CN105399821A - Single-domain heavy-chain antibody for aflatoxin - Google Patents
Single-domain heavy-chain antibody for aflatoxin Download PDFInfo
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- CN105399821A CN105399821A CN201510866664.0A CN201510866664A CN105399821A CN 105399821 A CN105399821 A CN 105399821A CN 201510866664 A CN201510866664 A CN 201510866664A CN 105399821 A CN105399821 A CN 105399821A
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- aflatoxin
- chain antibody
- domain heavy
- heavy chain
- single domain
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Abstract
The invention belongs to the technical field of biology, and relates to a single-domain heavy-chain antibody technology, in particular to a single-domain heavy-chain antibody for aflatoxin. The single-domain heavy-chain antibody has an amino acid sequence shown in SEQ ID NO.: 1. The antibody has the advantages of being acid and alkali resistant, high-temperature resistant and easy to produce. Significant practical value is achieved for an aflatoxin immunological detection method which is low in cost and capable of being used repeatedly.
Description
Technical field
The present invention relates to single domain heavy chain antibody technology (also known as nano antibody technology), and genetic engineering antibody technology, particularly for single domain heavy chain antibody or the polypeptide of aflatoxin.
Technical background
Aflatoxin (Aflatoxin) is by mycetogenetic secondary metabolites such as flavus (Aspergillusflavus), Aspergillus parasiticus (Aspergillusparasiticus), collection honeybee aspergillus (Aspergillusnomius) and Aspergillus tamariis (Aspergillustamarii), has strong carinogenicity and strong inhibitive ability of immunity.Aflatoxin is the two furocoumarin(e) derivatives that a class chemical structure is similar, mainly comprises AFB
1(AFB
1), B
2(AFB
2), G
1(AFG
1), G
2(AFG
1) and M
1(AFM
1) etc.In the aflatoxin found, AFB
1toxicity the strongest, delimited as I class carcinogens by the Agency for Research on Cancer of the World Health Organization.The agricultural-food such as Toxigenic fungi bacterial strain is extensively present in occurring in nature, grain oil plant all may be polluted in the processes such as production, processing and storage, cause aflatoxin to exceed standard.China is the area that aflatoxin contamination situation is comparatively serious.Therefore, develop stable, quick, highly sensitive, high-throughout Determination Methods of Aflatoxins, significant for the loss ensureing China's food safety, minimizing brings thus.
Existing Determination Methods of Aflatoxins mainly contains immunoassay, instrumental method and thin layer chromatography.Wherein immunoassay can avoid some defects that other two class methods exist, have highly sensitive, cost is low and can the advantage such as Site Detection.Immunoassay ratio juris is the specific recognition based on antigen-antibody, and the performance of antibody is the deciding factor of immune analysis method sensitivity, accuracy index.Therefore the antibody obtained for aflatoxin is prerequisite and the key of setting up related immunological detection technique.
Single domain heavy chain antibody (also known as nano antibody) is made up of the variable region of alpaca heavy chain antibody, is also called nano antibody, has acid and alkali-resistance, high temperature resistant and be easy to the characteristics such as production.These characteristics have important practical value for the low cost of aflatoxin, reusable immunological detection method.
Summary of the invention
The object of this invention is to provide the single domain heavy chain antibody for aflatoxin, reagent and the instrument of preparing detection aflatoxin can be used to.
The invention provides one for the single domain heavy chain antibody of aflatoxin, there is the aminoacid sequence shown in SEQIDNO.:1.The IMGT numbering of its aminoacid sequence and the division of structural domain comprise four framework regions (Frameworkregion, FR) and three complementary determining regions (Complementarity-determiningregion, CDR).
The invention provides a nucleic acid molecule, it is characterized in that the SEQIDNO.:1 that encodes, the concrete sequence of this nucleic acid molecule can be obtained by genetic codon at any time.
The present invention also provides a nucleic acid molecule, it is characterized in that coding SEQIDNO.:1 partial domain, can be obtained the concrete sequence of this nucleic acid molecule by genetic codon at any time.It can be SEQIDNO.:2 nucleic acid molecule.
Nucleotide sequence provided by the present invention or at least partly sequence can be undertaken expressing to obtain corresponding protein or polypeptide by suitable expression system.These expression systems comprise bacterium, yeast, filamentous fungus, zooblast, insect cell, vegetable cell, or Cell free expression system.
The present invention also provides a kind of carrier, comprises described nucleotide sequence.Because genetic codon has degeneracy, this nucleotide sequence can be different according to different application purposes.
The present invention also provides a kind of host cell, comprises described protein or expression vector.
