CN105037539B - Anti- Penicillium citrinum toxin single-chain antibody and preparation method and application - Google Patents
Anti- Penicillium citrinum toxin single-chain antibody and preparation method and application Download PDFInfo
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- CN105037539B CN105037539B CN201510499973.9A CN201510499973A CN105037539B CN 105037539 B CN105037539 B CN 105037539B CN 201510499973 A CN201510499973 A CN 201510499973A CN 105037539 B CN105037539 B CN 105037539B
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Abstract
The present invention provides a kind of single-chain antibody of anti-Penicillium citrinum toxin.The present invention carries out affine screening using artificial coupled antigen Penicillium citrinum toxin-BSA/BSA and Penicillium citrinum toxin-OVA/OVA antigen combinations to antibody ribosomes mRNA triplets complex, affine screening product is recycled using RT PCR methods in situ, the screening of single-chain antibody is carried out using ribosomal display technology, the genetic fragment screened carries out identification acquisition after prokaryotic expression using ELISA method.The present invention enormously simplifies screening and the preparation process of antibody, reduces the interference of protein carrier, improves the probability for obtaining specific single-chain antibody；Improve recycling yield.In view of Penicillium citrinum toxin possessed renal toxicity, teratogenesis and carcinogenicity in itself, the serious threat health of the mankind and domestic animal, anti-Penicillium citrinum toxin single-chain antibody provided by the invention can be used as and measure reagent needed for Penicillium citrinum endotoxin contamination residue detection in agricultural product and food samples.
Description
Technical field
The invention belongs to biotechnologys.It is related to a kind of single-chain antibody of anti-Penicillium citrinum toxin and preparation method and application.
Background technology
Penicillium citrinum toxin（Citrinin, CIT）It is by the mycetogenetic one kind such as Penicillium and aspergillus toxic secondary generation
It thanks to product, is widely present in a variety of agricultural product such as barley, red rice, oat, flour and food.Since it has strong kidney poison
Property and carcinogenicity, huge threat is constituted to the health of the mankind, so needing to establish high sensitivity, high specificity, easy to be fast
Prompt detection method is detected the Penicillium citrinum toxin in agricultural product and food, it is ensured that agricultural product and food security.
The detection of Penicillium citrinum toxin mainly has thin-layered chromatography and high performance liquid chromatography etc., toxin can be carried out qualitative
With quantitative detection, but instrument and equipment is expensive, complicated for operation, more demanding to sample treatment, and cost is also high, should not clinically push away
It is wide to use.And immunology detection such as enzyme linked immunosorbent assay, there are higher sensitivity and specificity, it is not high to sample requirement,
Testing cost is cheap, has a good application prospect.Therefore, it is to grind at present to establish Penicillium citrinum toxin residual immunological detection method
The hot spot studied carefully.
The key for establishing immunological detection method is antibody, phage single chain antibody（single chain
variable fragments, scFv）It is the minimum function active structure unit with antibody activity, is to utilize genetic engineering
Heavy chain of antibody variable region (VH), light chain variable region (VL) are formed by connecting by technical method, since it is with immunogenicity is relatively low, phase
, tissue penetration small to molecular mass is strong, can be expressed in prokaryotic system, is easy to the features such as large-scale production, extensively
It is general to be applied to the multinomial medical domain such as drug targeting and immune biological therapy.Phage display and ribosomes exhibition are mainly used at present
Show technology to screen and prepare single-chain antibody.Wherein, ribosomal display technology is that one kind carries out albumen or more in vitro completely
Peptide screening and the technology evolved, have structure high power capacity (>=1011More than), high quality gene library, be easy to screen and express spy
The albumen of the opposite sex, be easy to carry out Evolution in vitro and recombination, can to have cytotoxic molecule be shown and screen etc. it is special
Point, the extensive use of this technology and develop into preparation and screening small protein, polypeptide and antibody open one it is new
Approach has obtained a variety of high-affinity antibodies using this technology at present, and ribosomal display technology screening is utilized to be directed to tangerine
The single-chain antibody of mycotoxin of penicillium has not been reported.
Invention content
The purpose of the present invention is to provide a kind of single-chain antibodies of anti-Penicillium citrinum toxin, and amino acid sequence is respectively such as SEQ
ID No：1、SEQ ID No：2 and SEQ ID No：Shown in 3.
