CN103215652B - Aflatoxin positive antibody gene bank, establishing method and application thereof, and aflatoxin recombinant single-chain antibody 1A7 - Google Patents

Abstract

The invention relates to an aflatoxin positive antibody gene bank, an establishing method and an application thereof, and an aflatoxin recombinant single-chain antibody 1A7. According to the invention, Mixed light chain variable region genes and mixed heavy chain variable region genes of aflatoxin monoclonal antibodies 1C11, 2C9, 1C8, 10G4, 3G1 are randomly spliced with an overlap extension PCR method, such that single-chain antibody ScFv gene is obtained; and the gene is connected and transferred with a pCANTAB5E carrier, such that the aflatoxin positive antibody gene bank is obtained. The positive gene bank is completely composed of gene fragments (positive fragments) of antibodies with identification capacity against aflatoxin. Under a condition with a relatively small storage capacity, aflatoxin single-chain antibody fast screening can be realized. The aflatoxin recombinant single-chain antibody 1A7 is a general monoclonal antibody with a 50% inhibition concentration IC50 against aflatoxin B1 of 20pg/mL. Cross-reactivities of the antibody against aflatoxin B1, B2, G1, G2, and M1 are respectively 100%, 166%, 100%, 0.1% and 11.1%.

Description

Aflatoxin positive antibody gene pool, construction process, purposes and aflatoxin recombinant single chain antibody 1A7

Technical field

The present invention relates to aflatoxin positive antibody gene pool, construction process, purposes and aflatoxin recombinant single chain antibody 1A7.

Background technology

Aflatoxin is mainly the secondary metabolite being produced by flavus and Aspergillus parasiticus secretion, is the natural toxic compounds that can cause the various infringements of people and animals.Aflatoxin has found that more than 20 plant at present, and wherein the toxicity of aflatoxin B1 is the strongest, and its toxicity is 10 times of potassium cyanide, 68 times of arsenic.As far back as 1993, aflatoxin B1 was delimited as one of known the strongest carcinogenic chemical, i.e. I class carcinogenic substance by the cancer research mechanism of the World Health Organization.China belongs to the heavier area of aflatoxin contamination, at various food and agricultural-food, especially in corn, peanut and goods thereof, all probably has the pollution of aflatoxin.Therefore strengthen the detection of aflatoxin, particularly speed and survey, understand and grasp in time the health information of various food and agricultural-food, significant to ensureing China's food consumption safety.

The detection method of existing aflatoxin comprises chemical analysis, precision instrument analytical method and immune analysis method.Wherein chemical analysis is the most frequently used detection method of early detection aflatoxin, it does not need special plant and instrument, common laboratory all can be carried out, but reagent dosage is large, complex operation, other component serious interference, poor accuracy, can not accurate quantitative analysis, and larger to experimenter and surrounding environment pollution hazard, be unsuitable for field quick detection.Precision instrument analytical method comprises spectrophotofluorimetry and high performance liquid chromatography, and it is highly sensitive, and accuracy is good, but instrument costliness, requires aflatoxin Sample Purification on Single degree high, and sample pretreatment process is loaded down with trivial details, length consuming time, high to experimental situation requirement, be difficult to realize rapid detection.The immuno analytical method growing up has in recent years overcome the above two shortcoming, there is high specificity, highly sensitive, sample pre-treatments is simple, cost is low, little to the pollution hazard of experimenter and surrounding environment, be suitable for the advantages such as on-the-spot batch detection.Immunoassay is to utilize biology, physics or the chemical amplification of the marker on the specific association reaction of antigen and antibody and antibody, antigen to carry out qualitative and quantitative analysis to ultramicron residue, so study any immunology detection technology of setting up for aflatoxin, all must first make the antibody of aspergillus flavus resisting toxin.

Along with the development of antibody technique, recombinant single chain antibody is also employed gradually at the detection field of aflatoxin.Compare with traditional polyclonal antibody, monoclonal antibody, recombinant single chain antibody has its unique advantage, can be in prokaryotic expression system in a short period of time a large amount of produce and productive expense cheap, low cost, extensive detection to aflatoxin have important using value, can meet the need of production of growing aflatoxin antibody.And high-quality recombinant single chain antibody need to screen acquisition conventionally from specific phage display library, library storage capacity is larger conventionally, and the probability that screens object antibody is also just higher, but the also corresponding increase of the difficulty of library construction.Therefore, on the basis that does not increase library construction difficulty, the structure by positive antibody storehouse will improve the screening probability of specific antibody greatly.

Summary of the invention

Problem to be solved by this invention is to provide aflatoxin positive antibody gene pool, construction process, purposes and aflatoxin recombinant single chain antibody 1A7.

Aflatoxin positive antibody gene pool, it is characterized in that: it is by aflatoxin monoclonal antibody 1C11, 2C9, 1C8, 10G4, the mixing chain variable region gene of 3G1 and mixing heavy chain variable region gene adopt the random splicing of overlapping extension PCR method to obtain single-chain antibody ScFv gene, then be connected with pCANTAB 5E carrier and obtain with transforming, described aflatoxin monoclonal antibody 1C11, 2C9, 1C8, 10G4, 3G1 is CCTCC NO:C201013 by deposit number respectively, CCTCC NO:C201018, CCTCC NO:C201017, CCTCC NO:C201016, the hybridoma cell strain secretion of CCTCC NO:C201014 produces.