The present invention also provides a kind of method detecting aflatoxin, containing the single domain heavy chain antibody for aflatoxin of the present invention.Based on provided by the invention for the single domain heavy chain antibody of aflatoxin and the ability of aflatoxin specific binding, set up the detection method of aflatoxin.Wherein, preferred method comprises enzyme and connects immunoabsorption (Enzyme-linkedimmunosorbentassay, ELISA), fluorescent immune method (Fluoroimmunoassay, FIA), immuno-chip method, affinity chromatography and immunochromatographic method etc.
Aminoacid sequence provided by the present invention as precursor, can be transformed by random or site-directed mutagenesis technique, can obtain character (water-soluble, stability, avidity and specificity etc.) better mutant.
The invention still further relates to the aforementioned application of single domain heavy chain antibody in immunodetection, aflatoxin enrichment and purifying for aflatoxin.These immunodetection refer to the immunodetection of non-diseases diagnoses and treatment object.
The present invention some terms of describing there is following implication:
Structural domain: the fundamental structural unit of tertiary protein structure, has certain function usually.
IMGT numbers: the one in IMGT database (TheInternationalImMunoGeneTicsDatbase) is standardized antibody amino acids sequence method for numbering serial.Concrete method for numbering serial can reference (Ehrenman, F., Q.Kaas, et.al. (2010) .IMGT/3Dstructure-DBandIMGT/DomainGapAlign:adatabaseanda toolforimmunoglobulinsorantibodies, Tcellreceptors, MHC, IgSFandMhcSF.NucleicAcidsRes38 (Databaseissue): D301-307.Lefranc, M.P., C.Pommie, etal. (2003) .IMGTuniquenumberingforimmunoglobulinandTcellreceptorvar iabledomainsandIgsuperfamilyV-likedomainsDevcompImmunol2 7 (1): 55-77.) in description.
Codon (codon): be also called three disjunctor codons (tripletcode), refers to correspond to certain amino acid whose nucleotide triplet.In the process of translating, determine that this seed amino acid inserts the position of polypeptide chain in growth.
Embodiment
Below by preparation, the Analyzes and nurses of single domain heavy chain antibody (polypeptide), the present invention will be further described, and these specific embodiments should not be interpreted as limiting range of application of the present invention by any way.
Embodiment 1:
The structure of aspergillus flavus resisting toxin single domain heavy chain antibody (namely for the single domain heavy chain antibody of aflatoxin) non-immune libraries
By AFB
1with keyhole limpet hemocyanin (keyholelimpethemocyanin, KLH) covalent coupling, obtain aflatoxin artificial antigen AFB
1-KLH, gets 300 μ gAFB
1after-KLH and Freund's complete adjuvant emulsification, the immunity of subcutaneous multi-point injection is carried out to alpaca (Lamapacos).Booster immunization adopts 150 μ gAFB
1-KLH and Freund's incomplete adjuvant emulsification, interval is carried out for 2 weeks, and each immunity 7 days posterior veins get blood, adopts indirect elisa method to measure serum titer, selects the sample separation lymphocyte that serum titer is the highest, extract RNA.
The extraction of RNA is carried out with reference to TAKARA company RNAiso reagent specification sheets.Take RNA as template, oligodT is primer, with reference to TAKARA company ThermoScript II specification sheets synthesis cDNA first chain.
Adopt PrimeSTAR high-fidelity DNA polymerase, obtain the variable region encoding gene (primer of employing is in table 1) of heavy chain antibody through nest-type PRC.First round PCR is respectively with primer AlpVh-LD and CH2-R amplification cDNA, and reaction conditions is, 98 DEG C, 10s, 55 DEG C, 20s, 72 DEG C, 1min, 20 circulations, 98 DEG C, 10s, 68 DEG C, 1min, and 72 DEG C extend 10min.
By the first round PCR primer agarose gel electrophoresis of 1.2%, reclaim the DNA fragmentation of 600bp ~ 750bp, take turns the template of PCR as second, use primer AlpVh-SfiI and AlpVHHR1-NotI respectively, AlpVh-SfiI and AlpVHHR2-NotI, increases, and reaction conditions 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 extend 10min.Reclaim test kit through DNA fragmentation to reclaim, quantitatively, save backup in-20 DEG C.Phagemid pHEN1 and pcr amplification product are used SfiI, NotI double digestion respectively, reclaim through sepharose, quantitatively after, with 1: 3 mol ratio, at 16 DEG C, connection of spending the night.
