CN102653558B - Single-chain antibody and application thereof in detecting aflatoxin - Google Patents
Single-chain antibody and application thereof in detecting aflatoxin Download PDFInfo
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Abstract
The invention discloses a single-chain antibody and application thereof in detecting aflatoxin. The single-chain antibody is a polypeptide consisting of a light chain variable region, a connection peptide and a heavy chain variable region, wherein the connection peptide is positioned between the light chain variable region and the heavy chain variable region; the light chain variable region is represented by amino acid residues from 1st to 110th site of the tail ends of sequences from 1 to N of a sequence table; and the heavy chain variable region is represented by amino acid residues from 131st to 248th site of the tail ends of the sequences from 1 to N of the sequence table. The single-chain antibody has high cross reactivity, high affinity and high sensitivity to various types of aflatoxin, is suitable for quick immune detection for various types of multi-residue aflatoxin and has great significance for detecting the aflatoxin.
Description
Technical field
The present invention relates to a kind of single-chain antibody and the application in detecting aflatoxin thereof.
Background technology
Aflatoxin (Aflatoxin, AFT) be a kind of mycotoxins that human health and agriculture production is all had grave danger, its chemical structure is similar, be the derivative of dihydrofuran tonka bean camphor, molecular weight is about 312-346, is the secondary metabolite that is produced by flavus (Aspergillus flavus) Aspergillus parasiticus (A.parasiticus).People that mycotoxins brings out cancer nearly 250,000 is died from the whole world every year.The pollution of mycotoxins also causes a large amount of agricultural-food to be destroyed, and the financial loss that causes every year reaches multi-billion dollar.The probability that occurs aflatoxin in damp-heat area food and feed is the highest.
Aflatoxin is the extremely strong highly toxic substance of a kind of toxicity, and people and animal livers tissue are had destruction, can cause liver cancer even death when serious.It is 1 class carcinogens that aflatoxin in 1993 delimited by the cancer research mechanism of The World Health Organization (WHO).Kind surplus the aflatoxin that present isolation identification goes out reaches 20 mainly comprises B
1, B
2, G
1, G
2, M
1, M
2, P
1, Q, H
1, GM, B
2aWith malicious alcohol etc.In the food of natural contamination with AFB
1Much more to see that its toxicity and carinogenicity are also the strongest.Because the huge toxicity of aflatoxin, the requirement of limiting the quantity of has been done to the content of AFT in the food in existing more than 70 countries and regions.WHO Codex Committee on Food in 2006 recommends that aflatoxin exempt quantities standard is total amount (AFB in food, the feed
1, AFB
2, AFG
1, AFG
2Summation) less than 15 μ g/kg; AFM in the milk
1Exempt quantities be 0.5 μ g/kg.The European Union's level of mycotoxins in cereal and grain products of having reappraised in 2008 formulated more strict upper limit standard: AFB
1The 2 μ g/kg that limit the quantity of, the AFT total amount 4 μ g/kg that limit the quantity of.China Ministry of Health has issued " preventing aflatoxin contamination management of food hygiene way " November nineteen ninety, AFB in the regulation inhomogeneity sample
1Limit the quantity of and be 5-204 μ g/kg.
The method for quantitatively determining of AFT is divided into two big classes.One class is the physico-chemical analysis method that is based upon on the chromatogram basis, comprising use fluoroscopic examination dive method (LC), high performance liquid chromatography (HPLC) and liquid phase-mass spectrum of tlc (TLC), liquid phase look be used in conjunction detection (LC-MASS).Another kind of is immuno-chemical method, as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).In the above-mentioned detection technique, immunological method, especially elisa technique since its fast, low-cost, the characteristics such as high throughput testing that are fit to great amount of samples are by more and more widely the aflatoxin analyzing and testing field that is applied to.The used antibody of present domestic euzymelinked immunosorbent assay (ELISA) test kit mainly still is polyclonal antibody and monoclonal antibody.These two kinds of antibody production techniques are with high costs, and output is limited, greatly limited immunological method further developing in detection.
Summary of the invention
The purpose of this invention is to provide a kind of single-chain antibody and the application in detecting aflatoxin thereof.