The construction process of aflatoxin positive antibody gene pool is specific as follows: application PCR method increases respectively and obtains each aflatoxin monoclonal antibody 1C11, 2C9, 1C8, 10G4, the chain variable region gene VH of 3G1, heavy chain variable region gene VL, light by each antibody, the mole numbers such as heavy chain variable region gene mix collaboration template, then utilize overlapping extension PCR method that both are connected with the Linker fragment of the flexible short peptide sequence of coding (Gly4Ser) 3, be spliced into single-chain antibody scFv gene, the scFv gene building is connected with pCANTAB 5E carrier and transforms, structure obtains aflatoxin positive antibody gene pool.

Press such scheme, the primer light, heavy chain variable region gene that described amplification obtains its each aflatoxin monoclonal antibody is:

Variable region of heavy chain (Back): 5 '-AGG TGC AGC TGC AGC AGT CTG G-3 '

Variable region of heavy chain (For): 5 '-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CCA G-3 '

Variable region of light chain (Back): 5 '-GAC ATT GAG CTC ACC CAG TCT CCA-3 '

Variable region of light chain (For): 5 '-CCG TTT GAT TTC CAG CTT GGT GCC-3 '.

Press such scheme, the flexible short peptide sequence (Gly of amplification coding in described overlapping extension PCR method ₄ser) ₃the primer of linker fragment be:

Linker forward: 5 '-TCC GGC GGT GGT GGC AGC GGT GGC GGC GGT TCT GAC ATT GAG CTC ACC CAG TCT CCA-3 '

Linker is reverse: 5 '-ACC GCT GCC ACC ACC GCC GGA GCC ACC GCC ACC TGA GGA TGA GGA GAC GGT GAC CGT GGT-3 '.

Press such scheme, described by chain variable region gene V _hwith heavy chain variable region gene V _lwith the flexible short peptide sequence (Gly of coding ₄ser) ₃linker fragment assembly complete after, according near sequences Design pCANTAB 5E carrier cloning site, at scFv gene two ends, introduce respectively the primer of carrier pCANTAB 5E homologous sequence, and the end that makes every primer at least comprises the carrier pCANTAB 5E homologous site of 15bp, respectively to introduce at least carrier pCANTAB 5E homologous sequence of 15bp at scFv gene two ends, described primer is as follows:

RS（Back）: 5’- TCC TTT CTA TGC GGC CCA GCC GGC CAT GGC CCA GGT GCA GCT GCA GC A GTC TGG-3’

RS（For）: 5’- CGG CGC ACC TGC GGC CGC CCG TTT GAT TTC CAG CTT GGT GCC-3’

The primer sequence that wherein horizontal line part represents is and the site of carrier pCANTAB 5E homology.

Press such scheme, the construction process of described single-chain antibody scFv gene is: first in following pcr amplification reaction system:

10× Ex taq Buffer 5μl

dNTP(2.5 mmol/L) 4μl

V _hmix 50ng

V _lmix 50ng

Linker forward 1 μ l

The reverse 1 μ l of Linker

Ex taq DNA polysaccharase 1 μ l

DdH2O mends to total system 50 μ l;

PCR program is: 94 ℃ of 1min, 63 ℃ of 4min, 7 circulations of increasing;

Then in system, adding following material increases:

10× Ex taq Buffer 5μl

dNTP(2.5 mmol/L) 4μl

RS（Back） 1μl

RS（For） 1μl

Ex taq DNA polysaccharase 1 μ l

DdH2O mends to total system 50 μ l;

Amplification program is: 94 ℃ of 1min, 56 ℃ of 2min, 72 ℃ of 2min, 30 circulations of increasing.

Press such scheme, the method for attachment of described ScFv gene and pCANTAB 5 E carriers is: pCANTAB 5 E carriers, through the processing of Sfi I/Not I double digestion, are then carried out to In-fusion with ScFv gene and are connected; Described is converted into: aforementioned connection product is joined in e. coli tg1 Electroporation-competent cells, mix, electricity transforms, electricity conversion condition: 0.1 cm electricity transforms cup, 1.8 kV, 200 Ω, 25 μ F, electricity transforms after adding 2YT liquid nutrient medium piping and druming in cup and shifts at electricity immediately after transforming, 37 ℃ of slow joltings, 1 h that recovers.

Above-mentioned aflatoxin positive antibody gene pool is in the application of phage display method screening aflatoxin single-chain antibody.

