material.
5. it is mounted with the affinity column of the affine sorbing material of Aspergillus flavus toxin immuno described in claim 1.
6. the affine sorbing material of Aspergillus flavus toxin immuno according to claim 1 is in Aspergillus flavus toxin immuno detection, enrichment And the application in purifying.
7. the affine sorbing material of Aspergillus flavus toxin immuno according to claim 1 is removing aflatoxin, the yellow song of purification Application in mycotoxin contamination material.
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CN201710265811.8A Pending CN107308915A (en) 2015-12-02 2015-12-02 Based on specific bond AFB1The affine sorbing material of nano antibody
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