Single-chain antibody provided by the invention, the polypeptide of being formed by variable region of light chain, connection peptides and variable region of heavy chain; Described connection peptides is between described variable region of light chain and described variable region of heavy chain; Described variable region of light chain as the sequence 1 of sequence table from shown in N-terminal the 1st to the 110 amino acids residue; Described variable region of heavy chain as the sequence 1 of sequence table from shown in N-terminal the 131st to the 248 amino acids residue.
Described connection peptides can be as the sequence 1 of sequence table from shown in N-terminal the 111st to the 130 amino acids residue.
Described single-chain antibody specifically can be the polypeptide shown in the sequence 4 of the polypeptide shown in the sequence 1 of sequence table or sequence table.
The present invention also protects the gene of the described single-chain antibody of coding, is made up of the encoding sequence of the encoding sequence of described variable region of light chain, described connection peptides and the encoding sequence of described variable region of heavy chain.
The encoding sequence of described variable region of light chain can be as the sequence 2 of sequence table from shown in 5 ' terminal the 9th to 338 Nucleotide.The encoding sequence of described variable region of heavy chain can be as the sequence 2 of sequence table from shown in 5 ' terminal the 399th to 752 Nucleotide.
The encoding sequence of described connection peptides can be as the sequence 2 of sequence table from shown in 5 ' terminal the 339th to 398 Nucleotide.
Described gene specifically can be following (a) or (b): (a) sequence 2 of sequence table is from the dna molecular shown in 5 ' terminal the 9th to 752 Nucleotide; (b) sequence 2 of sequence table is from the dna molecular shown in 5 ' terminal the 9th to 791 Nucleotide.
Contain described expression of gene box, recombinant vectors, reorganization bacterium or transgenic cell line and all belong to protection scope of the present invention.
Described recombinant vectors specifically can be described gene is inserted the recombinant plasmid that pET-22b (+) plasmid obtains, and more specifically can be described gene is inserted the recombinant plasmid that obtains between the NcoI of pET-22b (+) plasmid and the XhoI restriction enzyme site.
Described reorganization bacterium specifically can be described recombinant plasmid is imported the reorganization bacterium that intestinal bacteria obtain, and more specifically can be described recombinant plasmid is imported the recombinant plasmid that e. coli bl21 (DE3) bacterial strain obtains.
The present invention also protects a kind of method for preparing described single-chain antibody, is the described reorganization of fermentation culture bacterium, obtains described single-chain antibody.
Described method adds IPTG and makes its concentration when comprising the steps: specifically that (1) is cultured to OD600=1.0 with described reorganization bacterium be 0.5mM, 25 ℃ then, 250rpm shaking culture 8 hours, centrifugal collection bacterial sediment; (2) step (1) is obtained thalline and carry out cytoclasis, centrifugal collection supernatant liquor; (3) supernatant liquor that step (2) is obtained is dialysed successively and is concentrated, and obtains single-chain antibody solution.
The present invention also protects the application of above arbitrary described single-chain antibody in detecting aflatoxin.
Described aflatoxin specifically can be AFB
1(AFB
1), AFB
2(AFB
2), AFM
1(aflatoxin M
1), AFM
2(aflatoxin M
2), AFG
1(AFG
1) or AFG
2(AFG
2).
Described single-chain antibody specifically can be the single-chain antibody that the above method of employing prepares.
Recombinant antibodies is the third generation antibody production techniques that new development is got up, by the efficient screening in antagonist library, and can the good antibody fragment of acquired character.Express this antibody fragment by expression system efficiently, can prepare this antibody fragment low-cost, in a large number.The present invention is directed to the deficiency of present monoclonal antibody and polyclonal antibody, with AFM
1The specific monoclonal antibody hybridoma is the source, and preparation single-chain antibody (scFv), and adopt the outer evolution technology of aleuroplast and phage displaying antibody storehouse technology that it is carried out performance optimization improves its avidity and to the cross reacting rate of different AFT.
Single-chain antibody provided by the invention has good cross reacting rate, high-affinity and highly sensitive to multiple aflatoxin, is applicable to that the tachysynthesis of multiple how residual aflatoxin detects, and will bring into play significant role in aflatoxin detects.
Description of drawings
Fig. 1 is aflatoxin scFv space-filling model; A:scFv backbone structure synoptic diagram; B:scFv solution accessible surface figure; Redness is HCDRs, and blueness is LCDRs.