Press such scheme, above-mentioned phage display method screening aflatoxin single-chain antibody is specifically achieved through the following technical solutions: the aflatoxin positive antibody gene pool successfully constructing is saved as after being illustrated in the form on phage capsid surface by M13KO7 helper phage, by two-step approach, screened: the first step adopts indirect elisa method to filter out aspergillus flavus resisting toxin and the positive hole of not anti-carrier proteins BSA; The positive hole nutrient solution that second step adopts indirect competitive ELISA method to filter out the first step detects, former as competition by aflatoxin B1, selects all higher holes of light absorption value and sensitivity, and final screening obtains aflatoxin recombinant single chain antibody.

Aflatoxin recombinant single chain antibody 1A7, is characterized in that: its aminoacid sequence is as shown in SEQ ID NO:1, and wherein weight chain variable region amino acid sequence is as shown in SEQ ID NO:2, and light chain variable region amino acid sequence is as shown in SEQ ID NO:3.

Press such scheme, the coding gene sequence of described aflatoxin recombinant single chain antibody 1A7 is as shown in SEQ ID NO:4, and wherein variable region of heavy chain coding gene sequence is as shown in SEQ ID NO:5, and variable region of light chain coding gene sequence is as shown in SEQ ID NO:6.

Beneficial effect of the present invention is:

(1) aflatoxin positive antibody gene pool provided by the invention (being called for short positive gene storehouse) is completely built and is formed by the gene fragment (positive fragment) aflatoxin to the antibody of recognition capability, in theory, the single-chain antibody that these positive fragment homologous recombination obtain also can have recognition capability to aflatoxin;

(2), in the situation that storage capacity is little relatively, can under the prerequisite that meets aflatoxin single-chain antibody screening requirement, realize the rapid screening of flavacin element single-chain antibody;

(3) aflatoxin recombinant single chain antibody 1A7 provided by the invention is general purpose single clonal antibody, to 50% inhibition concentration IC of aflatoxin B1 ₅₀be 20 pg/mL, the cross reacting rate of aflatoxin B1, B2, G1, G2, M1 is respectively to 100%, 166%, 100%, 0.1% and 11.1%.

Accompanying drawing explanation

Fig. 1 is the agarose gel electrophoresis detected result figure of scFv gene in the present embodiment.In figure, M:DNA Marker.

embodiment

The following aspergillus flavus resisting toxin monoclone antibody 1C11 used of embodiment 1,2C9,1C8,10G4,3G1 are respectively that employing deposit number is CCTCC NO:C201013, CCTCC NO:C201018, CCTCC NO:C201017, CCTCC NO:C201016, hybridoma cell strain 1C11, the 2C9 of CCTCC NO:C201014,1C8,10G4,3G1, are that the method for reporting in the patent of CN2010102450955, CN2011101082306, CN 2012101176204, CN2012101176149, CN201210117612X makes in advance respectively according to number of patent application.Concrete preparation method is as follows: apply mutually each hybridoma cell strain and inject the BALB/c mouse of processing with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, adopt caprylic acid-ammonium antibody purification, concrete operations are: with double-deck filter paper filtering mouse ascites, 4 ℃, the centrifugal 15min of 12000r/min, draw supernatant, gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes, under stirring, slowly add n-caprylic acid, every milliliter of required n-caprylic acid volume of ascites is 33 μ L, mixed at room temperature 30min, 4 ℃ of standing 2h, then 4 ℃, the centrifugal 30min of 12000r/min, abandon precipitation, by the supernatant liquor obtaining with after double-deck filter paper filtering, the volumetric molar concentration that adds 1/10 filtrate volume is the phosphate buffered saline buffer that 0.1mol/L and pH value are 7.4, with the sodium hydroxide solution of 2 mol/L, regulate the pH value to 7.4 of this mixed solution, 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL, 4 ℃ of standing 2h, then 4 ℃, the centrifugal 30min of 12000r/min, abandon supernatant, by resuspended with the 0.01mol/L phosphate buffered saline buffer of former ascites volume 1/10 gained precipitation, pack dialysis tubing into, pure water is dialysed, the protein solution of fully dialysing is put to-70 ℃ of refrigerator freezings, use afterwards freeze drier freeze-drying, collect lyophilized powder, obtain each aspergillus flavus resisting toxin monoclone antibody 1C11, 2C9, 1C8, 10G4, 3G1, antibody is put in-20 ℃ of refrigerators standby,

Described acetate buffer is 0.29g sodium-acetate, and 0.141mL acetic acid adds water and is settled to 100mL gained; The phosphate buffered saline buffer of described 0.1mol/L is 0.8g sodium-chlor, 0.29g disodium hydrogen phosphate, and 0.02g Repone K, potassium primary phosphate 0.02g, adds water and is settled to 100mL gained.