Second object of the present invention is to provide a kind of preparation method of anti-Penicillium citrinum toxin single-chain antibody, by following
Step prepares：
1. being taken under aseptic condition without any immune Balb/C mouse spleens, mouse spleen total serum IgE is extracted, through reversion
After record synthesis cDNA, PCR expands VH, Linker, VL and C kappa gene segment respectively.
2. VH, Linker and VL genetic fragment using purifying recycling are template, pass through Overlapping PCR
(Splicing by Overlap Extension, SOE) PCR method assembles single-chain antibody scFv gene libraries.
3. the single-chain antibody scFv gene libraries and C kappa genes using purifying recycling are template, pass through Overlapping PCR
PCR method builds ribosomal display gene library.
4. using constructed ribosomal display gene library as template, using TNT Quick Coupled
Transcription/Translation Systems in-vitro transcription translation kits, prepare antibody-ribosomal-mRNA three
Composite（ARM）.
5. using artificial coupled antigen Penicillium citrinum toxin-BSA/BSA and Penicillium citrinum toxin-OVA/OVA to antibody-core
Sugared body-mRNA triplet complexs（ARM）Carry out affine screening.
6. being recycled using RT-PCR method in situ to affine screening product, products therefrom is the DNA bases after screening
Because of segment.Outer transcription and translation-affine screening-original position the RT-PCR of repeat body recycles this process, after six wheel screenings, obtains high
The genetic fragment of abundance.
7. the genetic fragment obtained through sequencing and after comparing analysis, it is found that 32 sequences are complete, without lethal prominent
Become.All genetic fragments with expression vector pTIG-TRX are connected and form recombinant plasmid in e. coli bl21（DE3）Middle progress
Prokaryotic expression takes expression product to surpass the precipitation of the supernatant after splitting and carries out SDS-PAGE and WB identifications respectively.
8. coupled antigen CIT-BSA and carrier protein BSA are used respectively as envelope antigen, the induction of empty carrier bacterium colony
Product surpasses expression product using indirect ELISA detection method and splits the combination activity of rear supernatant and identify as blank control.
The super Competitive assays effect for splitting rear supernatant is identified using racing ELISA detecting method.It is green that 3 anti-tangerines are obtained altogether
The single-chain antibody of mould toxin is scFv 23, scFv 68 and scFv 109 respectively.
9. utilize Penicillium patulum toxin（PAT）, aflatoxin B1（AFB1）, fumonisin B1（FB1）And ochratoxin
A（OTA）The specificity of 3 anti-Penicillium citrinum toxin single-chain antibodies obtained is identified.
Third object of the present invention is to provide use of the anti-Penicillium citrinum toxin single-chain antibody in detection kit is prepared
On the way.Application especially in measure agricultural product and food samples are prepared in the remaining detection kit of Penicillium citrinum endotoxin contamination.
In view of Penicillium citrinum toxin possessed renal toxicity, teratogenesis and carcinogenicity in itself, the serious threat mankind and domestic animal
Health, establish Penicillium citrinum endotoxin contamination residue detection kit and be very necessary.A kind of anti-Penicillium citrinum provided by the invention
Toxin single-chain antibody can be used as and measure reagent needed for Penicillium citrinum endotoxin contamination residue detection in agricultural product and food samples.
Invention utilizes molecule by extraction without any immune Balb/C mouse spleen mRNA, reverse transcription cDNA
Biological Principles amplify antibody heavy chain variable region gene library and chain variable region gene library respectively, build ribosomal display
Gene library.In-vitro transcription and translation are carried out using ribosomal display technology, it is manually even using Penicillium citrinum toxin-protein carrier
Associated antigen carries out affine screening, and the genetic fragment screened is identified after prokaryotic expression using ELISA method, obtains anti-tangerine
The specific single-chain antibody of mycotoxin of penicillium.