Fig. 2 is scFv and AFM
1The interaction computer simulation; A:AFM
1Combining site (yellow is HCDR3, and orange is HCDR2, and redness is HCDR3, and blueness is LCDR3); B: antigen antibody interaction 2D synoptic diagram (red-purple is the amino-acid residue of polarization contact, and green for producing the amino-acid residue of nonpolar contact, blue dotted line is hydrogen bond action).
Fig. 3 is for adopting AFB
1The graphic representation of making.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all arranges repeated experiments three times, results averaged.AFB
1(AFB
1), AFM
1(aflatoxin M
1), AFM
2(aflatoxin M
2), AFB
2(AFB
2), AFG
1(AFG
1) and AFG
2(AFG
2) all available from U.S. Sigma company.
Used PBS damping fluid among the embodiment if no special instructions, is the PBS damping fluid of pH7.4,0.01M.
Used carbonate buffer solution is the sodium carbonate buffer of pH9.6,0.05mol/L among the embodiment.
Bovine serum albumin is called for short BSA.
N, dinethylformamide (DMF) is shown in formula I.
Formula I
N-hydroxy-succinamide (NHS) is shown in formula II.
Formula II
1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is shown in formula III.
Formula III
Carboxymethyl azanol half hydrochloride is shown in formula IV.
Formula IV
The discovery of embodiment 1, single-chain antibody
1, the extraction of hybridoma mRNA and total length scFv are gene constructed
Get homogeneity, good hybridoma (the secretion AFM of purity
1Monoclonal antibody specific) 1 * 10
6Individual, extract total RNA and purifying and obtain mRNA, reverse transcription obtains cDNA.Use a complete set of primer, be that template increases respectively and obtains variable region of heavy chain (VH), variable region of light chain (VL) gene with cDNA, be cloned in pCR 2.1 carriers respectively and order-checking, after machine analysis and the database BLAST comparison, identify VH and VL gene with potential immunocompetence and correct expression cassette as calculated.Design overlapping extension PCR (SOE-PCR) primer according to above-mentioned through genes identified, and splicing makes up total length scFv gene.This scFv gene is checked order and sequential analysis, and be cloned into and preserve in pCR 2.1 carriers and expand numerous.
2, scFv expresses and identifies
Above-mentioned scFv being carried out the scFv expression by being cloned in pCR 2.1 carriers in the pET serial carrier, adjust expression condition, is that purpose scFv albumen is positioned thoughtful chamber.Adopt gentle osmotic shock method to extract the thoughtful chamber albumen of expressive host bacterium, and it is carried out SDS denaturing polyacrylamide gel electrophoresis (SDS page) and immunoblotting assay (immunoblotting), evaluation expression product and expression efficiency thereof.Be envelope antigen with AFM1-BSA, thoughtful chamber scFv is carried out the indirect competitive ELISA analysis, estimate the immunocompetence of this scFv.
3, the modeling of aflatoxin scFv homology and molecular docking
(1) homology modeling
VL in the scFv sequence and VH sequence are committed to Protein Data Bank (Protein Data Bank, BrookhavenNational Laboratory) respectively, obtain the highest protein structures of homology by the BLAST comparison.Use among the Discovery Studio 2.5.5 (DS 2.5.5) the Modeler algorithm that above-mentioned VL, VH sequence and its homology crystalline structure are compared (Alignment), and be the fundamental construction homology model with the antibody variable region framework region in the crystalline structure template.Because antibody CDR zone variability is higher, therefore utilize Looper algorithm among the DS 2.5.5 to define CDR district in gained VL, the VH model on above-mentioned homology model basis, and carry out the PDB database retrieval at the CDR district, obtain the highest CDR crystalline structure template of homology and rebuild the CDR district.At the highest H CDR3 district of variability, because the homology of its optimal Template is lower than 50%, therefore adopts the accent of de novo algorithm to calculate and obtain its reasonable conformation.Behind the homology model that makes up, obtains VH and VL respectively, it is overlapping with final structure Fv structure to carry out the protein main chain structure by method of least squares, and carries out 200 step method of steepest descent energy minimizations (steepest descent energy minimization) and 200 and go on foot conjugate gradient energy minimizations (Cojugate Gradient energy minimization).Final gained scFv space-filling model such as Fig. 1.
(2) molecular docking
The aflatoxin chemical structure is obtained by NCBI PubChem database retrieval.Use DS 2.5.5 and CCDCGold suite 4.2 to carry out above-mentioned flavacin specificity scFv and AFM
1Flexible docking.Obtain 10 butt joint results, adopt Gold score that the small molecules conformation among all butt joint results is assessed, and ligand analysis module (Analyze biding site tool) is further analyzed result such as Fig. 2 among the use DS 2.5.5 to the result.