The structure of aflatoxin positive antibody gene pool

Aflatoxin positive antibody gene pool build in required primer synthetic

(1) weight of monoclonal antibody 1C11,2C9,1C8,10G4,3G1, the required primer of chain variable region gene sequence amplification:

Variable region of heavy chain (Back): 5 '-AGG TGC AGC TGC AGC AGT CTG G-3 '

Variable region of heavy chain (For): 5 '-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CCA G-3 '

Variable region of light chain (Back): 5 '-GAC ATT GAG CTC ACC CAG TCT CCA-3 '

Variable region of light chain (For): 5 '-CCG TTT GAT TTC CAG CTT GGT GCC-3 '

(2) (connection peptides of amplification connection weight, chain variable region gene sequence is encoded flexible short peptide sequence (Gly ₄ser) ₃) linker primer:

Linker forward primer: 5 '-TCC GGC GGT GGT GGC AGC GGT GGC GGC GGT TCT GAC ATT GAG CTC ACC CAG TCT CCA-3 '

Linker reverse primer: 5 '-ACC GCT GCC ACC ACC GCC GGA GCC ACC GCC ACC TGA GGA TGA GGA GAC GGT GAC CGT GGT-3 '

(3) in the two ends amplification of scFv gene fragment, at least comprise the primer RS Back/For of the carrier pCANTAB 5E homologous sequence of 15bp;

RS（Back）：5’- TCC TTT CTA TGC GGC CCA GCC GGC CAT GGC CCA GGT GCA GCT GCA GC A GTC TGG-3’

RS（For）：5’- CGG CGC ACC TGC GGC CGC CCG TTT GAT TTC CAG CTT GGT GCC-3’

Wherein: draw horizontal line part and represent the homologous site with phage vector pCANTAB 5E;

2. the acquisition of each monoclonal anti body weight, chain variable region gene fragment and quantitative:

(1) extract total RNA: adopt the total RNA extraction reagent box of Tian Gen company and extract to specifications total RNA that can produce hybridoma strain 1C11,2C9,1C8,10G4,3G1;

(2) synthetic cDNA: total RNA of the each hybridoma strain obtaining take step 1 is template, oligo (dT) ₁₅for primer, according to SuperScript ^tM-2 II ThermoScript II specification sheetss carry out reverse transcription, synthetic cDNA the first chain; Primer oligo (dT) ₁₅by Invitrogen, buied;

(3) PCR method clone variable region gene: take the cDNA of each hybridoma strain as template, correspondingly respectively get each cDNA the first chain reaction thing 2 μ l, 10 × PCR Buffer, 5 μ l, dNTP (2.5mmol/L) 4 μ l, VH (or VL) is primer (10 μ mol/L) 1 μ l (Back), VH (or VL) is primer (10 μ mol/L) 1 μ l (For), sterile pure water 37 μ l to 50 μ l, vortex mixes, of short duration centrifugal after, carry out pcr amplification reaction, reaction conditions is 94 ℃ of sex change 2min, add after 0.5 μ l Pyrobest DNA enzyme, 94 ℃ of sex change 30s, 55 ℃ of (if amplification variable region of light chain annealing temperature changes 60 ℃ into) annealing 1min, 72 ℃ are extended 1min, 30 circulations, 72 ℃ are extended 5min.PCR product, after 1% agarose gel electrophoresis separates, is purified and is reclaimed DNA fragmentation with test kit.

(4) prepare 1.0% sepharose, light, the heavy chain variable region gene of the 1C11 that clone is obtained, 2C9,1C8,10G4,3G1 are respectively got respectively 2 μ l and are joined in 3 μ l TE damping fluids, and respectively add 1 μ l 6 × loading buffer, mix standby, above-mentioned all ready samples are loaded on sepharose successively, at two swimming lanes of centre, add respectively DL2000 DNA marker 125ng and 250ng simultaneously, electrophoresis is until tetrabromophenol sulfonphthalein migrates to 2/3 place of gel, after EB dyeing, gel imaging system imaging, fully exposure make different antibodies variable region gene fragment and DNA marker all high-visible, then by with the comparison of different concns DL2000 DNA marker staining power, estimation obtains heavy chain variable region gene fragment and the definite amount of chain variable region gene fragment of each antibody, according to estimation result, light by each antibody, heavy chain variable region gene respectively get 50ng respectively correspondence join in two EP pipes, be labeled as respectively chain variable region gene V _hmix and heavy chain variable region gene V _lmix, as the hybrid template of next step PCR reaction.

3. gently, the mixing connection of heavy chain variable region gene and the structure of scFv gene

Use overlapping extension (splicing by overlap extension, SOE) method of PCR is spliced antibody heavy chain variable region gene mixture and antibody chain variable region gene mixture random combine, and introduces connection peptides (Linker) encoding sequence ((Gly ₄ser) ₃), to complete the structure of scFv gene.Separately, for the ease of the structure in next step library, also the scFv fragment two ends after splicing completes respectively add at least carrier pCANTAB 5E homologous sequence of 15bp.Concrete grammar is as follows:

PCR system is as follows:

10× Ex taq Buffer 5μl

dNTP(2.5 mmol/L) 4μl

V _hmix 50ng

V _lmix 50ng

Linker forward 1 μ l

The reverse 1 μ l of Linker

Ex taq DNA polysaccharase 1 μ l

DdH2O mends to total system 50 μ l

PCR program is: 94 ℃ of 1min, 63 ℃ of 4min, and 7 circulations of coamplification, to complete the structure of scFv gene;

Then in system, add following material:

10× Ex taq Buffer 5μl

dNTP(2.5 mmol/L) 4μl

RS（Back） 1μl

RS（For） 1μl

Ex taq DNA polysaccharase 1 μ l

DdH2O mends to total system 50 μ l

PCR program is: 94 ℃ of 1min, 56 ℃ of 2min, 72 ℃ of 2min, then increase 30 and circulate, at least to comprise the carrier pCANTAB 5 E homologous sequences of 15bp in the each introducing in scFv gene fragment two ends.