The beneficial effects of the present invention are：（1）The screening of single-chain antibody is carried out using ribosomal display technology, is greatly simplified
The screening of antibody and preparation process, using artificial coupled antigen Penicillium citrinum toxin-BSA/BSA and Penicillium citrinum toxin-OVA/
OVA antigen combinations are to antibody-ribosomal-mRNA triplet complexs（ARM）Affine screening is carried out, is reduced to the maximum extent
The interference of protein carrier improves the probability for obtaining specific single-chain antibody；（2）Using RT-PCR method in situ to affine screening
Product is recycled, and is reduced influences of the RNase to ribosomes composite construction, is improved recycling yield；（3）3 are obtained altogether
The single-chain antibody gene segment of anti-Penicillium citrinum toxin is scFv 23, scFv 68 and scFv 109 respectively, and all has preferable
Sensibility and specificity, wherein scFv 23 is to the IC of CIT50It is worth for 3.2705 μ g/mL, minimum detection limit LOD:IC10For
0.0111 μg/mL；The IC of scFv 6850It is worth for 1.2422 μ g/mL, minimum detection limit LOD:IC10For 0.0031 μ g/mL；
The IC of scFv 10950It is worth for 2.7626 μ g/mL, minimum detection limit LOD:IC10For 0.0061 μ g/mL；ScFv 68 is green with exhibition
Mould toxin, aflatoxin B1, fumonisin B1 and ochratoxin A cross reacting rate be below 0.01%；（4）Both at home and abroad
Anti- Penicillium citrinum toxin single-chain antibody is prepared for the first time, and prepares Penicillium citrinum endotoxin contamination in agricultural product and food using single-chain antibody
Residue detection kit.
Description of the drawings
Fig. 1 is that VH, Linker, VL, C κ, single-chain antibody scFv gene libraries and ribosomal display gene library PCR expand
Increase result.Wherein A schemes：1：VH；2：VL；B schemes：1：Cκ；C schemes：1：Linker；D schemes：1：Single-chain antibody scFv gene libraries；2：
Ribosomal display gene library；M：100bp DNA ladder marker.
Fig. 2 is that constructed single-chain antibody gene library sequence compares analysis result.
Fig. 3 is the genetic fragment PCR amplification result obtained after six wheel ribosomal display-affine screenings.M in figure：
100bp DNA ladder marker；1：The genetic fragment PCR amplification obtained after first round ribosomal display-affine screening
As a result；2：The genetic fragment PCR amplification result obtained after second wheel ribosomal display-affine screening；3：Third round ribosomes
The genetic fragment PCR amplification result obtained after displaying-affine screening；4：It is obtained after fourth round ribosomal display-affine screening
The genetic fragment PCR amplification result obtained；5：The genetic fragment PCR amplification obtained after 5th wheel ribosomal display-affine screening
As a result；6：The genetic fragment PCR amplification result obtained after 6th wheel ribosomal display-affine screening.
Fig. 4 is SDS-PAGE the and WB qualification results of single-chain antibody prokaryotic expression product.1 and 4 in figure:Expression product surpasses
Split rear supernatant qualification result；2 and 5:The full dientification of bacteria result of expression product；3 and 6:Expression product surpasses split after precipitate qualification result；
7 and 8:Expression product, which surpasses, splits rear supernatant WB qualification results；M: protein standards.
Fig. 5 is that indirect ELISA detection single-chain antibody expression product surpasses the combination activity for splitting rear supernatant.
Fig. 6 is that competitive ELISA detection single-chain antibody expression product surpasses the inhibition for splitting rear supernatant.
Specific embodiment
The present invention is described further with reference to the drawings and specific embodiments.It should be understood that these embodiments are merely to illustrate
Purpose rather than the limitation scope of the invention.
Embodiment 1：The structure of ribosomal display gene library
1. the extraction of RNA and the synthesis of cDNA：It is taken under aseptic condition without any immune mouse spleen, according to examination
Agent illustrates to operate, and mouse spleen total serum IgE is extracted with Trizol methods.According to ReverTra Ace- α-reverse transcription reagent box explanation
Operation synthesizes cDNA using mouse spleen total serum IgE through reverse transcription.
2. the design and synthesis of primer：Separately design VH, Linker, VL, C kappa gene segment and structure ribosomal display
PCR primer needed for gene library, primer sequence are as shown in table 1.