4, scFv hypervariable region (CDRs) rite-directed mutagenesis and screening
In antibody and haptenic combination, the CDRs zone in the antibody structure, especially heavy chain CDR3(H CDR3) and light chain CDR3 (L CDR3) bringing into play of paramount importance effect.Simultaneously because the volume of hapten molecule is minimum, so often its be merely able to 6 CDR districts in a few come in contact.Therefore, need instruct follow-up optimization by molecular simulation, choose relatively effectively suddenly change zone and evolution strategy.
Above-mentioned computer molecular simulation result shows, at the AFM of scFv
1Combination in, the H59Tyr in heavy chain CDR2 district is bringing into play of paramount importance effect, has formed hydrogen bond and stronger π-π and has interacted.In addition, light chain CDR3 has made main relatively contribution for the hydrophobic interaction cave.Analyzing with this is guidance, according to known scFv gene order design degenerated primer, introduces sudden change at H CDR3 and L CDR3 respectively by round pcr.Can farthest keep the hydrogen bond in the Proantigen antibodies unaffected like this.Simultaneously, for avoiding producing too much non-activity or clone that can not normal expression, adopt " NNS " degenerate codon through adjusting to introduce sudden change, at the random mutation probability of each amino acid mutation point generation 50%.(be about 10 because the storage capacity of phage display library is restricted
9), therefore carry out 6-7 amino acid whose random mutation at every turn, produce 6.4 * 10 approximately
7Individual muton.Therefore, for 13 amino acid that come in contact with haptens in the above-mentioned binding analysis, (storage capacity is respectively 1.0 * 10 need to make up two bacteriosphage mutation display libraries respectively
7～8), carry out twice screen mutation continuously.
5, active clonal expression and evaluation
After making up the sudden change phage display library, use the nanometer magnetic bead of biotin labeled haptens and Streptavidin bag quilt that the clone of the activity in the library is carried out the affine enrichment screening of pure liquid phase immunity.Carry out the 4-5 wheel altogether, progressively reduce the used antigen concentration of screening in the enrichment every the wheel, and change different evolution targets enrichment screening (seeing Table 1) is carried out in the sudden change library, to improve antibody to cross reacting rate and the avidity of medicine of the same clan.
Enrichment and the screening of table 1 bacteriosphage mutation display libraries
| The enrichment number of times | Antigen | Concentration | The phage input | The phage work output |
| 1 | AFM 1 | 1μg/mL | 1.0×10 12 | 2.3×10 5 |
| 2 | AFM 1 | 0.1μg/mL | 1.0×10 12 | 3.8×10 6 |
| 3 | AFB 1 | 10μg/mL | 1.0×10 12 | 1.9×10 4 |
| 4 | AFB 1 | 1μg/mL | 1.0×10 12 | 3.4×10 5 |
| 5 | AFB 1 | 0.1μg/mL | 1.0×10 12 | 6.1×10 6 |
In the end one take turns enrichment and finish after, the independent colony clone of random choose carries out the secreting, expressing of scFv, and prepares thoughtful chamber extract, carries out SDS-Page and ELISA and identifies.
Gained scFv sequence is shown in the sequence 1 of sequence table.In the sequence 1, be variable region of light chain from N-terminal the 1st to 110 amino acids residue, the 111st to 130 amino acids residue is connection peptides, and the 131st to 248 amino acids residue is variable region of heavy chain.
The preparation of embodiment 2, single-chain antibody
One, construction of recombinant plasmid
1, the double chain DNA molecule shown in the sequence 2 of composition sequence table (is the NcoI restriction endonuclease recognition sequence from 5 ' terminal the 1st to 6 Nucleotide, the the 9th to 338 Nucleotide is the encoding sequence of variable region of light chain, the the 339th to 398 Nucleotide is the encoding sequence of connection peptides, the the 399th to 752 Nucleotide is the encoding sequence of variable region of heavy chain, the the 762nd to 791 Nucleotide is the encoding sequence of C-myc tag label, and the 792nd to 797 Nucleotide is the XhoI restriction endonuclease recognition sequence).