After completion of the reaction, take out the 1% agarose gel electrophoresis detection of 2 μ l PCR products and confirm to obtain the splicing DNA fragmentation that object size is 750bp, see Fig. 1.

In order to obtain more diversity, by V _hmix EP pipe and V _lthe sample mixing in EP pipe all carries out pcr amplification in batches, and reaction system and reaction conditions are the same.After having increased, all amplified productions are reclaimed with sepharose DNA purification kit.

4. scFv mixes being connected of pCANTAB 5 E carriers of fragment and double digestion processing

(1) double digestion is processed pCANTAB 5 E:

Sfi I single endonuclease digestion:

According to following system preparation reaction solution: pCANTAB 5 E vector 30 μ l

SfiⅠ 1μl

10×M Buffer 10μl

DdH ₂o mends to total system 100 μ l

After 50 ℃ of water-bath 2 h, with sepharose DNA purification kit, reclaim.

Not I enzyme is cut:

According to following system preparation reaction solution: pCANTAB 5 E Sfi I single endonuclease digestions, reclaim product 30 μ l

NotⅠⅠ 1μl

10×H Buffer 10μl

DdH ₂o mends to total system 100 μ l

After 37 ℃ of water-bath 4 h, with sepharose DNA purification kit, reclaim;

(2) according to following system, carry out In-Fusion connection:

The pCANTAB 5 E vector 120ng of Sfi I/Not I double digestion processing

ScFv mixes fragment 40ng

5×In-Fusion buffer 2μl

In-Fusion Enzyme 1μl

DdH ₂o mends to total system 10 μ l

After 37 ℃ of water-bath 15min, put into 50 ℃ of water-bath 15min, be placed on immediately 5min on ice after water-bath, add 40 μ l TE damping fluids, after mixing ,-20 ℃ of preservations are stand-by.

5. scFv mixes the electricity conversion of the connector of fragment and pCANTAB 5 E carriers

ScFv being mixed to the product that is connected of fragment and pCANTAB 5 E carriers gets 5 μ l and joins 50 μ l e. coliin TG1 Electroporation-competent cells, after mixing, the 0.1 cm electricity that joins precooling transforms in cup (Bio-RAD), place 10min on ice, be then placed on Bio-rad electricity conversion instrument and carry out electricity conversion, electric conversion condition: 1.8 kV, 200 Ω, 25 μ F, electricity transforms after adding 0.5 mL 2YT liquid nutrient medium piping and druming in cup and is transferred in the clean 1.5 mL EP pipes after a sterilizing at electricity immediately after transforming, 37 ℃ of slow joltings, 1 h that recovers.

For guaranteeing storage capacity, the sample for building storehouse is adopted as the method for transformation that powers on carries out electricity under same electric conversion condition in batches and transformed, so that whole sample electricity are transformed.Electricity can transform the sample obtaining by electricity and all be coated with several piece SOBAG flat board after transforming, and in 30 ℃ of constant incubators, is inverted overnight incubation, and scrape the lawn growing in sample plate next day, adds after glycerine, and-70 ℃ save backup.

Embodiment 2: utilize aflatoxin recombinant single chain antibody 1A7, CHARACTERISTICS IDENTIFICATION and the application thereof of above-mentioned aflatoxin positive antibody gene pool screening aflatoxin recombinant single chain antibody, acquisition

1. the screening of aflatoxin recombinant single chain antibody

(1) rescue of aflatoxin positive antibody gene pool:

By coating SOBAG flat board after the bacterium liquid dilution of the phage display aflatoxin positive antibody gene pool building, in 30 ℃ of constant incubators, be inverted overnight incubation; Next day, by the random picking of single bacterium colony on flat board, in 96 hole microbial culture plates, every hole adds 200 μ l 2YT-AG, 225 rpm, 30 ℃ of overnight incubation, this plate is labeled as master plate, and is placed on moisturizing in the box that 96 hole microbial culture plates are lined with wet filter paper; Next day, prepare 96 orifice plates that another piece is new, the inside adds and has added in advance 2.5 × 10 ¹⁰the 2YT-AG substratum of the M13KO7 helper phage of pfu, every hole 180 μ l, this plate is numbered plate P1, then the bacterium liquid that 20 μ l incubated overnight are got in every hole in master plate joins in the corresponding aperture of plate P1, and 37 ℃, slow jog 2 h of 150 rpm, now bacterium liquid should become muddy, then centrifugal 20 min of 1500rpm, carefully suck substratum supernatant with rifle head, and every hole adds 200 μ l 2YT-AK substratum (100 μ g/ml penbritins; 50 μ g/ml kantlex) resuspended bacterial sediment, 250 37 ℃ of rpm overnight incubation; Next day, by plate P1, with centrifugal 20 min of 1500 rpm, supernatant is transferred in 96 new hole microbial culture plates, and 4 ℃ save backup.