Table 1 library construction and identify primer used
Note: underscore represents restriction endonuclease recognition sequence
Connect product after alcohol settling, be dissolved in 10 μ L sterilized waters, divide and carry out Electroporation Transformation e. coli tg1 ten times.Get the bacterium liquid doubling dilution after 10 μ L electric shocks, cultivation, coating penbritin 2 × YT culture plate, 37 DEG C, is inverted cultivation 12 ~ 16h, adopts primer M13-R and pHEN-R to carry out bacterium colony PCR, calculating storage capacity; Rest part all coats 24cm × 24cm penbritin 2 × YT culture plate, 37 DEG C, is inverted cultivation 12 ~ 16h.With 10mL, after the lawn on culture plate scrapes by 2 × YT substratum, add final concentration 15 ~ 30% glycerine, packing ,-80 DEG C save backup.
According to the storage capacity result calculated, the viable cell of inoculation 10 times of storage capacities in the 2 × YT of 20mL (containing 2% glucose, 100 μ g/mL penbritins), 30 DEG C, 220r/min is cultured to OD600 and reaches 0.5, adds helper phage by infection multiplicity 20: 1,37 DEG C, 220r/min, 60min.Culture is centrifugal, by 2 × YT (containing 100 μ g/mL penbritins and 50 μ g/mL kantlex) the resuspended precipitation of 50mL, 30 DEG C, after 220r/min incubated overnight, 3000g centrifuging and taking supernatant, add 5 × PEG/NaCl solution, place 1h or 4 DEG C on ice to spend the night, the centrifugal 30min of 12000rpm, resuspended phosphoric acid buffer (PBS, the 0.01M be deposited in containing 10% glycerine, pH7.4), namely obtain aspergillus flavus resisting toxin single domain heavy chain antibody non-immune libraries, get 10 μ L and measure titre, all the other are sub-packed in-80 DEG C and save backup.
Embodiment 2:
The elutriation of aspergillus flavus resisting toxin single domain heavy chain antibody and qualification
Adopt the method for solid phase affine elutriation from embodiment 1 gained aspergillus flavus resisting toxin single domain heavy chain antibody non-immune libraries library elutriation for the single domain heavy chain antibody of aflatoxin.By AFB
1with oralbumin (albumin, OVA) covalent coupling, obtain artificial antigen AFB
1-OVA.Every hole adds the artificial antigen AFB that 100 μ L PBS dilute
1-OVA, 4 DEG C, wrap and spent the night, the bag of often taking turns elutriation is respectively 100 by concentration, 75,50 μ g/mL; Sucking-off coating buffer, PBS washes plate 3 times, and every hole adds 300 μ L3%BSA-PBS, 37 DEG C, closes 2h; PBS washes plate 6 times, adds 100 μ L phage antibody libraries (about containing 2 × 10
11cFU), 37 DEG C, 1.5h is hatched; The unconjugated phage of sucking-off, washes plate 5 times (by wheel increase by 5 times) with PBST (containing 0.5%Tween-20), then washes plate 10 times (washing plate number of times by wheel increase by 5 times) with PBS; With 100 μ L elutriant (glycine-HCI, pH2.2) wash-out is adsorbed on the phage in enzyme mark hole, with in 50 μ LTris-HCl (1mol/L, pH8.0) and eluate, get 10 μ L for titer determination, for next round elutriation after the amplification of all the other eluates.Second takes turns and third round elutriation employing competitive elution, uses the AFB of 50 and 25ng/mL respectively
1solution, at 37 DEG C, hatches 1h.
After three-wheel elutriation, the mono-clonal of helper phage KM13 to random picking is adopted to rescue, obtain the phage particle showing antibody variable region respectively, binding activities and the specificity of phage particle is measured again with indirect phage-ELISA and indirect competition phage-ELISA, experiment setting negative control and ground control, concrete load procedure is in table 2.