2, be template with the synthetic double chain DNA molecule of step 1, to carrying out pcr amplification, obtain pcr amplification product with the primer of F1 and R1 composition.
F1：5’-
CCATGGCACAGACGGTTG-3’；
R1：5’-
CTCGAGCAGGTCTTCTTC-3’。
3, with the pcr amplification product of restriction enzyme NcoI and XhoI double digestion step 2, reclaim enzyme and cut product.
4, with restriction enzyme NcoI and XhoI double digestion pET-22b (+) plasmid (Novagen, Beijing), reclaim carrier framework (about 5424bp).
5, the carrier framework of the enzyme of step 3 being cut product and step 4 is connected, and obtains recombinant plasmid.According to sequencing result, it is as follows that recombinant plasmid is carried out structrual description: inserted the double chain DNA molecule shown in the sequence 2 of sequence table between the NcoI of pET-22b (+) plasmid and XhoI restriction enzyme site.In recombinant plasmid, the encoding sequence fusion of the His-tag label on encoding sequence fusion, 3 ' end and the carrier framework of the guiding peptide pelB on the double chain DNA molecule 5 ' end shown in the sequence 2 of sequence table and the carrier framework, expressed fusion protein.
Two, the structure of reorganization bacterium
The recombinant plasmid that step 1 is made up imports e. coli bl21 (DE3) bacterial strain (Novagen, Beijing), obtains the bacterium of recombinating.
Three, the expression of single-chain antibody and purifying
1, the reorganization bacterium that step 2 is made up is seeded to Super Broth substratum, and making the starting point concentration of reorganization bacterium is OD600=0.1, and 37 ℃, 250rpm shaking culture are to OD600=1.0; Adding IPTG and making its concentration is 0.5mM, 25 ℃, 250rpm shaking culture 8 hours; Then with the centrifugal 15min of bacterium liquid 6000g, collect bacterial sediment, (parameter of ultrasonication: ultrasound probe is 6 Φ, power 400W, omnidistance 30 minutes working hours to carry out cytoclasis, wherein, each circulating ultrasonic 3 seconds is 5 seconds at interval), centrifugal (12000g, 30min) collects supernatant liquor, then supernatant liquor dialysed in the PBS damping fluid, dialysis finishes with 0.22 μ m membrane filtration, collects 4 ° of C of filtrate and preserves standby.
2,1g powdery Ni-IDA resin (German MACHEREY-NAGEL company) is dissolved in the 5mL ultrapure water, adds chromatography column then, make it form stable post bed in the action of gravity deposit.When treating that water liquid level in the chromatography column is down to the post bed surface, the pH 8.0PBS damping fluid that slowly adds 5 times of column volumes carries out the purification column balance.Add the filtrate that step 1 is collected subsequently, make it pass through purification column under action of gravity, when its liquid level was down to the post bed surface, the pH 8.0PBS damping fluid that contains the 20mM imidazoles that adds 10 times of column volumes washed, and removed foreign protein.The PBS damping fluid that adds the pH8.0 that contains the 100mM imidazoles of 3 times of column volumes at last, the wash-out target protein was collected elutriant behind the post.The PEG8000 aqueous solution with 40g/100mL concentrates (4 ℃) to elutriant after crossing post at last, obtains single-chain antibody solution.
Four, the preparation of contrast solution
1, the double chain DNA molecule shown in the sequence 3 of composition sequence table (is the NcoI restriction endonuclease recognition sequence from 5 ' terminal the 1st to 6 Nucleotide, the the 18th to 47 Nucleotide is the encoding sequence of C-myc tag label, and the 48th to 53 Nucleotide is the XhoI restriction endonuclease recognition sequence).
Step 2 is to 4 with 2 to 4 of step 1.
5, the carrier framework of the enzyme of step 3 being cut product and step 4 is connected, and obtains control plasmid.According to sequencing result, it is as follows that control plasmid is carried out structrual description: inserted the double chain DNA molecule shown in the sequence 3 of sequence table between the NcoI of pET-22b (+) plasmid and XhoI restriction enzyme site.
6, the control plasmid that step 5 is obtained imports e. coli bl21 (DE3) bacterial strain (Novagen, Beijing), obtains contrasting bacterium.
7, will contrast bacterium and replace the reorganization bacterium to carry out step 3, obtain contrast solution.