(2) screening of aflatoxin recombinant single chain antibody:

Use AFB1-BSA(400ng/ hole) and BSA(400ng/ hole) (as negative control) coated ELISA plate respectively, 4 ℃ are spent the night; Next day, incline and fall after coating buffer, PBST washes plate 3 times, then with 4% PBSTM, seals 1 h; PBST washes plate 3 times, and every hole adds 50 ul 4% PBSTM after adding the supernatant 50 μ l that obtain in the rescue of aflatoxin positive antibody gene pool in above-mentioned steps (1) again, rocks gently elisa plate, after mixing, and 37 ℃ of insulation 1 h; PBST washes after plate 3 times, and every hole adds with confining liquid by the HRP/ANTI-M13 of 1:5000 dilution proportion 100 μ l, 37 ℃ of insulation 1 h; PBST washes plate 6 times, and every hole adds the freshly prepared TMB substrate solution of 100 μ l, 37 ℃ of insulation 15 min; Add 2mol/L H ₂sO ₄, every hole 50 μ l stopped reactions, measure respectively the OD450 value of AFB1-BSA plate and BSA plate by microplate reader, and the hole of P/N>=2.1 is judged to positive colony hole.

With coating buffer preparation AFB1-BSA, to final concentration 2 μ g/ml, be coated with 96 hole ELISA plates, every hole 100 μ l, 4 ℃ of coated spending the night; Next day, incline and fall after coating buffer, PBST washes plate 3 times, then with 4% PBSTM, seals 1 h; Get AFB ₁standard substance storage liquid, with confining liquid gradient dilution to a ten different working concentration, by the ascending order of aflatoxin B1 concentration, join successively in ten row in the middle of ELISA plate (50 μ l/hole), every hole adds the phage of the suitable extension rate of 50 μ l again, and each toxin concentration repeats 3 times; Last row of elisa plate only add PBST as blank, and the positive colony supernatant that first row adds 50 μ l PBST and the above-mentioned indirect ELISA method of 50 μ l to screen, as B ₀, after jog plank mixes, put 37 ℃ of incubators and react 1 h; PBST washes after plate 3 times, and every hole adds 100 ul confining liquid by the HRP/ANTI-M13 of 1:5000 dilution proportion, 37 ℃ of insulation 1 h; PBST washes plate 6 times, and every hole adds the freshly prepared TMB substrate solution of 100 μ l, 37 ℃ of insulation 15 min; Add 2 mol/L H ₂sO ₄, every hole 50 μ l stopped reactions, measure respectively OD by microplate reader ₄₅₀value; According to aflatoxin B1 concentration to B/B ₀value is drawn competition curve, calculates IC ₅₀.Screening obtains all higher holes (first A7 hole above elisa plate) of light absorption value and sensitivity thus, in picking master plate corresponding bacterium liquid in addition enlarged culturing be aflatoxin recombinant single chain antibody 1A7.

Particularly, described aflatoxin recombinant single chain antibody 1A7 can obtain by the following method:

1) obtain and can secrete the bacterium liquid of aflatoxin recombinant single chain antibody 1A7, streak culturely on SOBAG flat board isolate single bacterium colony, random single bacterium colony of picking adds in 3 ml 2YT-AG liquid nutrient mediums, 225 rpm, 30 ℃ of overnight incubation;

2) good 1A7 sample is inoculated in 100 ml 2 × YT-AG liquid nutrient mediums to get 1 ml incubated overnight, in 37 ℃ of constant-temperature tables, is cultured to OD600=0.5-0.8;

3) in intestinal bacteria: helper phage number joins helper phage M13KO7 in the bacterium liquid that step (2) shakes than the ratio that is 1:5, and 37 ℃ of constant-temperature tables are cultivated 1 h;

4) 4 ℃, 5 000 rpm, frozen centrifugation 10 min, the careful supernatant of removing in aseptic operating platform, bacterial sediment is resuspended with 100 ml 2YT-AK liquid nutrient mediums, and 37 ℃, 225 rpm overnight incubation;

5) overnight culture is with 12000 rpm, and 4 ℃ of frozen centrifugation 20 min, carefully move to supernatant in new centrifuge tube in aseptic operating platform, add the PEG/NaCl aqueous solution of 1/5 volume, ice bath 1 h, the phage in precipitation supernatant;

6) 4 ℃, 12000 rpm, frozen centrifugation 20 min, outwell supernatant, and by resuspended the phage 10ml PBS that is deposited in centrifuge tube bottom, 4 ℃ save backup.