Table 2 is phage-ELISA application of sample table indirectly
Send biotechnology service company to carry out sequencing ELISA positive colony, obtain the DNA sequence dna of Insert Fragment, it encodes for the single domain heavy chain antibody of aflatoxin, (SEQIDNO.:2) specific as follows:
CAGTTGCAGCTCGTGGAGTCGGGTGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGAAGCATCTATAGCCTCGTTGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTGGGTCGCAGATATTACTCGTGTTGGTAACACAAACCATGCGAACTCCAGGGAGGACCGATTCACCATCTCGACAGGTGTCCCCTGGAACACGGTGATTCTGTCTATGAACAGCCTGGAACCTGAGGACACGGCCGTCTATTATTGTAATGCCCGAGTTACCTCCGGGCTTAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
The aminoacid sequence (SEQIDNO.:1) for the single domain heavy chain antibody of aflatoxin can be obtained according to DNA sequencing result and password sublist:
QLQLVESGGGLVQPGGSLRLSCTASGSIYSLVAMGWYRQAPGKQREWVADITRVGNTNHANSREDRFTISTGVPWNTVILSMNSLEPEDTAVYYCNARVTSGLNYWGQGTQVTVSS
Indirect competition phage-ELISA method is adopted to measure, by AFB the cross reacting rate of positive colony from several different aflatoxin hypotype
1, AFB
2, AFG
1, AFG
2and AFM
1five kinds of standard substance are diluted to 12 different working concentrations, carry out indirect competition phage-ELISA mensuration under identical condition, draw competitive ELISA curve respectively, standard concentration (IC when to calculate inhibiting rate be 50%
50), according to formula: cross reacting rate (%)=(AFB
1iC
50/ analogue IC
50) × 100%, described analogue is AFB
2, AFG
1, AFG
2or AFM
1, obtain positive colony of the present invention (the single domain heavy chain antibody for aflatoxin) for AFB
150% inhibition concentration.Result shows, positive colony of the present invention (the single domain heavy chain antibody for aflatoxin) is for AFB
1there is good specificity, to AFG
1and AFG
2also certain binding ability is had.
Embodiment 3:
The scale preparation of aspergillus flavus resisting toxin single domain heavy chain antibody
Encode anti-AFB
1the acquisition of the DNA fragmentation of single domain heavy chain antibody: 1. adopt restriction enzyme SfiI/NotI, the anti-AFB of double digestion phagemid pHEN-
1single domain heavy chain antibody genes, agarose gel electrophoresis reclaims anti-AFB
1single domain heavy chain antibody genes; 2. directly by anti-AFB
1single domain heavy chain antibody encoding sequence send biotechnology service company to carry out chemosynthesis; 3. design Auele Specific Primer, increased from the cDNA storehouse that alpaca (Lamapacos) is originated by round pcr.
By the anti-AFB obtained
1single domain heavy chain antibody genes fragment is cloned into expression vector pET25, cuts qualification through PCR and enzyme, has built anti-AFB
1the colibacillus expression plasmid of single domain heavy chain antibody.
Expression plasmid is converted into e. coli bl21, and picking list bacterium colony carries out abduction delivering.By in single bacterium colony access 4mLLBA (Luria-Bertanibrothwith100 μ g/mLampicillin) liquid nutrient medium, 37 DEG C, 250r/min shaking culture 12h; Be transferred in 50mLLBA liquid nutrient medium with the inoculum size of 1% culture volume, 37 DEG C, 250r/min shaking culture is to OD
600reach 0.5 (about needing 2.5 ~ 3h), add the IPTG of final concentration 0.1mM, 30 DEG C, 200r/min inducing culture.
Induced cultures 8000r/min is centrifugal, and in cell precipitation, add 20mL phosphoric acid buffer (pH7.4) mixing, 8000r/min is centrifugal, removes supernatant, retains cell precipitation; 10mL same buffer is added in cell precipitation, mixing, ultrasonic cell-break process on ice, ultrasonication condition is 200W, broken 2s, interval 3s, totally 240 circulations, to the centrifugal 20min of cytoclasis thing 12000r/min at 4 DEG C, get supernatant and carry out affinitive layer purification and SDS-PAGE electrophoretic analysis, or in supernatant, adding the glycerine of final concentration 30%, mixing, is stored in-20 DEG C of refrigerator-freezers stand-by.
By optimizing abduction delivering condition (as Host Strains, expression vector, inducing culture time, temperature and IPTG concentration etc.), target protein (single domain heavy chain antibody) expression amount can be improved further, for preparing anti-AFB in a large number
1single domain heavy chain antibody provides approach.
Embodiment 4:
The amalgamation and expression of aspergillus flavus resisting toxin single domain heavy chain antibody
By anti-for the present invention AFB
1single domain heavy chain antibody genes is cloned into fusion expression vector pAP, cuts qualification through PCR and enzyme, has built anti-AFB
1the alkaline phosphatase fusion expression plasmid of single domain heavy chain antibody.
Alkaline phosphatase non-specific catalytic phosphatase monoesters hydrolysis can generate inorganic phosphate and corresponding alcohol, phenol or saccharide compound.This enzyme Chang Zuowei signal label is used for the detection methods such as ELISA, immunoblotting, histological chemistry.Fusion expression plasmid is by anti-AFB
1single domain heavy chain antibody is blended in the N end of alkaline phosphatase, with reference to the expression method in application example 2, can at expression in escherichia coli, be purified into the anti-AFB of fusion rotein AP-
1single domain heavy chain antibody genes.