The application of embodiment 3, single-chain antibody
One, AFB
1The preparation of-BSA
1, AFB
1-BSA's is synthetic
(1) with 5mg flavacin B
1Be dissolved in the 5ml pyridine, add 25mg carboxymethyl azanol half hydrochloride, room temperature lucifuge stirring reaction spends the night, and 40 ℃ are spin-dried for; With the small amount of methanol dissolving, the TLC scraper plate separates, and developping agent is methyl alcohol: chloroform=1:9, gets the product fluorescence band of RF=0.2, uses the 2ml ethyl acetate extraction, adds 500 μ l methyl alcohol and 200 μ l DMF assisted extraction, filters, and filtrate is revolved steaming for 40 ℃, gets product liquid 300 μ l.
(2) get the said products liquid 300 μ l, add 300 μ l DMF and 300 μ l water again, add 8mg EDC and 8mgNHS, activation 1h, the reaction lucifuge obtains the solution I;
(3) (0.1M PH=9.51), fully dissolves, and stirring at room is the solution II 10mg BSA to be dissolved in the 1ml carbonate buffer solution.
(4) the solution I is added in the solution II, slowly stirs the dialysis tubing of packing into behind the 24h, in physiological saline, change water 6 times in the middle of 4 ℃ of dialysis 72h(), under 4 ℃ of conditions, the centrifugal 30min of 8000rmp gets supernatant, i.e. AFB then
1-BSA solution is sub-packed in the ampere bottle-20 ℃ of preservations.
(5) with AFB
1After-BSA solution dilutes with the PBS damping fluid, measure the spectrophotometric value of 280nm and 260nm, by formula calculate the protein concentration in the diluent, the protein concentration that records is on duty to be former AFB behind its extension rate
1AFB in the-BSA solution
1-BSA concentration.Protein concn (mg/ml)=1.45 * OD
280-0.74 * OD
260AFB
1AFB in the-BSA solution
1-BSA concentration is 6.1mg/ml.
2, AFB
1The sign of-BSA
With AFB
1-BSA solution (makes AFB with the dilution of PBS damping fluid
1The concentration of-BSA is 5mg/mL), as the solution first; To contain 5mg/mL AFB
1The PBS damping fluid as solution second; To contain the PBS damping fluid of 5mg/mL BSA as solution third.Respectively solution first, solution second and solution third are carried out ultraviolet (200-380nm) spectral scan.Considerable change has taken place in the uv-spectrogram of comparing the solution first with solution third, and compound and BSA success coupling is described.
Two, the application of single-chain antibody
1, adopts the AFB of embodiment 2 preparations
1-BSA(employing carbonate buffer solution adjusting concentration) wrap quilt, 100 μ L/ holes, AFB
1The bag of-BSA is 0.5 μ g/mL by concentration.
2, hatched 16 hours for 4 ℃.
3, seal and wash plate.
4, every hole adds 50 μ L AFBs
1(by AFB
1Form with the PBS damping fluid; AFB
1Concentration be respectively 0.0001ng/ml, 0.001ng/ml, 0.01ng/ml, 0.05ng/ml, 0.1ng/ml, 0.5ng/ml, 0.8ng/ml, 1ng/ml, 5ng/ml, 10ng/ml; With hole in contrast, the hole that only adds the PBS damping fluid); Each concentration arranges 3 multiple holes.
5, every hole adds the single-chain antibody solution of 50 μ L embodiment, 2 preparations.
6, incubated at room 2h washes plate.
7, every hole add 100 μ L horseradish peroxidase-labeled anti-C-myc antibody (Bioisystech Co., Ltd of China fir Golden Bridge in Beijing, TA-1), incubated at room 2h.
8, wash plate.
9, add TMB colour developing liquid, lucifuge colour developing 15min.
10, every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD
450Value.
The AFB of each concentration will be adopted
1The light absorption value that the obtains mean values of holes (three multiple) divided by the light absorption value of control wells as ordinate zou, with AFB
1Concentration (ng/ml) is X-coordinate curve plotting figure.The results are shown in Figure 3.Control curve figure obtains the AFB that Y value equals 50% o'clock correspondence
1Concentration (μ g/L), i.e. IC
50Value.
Single-chain antibody detects AFB
1IC
50Value is 0.125ng/ml, detects and is limited to 0.01ng/ml.
Three, controlled trial
Contrast solution is replaced single-chain antibody solution, and other are same step 2 all, adopts each AFB
1The light absorption value of concentration all equates, enzyme linked immunoassay does not all take place.