(3) characteristic of aflatoxin recombinant single chain antibody 1A7 and sequencing analysis result are as follows:

Adopt indirect competitive ELISA method to measure the antibodies specific of aflatoxin recombinant single chain antibody 1A7, specifically with cross reacting rate, describe, testing method is as follows: by AFB ₁, AFB ₂, AFG ₁, AFG ₂and AFM ₁five kinds of different standards product storage liquid, with confining liquid gradient dilution to a ten different working concentration, under equal conditions, adopt indirect competitive ELISA method to measure, draw successively the competitive ELISA curve of five kinds of aflatoxin, obtain standard substance concentration when inhibiting rate is 50% separately, use IC ₅₀represent, and calculate cross reacting rate according to following calculation formula: cross reacting rate (%)=(AFB ₁iC ₅₀/ analogue IC ₅₀) × 100%.Obtain the 50% inhibition concentration IC of aflatoxin single-chain antibody 1A7 for aflatoxin B1 ₅₀be 20 pg/mL, its cross reacting rate for five kinds of main aflatoxin B1, B2, G1, G2, M1 is respectively 100%, 166%, 100%, 0.1% and 11.1%, and this aflatoxin recombinant single chain antibody 1A7 is the general recombinant antibodies of a kind of aflatoxin as seen.

The characteristic sequence of aflatoxin recombinant single chain antibody 1A7 is measured: the clone bacterium liquid of aflatoxin recombinant single chain antibody 1A7 is delivered to Shanghai Invitrogen company and carry out sequencing analysis, sequencing primer is phage vector universal primer R1:5 '-CCA TGA TTA CGC CAA GCT TTG GAG CC-3 ', and sequencing result adopts GENETOOL software and uploads website IGMT/V-QUEST (http://www.imgt.org/IMGT_vquest/) and carry out antibody variable region sequential analysis.Obtaining its aminoacid sequence is shown in SEQ ID NO:1, wherein weight chain variable region amino acid sequence is shown in SEQ ID NO:2, light chain variable region amino acid sequence is shown in SEQ ID NO:3, coding gene sequence is shown in SEQ ID NO:4, wherein variable region of heavy chain coding gene sequence is shown in SEQ ID NO:5, and variable region of light chain coding gene sequence is shown in SEQ ID NO:6.

Compared with traditional monoclonal antibody or polyclonal antibody, recombinant single chain antibody can be produced in a short period of time in a large number.The every 500ml inoculum of recombinant single chain antibody approximately can obtain 5mg-50mg single-chain antibody within 2 day time; And conventional hybridization

Every mouse of knurl monoclonal antibody needs the time in 2-8 week just can obtain about 5mg-10mg monoclonal antibody.

Aflatoxin recombinant single chain antibody 1A7 can effectively identify aflatoxin B1, B2, G1 etc., can be applicable to the immunology detection field of aflatoxin B1, B2, G1 etc., comprise for the ELISA of the total aflatoxin contents such as the agricultural-food of food safety domain-specific, food detect, liquid phase chromatography detects sample pre-treatments in aflatoxin toxin etc., significant to the monitoring of food safety field aflatoxin.

Sequence table

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

<120> aflatoxin positive antibody gene pool, construction process, purposes and aflatoxin recombinant single chain are anti-

Body 1A7

<160> 6

<210> 1

<211> 261

<212> PRT

<213> mouse

<400> 1

Met Ala Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys

1 5 10 15

Pro Gly Gly Pro Leu Lys Leu Ser Cys Ser Ala Ser Gly Phe Thr

20 25 30

Phe Ser Asn Tyr Gly Met Ser Trp Leu Arg Gln Thr Ala Glu Lys

35 40 45

Arg Lle Glu Trp Val Ala Ser Ile Ser Gly Gly Ser Tyr Ser Tyr

50 55 60

Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75

Ala Lys Asn Asn Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu

80 85 90

Asp Thr Ala Lys Tyr Phe Cys Ala Ser His Asp Tyr Ala Trp Ser

95 100 105

Phe Gly Val Trp Gly Ala Gly Ala Thr Val Thr Val Ser Ser Ser

110 115 120

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

125 130 135

Ser Ala Leu Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly

140 145 150

Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Ser Gly Ala Val Thr

155 160 165

Thr Ser Asn Ser Ala Asn Trp Ile Gln Glu Lys Ala Asp His Leu

170 175 180

Phe Thr Gly Leu Ile Gly Gly Ile Asn Lys Arg Ala Pro Gly Val

185 190 195

Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu

200 205 210

Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Val Tyr Phe Cys

215 220 225

Ala Leu Trp Asp Ser Asn His Leu Val Phe Gly Gly Gly Thr Lys

230 235 240

Leu Thr Val Leu Gly Ala Ala Ala Gly Ala Pro Val Pro Tyr Pro

245 250 255

Asp Pro Leu Glu Pro Arg

260

<210> 2

<211> 121

<212> PRT

<213> mouse

<400> 2

Met Ala Gln Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys

1 5 10 15

Pro Gly Gly Pro Leu Lys Leu Ser Cys Ser Ala Ser Gly Phe Thr

20 25 30

Phe Ser Asn Tyr Gly Met Ser Trp Leu Arg Gln Thr Ala Glu Lys

35 40 45

Arg Lle Glu Trp Val Ala Ser Ile Ser Gly Gly Ser Tyr Ser Tyr

50 55 60

Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

65 70 75

Ala Lys Asn Asn Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu

80 85 90

Asp Thr Ala Lys Tyr Phe Cys Ala Ser His Asp Tyr Ala Trp Ser

95 100 105

Phe Gly Val Trp Gly Ala Gly Ala Thr Val Thr Val Ser Ser Ser

110 115 120

Ser

<210> 3

<211> 125

<212> PRT

<213> mouse

<400> 3

Ala Leu Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Ser Gly Ala Val Thr Thr