Embodiment 5:
Aspergillus flavus resisting toxin single domain heavy chain antibody is used for AFB
1detection
Preparation of samples: take not containing peanut, each three parts of the corn sample of aflatoxin, add AFB respectively
1standard substance 10 μ g/kg, 50 μ g/kg, 100 μ g/kg, with the methyl alcohol-centrifugal 10min of PBS solution vortex oscillation 15min, 9000g of 25mL60%, supernatant is stand-by after PBS dilution.
Indirect competitive enzyme-linked immunosorbent detects:
With PBS (0.01M, pH7.4) by AFB
1artificial antigen is diluted to 0.25 μ g/mL, 100 μ L/ holes, be coated in enzyme plate, 4 DEG C are spent the night, phosphoric acid buffer (PBST) containing 0.5%Tween-20 (V/V) washes plate 5 times, pats dry lath, adds 3% skimmed milk (W/V), 300 μ L/ holes, 37 DEG C of closed 2h.After PBST washes plate 3 times, every hole adds 50 μ L and adds and the present invention is directed to AFB
1single domain heavy chain antibody and 50 μ LAFB
1standard solution or testing sample, horizontal direction mixes gently, 37 DEG C of incubation 1h.PBST washes plate 5 times, pats dry, and adds the anti-HisTag tag antibody of rabbit of horseradish peroxidase-labeled, 100 μ L/ holes, 37 DEG C of incubation 1h.PBST washes plate 5 times, pats dry, and adds 100 μ L/ hole TMB nitrite ions, 37 DEG C of lucifuge colour developing 5min.Add 50 μ L/ hole stop buffer (2MH
2sO
4), microplate reader reading.According to AFB in the light absorption value calculation sample measured
1content.
Claims (7)
1., for the single domain heavy chain antibody of aflatoxin, there is the aminoacid sequence shown in SEQIDNO.:1.
2. a nucleic acid molecule, is characterized in that aminoacid sequence described in coding claim 1.
3. comprise the carrier of nucleotide sequence according to claim 2.
4. comprise the host cell of carrier described in claim 3.
5. the application of single domain heavy chain antibody in immunodetection, aflatoxin enrichment and purifying for aflatoxin according to claim 1.
6. the single domain heavy chain antibody for aflatoxin according to claim 1 is preparing the application in Aspergillus flavus toxin immuno detection, enrichment and purified reagent or material.
7. the single domain heavy chain antibody for aflatoxin according to claim 1 by random or site-directed mutagenesis technique carry out house of correction acquisition can with the antibody of aflatoxin specific binding.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103204937A (en) * | 2013-04-26 | 2013-07-17 | 山西德丰信成生物科技有限公司 | Single-domain heavy chain antibody T10 aiming at immune globulin Fc segment |
| CN103497252A (en) * | 2013-09-22 | 2014-01-08 | 南昌大学 | Single-domain heavy chain antibody L5-78 for Listeria monocytogenes |
| CN103866401A (en) * | 2014-03-28 | 2014-06-18 | 中国农业科学院油料作物研究所 | Aflatoxin nano antibody gene pool, construction method and application of aflatoxin nano antibody gene pool as well as aflatoxin B1 nano antibody 2014AFB-G15 |
| CN103861569A (en) * | 2014-03-28 | 2014-06-18 | 中国农业科学院油料作物研究所 | Aflatoxin nano-antibody immunosorbent, immunoaffinity column and preparation methods and application of aflatoxin nano-antibody immunosorbent and immunoaffinity column |
-
2015
- 2015-12-02 CN CN201510866664.0A patent/CN105399821A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103204937A (en) * | 2013-04-26 | 2013-07-17 | 山西德丰信成生物科技有限公司 | Single-domain heavy chain antibody T10 aiming at immune globulin Fc segment |
| CN103497252A (en) * | 2013-09-22 | 2014-01-08 | 南昌大学 | Single-domain heavy chain antibody L5-78 for Listeria monocytogenes |
| CN103866401A (en) * | 2014-03-28 | 2014-06-18 | 中国农业科学院油料作物研究所 | Aflatoxin nano antibody gene pool, construction method and application of aflatoxin nano antibody gene pool as well as aflatoxin B1 nano antibody 2014AFB-G15 |
| CN103861569A (en) * | 2014-03-28 | 2014-06-18 | 中国农业科学院油料作物研究所 | Aflatoxin nano-antibody immunosorbent, immunoaffinity column and preparation methods and application of aflatoxin nano-antibody immunosorbent and immunoaffinity column |
Non-Patent Citations (1)
| Title |
|---|
| 《食品安全质量检测学报》 * |
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Application publication date: 20160316 |