Four, the cross reacting rate of single-chain antibody
1 to 3 with 1 to 3 of step 2.
4, every hole adds 50 μ L aflatoxin and (is made up of aflatoxin and PBS damping fluid; The concentration of aflatoxin is respectively 0.0001ng/ml, 0.001ng/ml, 0.01ng/ml, 0.05ng/ml, 0.1ng/ml, 0.5ng/ml, 0.8ng/ml, 1ng/ml, 5ng/ml, 10ng/ml; With hole in contrast, the hole that only adds the PBS damping fluid); Each concentration arranges 3 multiple holes.
Adopt following several aflatoxin: AFB respectively
1, AFM
1, AFM
2, AFB
2, AFG
1, AFG
2
5 to 10 with 5 to 10 of step 2.
The light absorption value (mean values in three multiple holes) that the concrete aflatoxin that adopts each concentration is obtained as ordinate zou, is X-coordinate curve plotting figure with concrete aflatoxin concentration (ng/ml) divided by the light absorption value of control wells.Control curve figure obtains the concrete aflatoxin concentration (μ g/L) that Y value equals 50% o'clock correspondence, i.e. IC
50Value.
Calculate single-chain antibody to the cross reacting rate of aflatoxin with following formula.
Single-chain antibody detects aflatoxin M
1Cross reacting rate be 91.3%.Single-chain antibody detects aflatoxin M
2Cross reacting rate be 45%.Single-chain antibody detects AFB
2Cross reacting rate be 95%.Single-chain antibody detects AFG
1Cross reacting rate be 136%.Single-chain antibody detects AFG
2Cross reacting rate be 50%.Satisfy the requirement that the flavus total amount detects.
Claims (10)
1. single-chain antibody, the polypeptide of being formed by variable region of light chain, connection peptides and variable region of heavy chain; Described connection peptides is between described variable region of light chain and described variable region of heavy chain; Described variable region of light chain as the sequence 1 of sequence table from shown in N-terminal the 1st to the 110 amino acids residue; Described variable region of heavy chain as the sequence 1 of sequence table from shown in N-terminal the 131st to the 248 amino acids residue.
2. single-chain antibody as claimed in claim 1 is characterized in that: described connection peptides as the sequence 1 of sequence table from shown in N-terminal the 111st to the 130 amino acids residue.
3. single-chain antibody as claimed in claim 2 is characterized in that: described single-chain antibody is the polypeptide shown in the sequence 4 of the polypeptide shown in the sequence 1 of sequence table or sequence table.
4. the gene of the described single-chain antibody of coding claim 1 is made up of the encoding sequence of the encoding sequence of described variable region of light chain, described connection peptides and the encoding sequence of described variable region of heavy chain.
5. gene as claimed in claim 4 is characterized in that: the encoding sequence of described variable region of light chain as the sequence 2 of sequence table from shown in 5 ' terminal the 9th to 338 Nucleotide; The encoding sequence of described variable region of heavy chain as the sequence 2 of sequence table from shown in 5 ' terminal the 399th to 752 Nucleotide.
6. gene as claimed in claim 5 is characterized in that: the encoding sequence of described connection peptides as the sequence 2 of sequence table from shown in 5 ' terminal the 339th to 398 Nucleotide.
7. gene as claimed in claim 6 is characterized in that: described gene is following (a) or (b):
(a) sequence 2 of sequence table is from the dna molecular shown in 5 ' terminal the 9th to 752 Nucleotide;
(b) sequence 2 of sequence table is from the dna molecular shown in 5 ' terminal the 9th to 791 Nucleotide.
8. contain arbitrary described expression of gene box, recombinant vectors, reorganization bacterium or transgenic cell line in the claim 4 to 7.
9. the method for preparing arbitrary described single-chain antibody in the claim 1 to 3 is the described reorganization of fermentation culture claim 8 bacterium, obtains described single-chain antibody.
10. the application of arbitrary described single-chain antibody in detecting aflatoxin in the claim 1 to 3.
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| CN104693304A (en) * | 2015-02-13 | 2015-06-10 | 深圳雅臣生物科技有限公司 | Genetic engineering recombinant high-purity anti-mycotoxin monoclonal antibody for inhibiting and killing mycotoxin as well as composite monoclonal antibody preparation method and combined preparation thereof |
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