20 25 30

Ser Asn Ser Ala Asn Trp Ile Gln Glu Lys Ala Asp His Leu Phe

35 40 45

Thr Gly Leu Ile Gly Gly Ile Asn Lys Arg Ala Pro Gly Val Pro

50 55 60

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

65 70 75

Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Val Tyr Phe Cys Ala

80 85 90

Leu Trp Asp Ser Asn His Leu Val Phe Gly Gly Gly Thr Lys Leu

95 100 105

Thr Val Leu Gly Ala Ala Ala Gly Ala Pro Val Pro Tyr Pro Asp

110 115 120

Pro Leu Glu Pro Arg

125

<210> 4

<211> 783

<212> DNA

<213> mouse

<400> 4

atggcccagg tcaaactgca gcagtcaggg ggaggcttag tgaagcctgg agggcccctg 60

aaactctcct gttcagcctc tggattcact ttcagtaact atggcatgtc ttggcttcgc 120

cagaccgcgg agaagaggct ggagtgggtc gcaagtatta gtggtggtag ttatagctac 180

tatccagaca gtgtgaaggg gcgattcacc atctccagag acaatgccaa gaacaacctg 240

tacctacaaa tgagtagtct gaagtctgag gacacggcct tgtatttctg tgcaagtcat 300

gattacgcct ggtccttcgg tgtctggggc gcaggggcca cggtcaccgt ctcctcatcc 360

tcaggtggcg gtggctccgg cggtggtggc agcggtggcg gcggttctgc tcttgtgact 420

caggaatctg cactcaccac atcacctggt gaaacagtca cactcacttg tcgctcaagt 480

agtggggctg ttacaactag taactctgcc aactggatcc aagaaaaagc agatcattta 540

ttcactggtc taataggtgg cattaacaag cgagctccag gtgttcctgc cagattctca 600

ggctccctga ttggagacaa ggctgccctc accatcacag gggcacagac tgaggatgag 660

gcagtatatt tctgtgctct atgggacagc aaccatttgg tgttcggtgg aggaaccaaa 720

ctgactgtcc taggtgcggc cgcaggtgcg ccggtgccgt atccggatcc gctggaaccg 780

cgt 783

<210> 5

<211> 363

<212> DNA

<213> mouse

<400> 5

atggcccagg tcaaactgca gcagtcaggg ggaggcttag tgaagcctgg agggcccctg 60

aaactctcct gttcagcctc tggattcact ttcagtaact atggcatgtc ttggcttcgc 120

cagaccgcgg agaagaggct ggagtgggtc gcaagtatta gtggtggtag ttatagctac 180

tatccagaca gtgtgaaggg gcgattcacc atctccagag acaatgccaa gaacaacctg 240

tacctacaaa tgagtagtct gaagtctgag gacacggcct tgtatttctg tgcaagtcat 300

gattacgcct ggtccttcgg tgtctggggc gcaggggcca cggtcaccgt ctcctcatcc 360

tca 363

<210> 6

<211> 375

<212> DNA

<213> mouse

<400> 6

gctcttgtga ctcaggaatc tgcactcacc acatcacctg gtgaaacagt cacactcact 60

tgtcgctcaa gtagtggggc tgttacaact agtaactctg ccaactggat ccaagaaaaa 120

gcagatcatt tattcactgg tctaataggt ggcattaaca agcgagctcc aggtgttcct 180

gccagattct caggctccct gattggagac aaggctgccc tcaccatcac aggggcacag 240

actgaggatg aggcagtata tttctgtgct ctatgggaca gcaaccattt ggtgttcggt 300

ggaggaacca aactgactgt cctaggtgcg gccgcaggtg cgccggtgcc gtatccggat 360

ccgctggaac cgcgt 375

Claims (2)

1. aflatoxin recombinant single chain antibody 1A7, it is characterized in that: the aminoacid sequence of described aflatoxin recombinant single chain antibody 1A7 is as shown in SEQ ID NO:1, wherein weight chain variable region amino acid sequence is as shown in SEQ ID NO:2, and light chain variable region amino acid sequence is as shown in SEQ ID NO:3.

2. aflatoxin recombinant single chain antibody 1A7 according to claim 1, it is characterized in that: the coding gene sequence of described aflatoxin recombinant single chain antibody 1A7 is as shown in SEQ ID NO:4, wherein variable region of heavy chain coding gene sequence is as shown in SEQ ID NO:5, and variable region of light chain coding gene sequence is as shown in SEQ ID NO:6.

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