3. the amplification of VH, Linker, VL, C kappa gene segment：Using the mouse cDNA of synthesis for template, PCR amplification VH,
Linker, VL and C kappa gene segment.Reaction condition is respectively：
VH：95 ℃ 3 min；95 DEG C of 30 s, 59 DEG C of 1 min, 72 DEG C of 1 min, totally 30 recycle；72 ℃ 10
min；
Linker：95 ℃ 3 min；95 DEG C of 30 s, 61 DEG C of 1 min, 72 DEG C of 1 min, totally 30 recycle；72
℃ 10 min；
VL：95 ℃ 3 min；95 DEG C of 30 s, 53 DEG C of 1 min, 72 DEG C of 1 min, totally 30 recycle；72 ℃ 10
min；
Cκ：95 ℃ 3 min；95 DEG C of 30 s, 58 DEG C of 1 min, 72 DEG C of 1 min, totally 30 recycle；72 ℃ 10
min；After reaction, all pcr amplification products are identified through 1 % agarose gel electrophoresis, and purpose item is observed under ultraviolet lamp
Band, as shown in Figure 1, the VH genetic fragments of visible about 360 bp, the Linker genetic fragments of about 86 bp, about 330 respectively
The C kappa gene segments of the VL genetic fragments of bp and about 338 bp, it is consistent with expected clip size.Utilize Wizard SV
Gel and PCR Clean-up System gel reclaims kit specifications are operated, to purpose band carry out purifying and
Recycling.
4. the structure of single-chain antibody scFv gene libraries：VH, Linker and VL genetic fragment using purifying recycling is mould
Plate assembles single-chain antibody by Overlapping PCR (Splicing by Overlap Extension, SOE) PCR method
ScFv gene libraries, reaction are carried out in two steps：
First step reaction is carried out under conditions of primer is not added with, and reaction condition is：95 ℃ 3 min；95 DEG C of 30 s,
62 DEG C of 30 s, 72 DEG C of 2 min, totally 8 recycle；72 ℃ 10 min；
Second step reaction only needs to add in primer V in original PCR pipeHR and VLF, reaction condition are：95 ℃ 3
min；95 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 2 min, totally 25 recycle；72 ℃ 10 min；
After reaction, pcr amplification product is identified through 1 % agarose gel electrophoresis, purpose band is observed under ultraviolet lamp, such as
Shown in Fig. 1, it is seen that the genetic fragment of about 750 bp, it is consistent with expected clip size.Utilize Wizard SV Gel and
PCR Clean-up System gel reclaims kits recycle purpose band.By recovery product and pMD18-T carriers into
Row connection overnight, connection product are transformed into 10 competent cells of Escherichia coli Top, monoclonal are selected at random after overnight growth
Bacterium colony carries out PCR identifications, and the positive colony for identifying gained is sent to Bo Shang Bioisystech Co., Ltd and carries out sequence, and profit
Obtained positive colony gene order is compared with NCBI and V-BASE DNAPLOT databases, to constructed scFv
The integrality and diversity of gene library are analyzed, as shown in Fig. 2, the sequencing result warp of the positive monoclonal bacterium colony obtained
Analysis shows that constructed scFv gene libraries size is correct, and variable region gene sequence is different, in most of sequence not
There are terminator codons and lethal mutation, have preferable diversity and integrality, available for ribosomal display.
5. the structure of ribosomal display gene library：Utilize the single-chain antibody scFv gene libraries and C κ bases of purifying recycling
Because of template, ribose is built by Overlapping PCR (Splicing by Overlap Extension, SOE) PCR method
Body display gene library, reaction are carried out in two steps：
First step reaction is carried out under conditions of primer is not added with, and reaction condition is：95 ℃ 3 min；95 DEG C of 30 s,
60 DEG C of 1 min, 72 DEG C of 2 min, totally 8 recycle；72 ℃ 10 min；
Second step reaction only needs to add in primer T7R and C κ F in original PCR pipe, and reaction condition is：95 ℃ 3
min；95 DEG C of 30 s, 50 DEG C of 1 min, 72 DEG C of 2 min, totally 25 recycle；72 ℃ 10 min.
After reaction, pcr amplification product is identified through 1 % agarose gel electrophoresis, purpose band is observed under ultraviolet lamp, such as
Shown in Fig. 1, it is seen that the genetic fragment of about 1100 bp, it is consistent with expected clip size.Utilize Wizard SV Gel and
PCR Clean-up System gel reclaims kits recycle purpose band.By recovery product and pMD18-T carriers into
Connection, connection product are transformed into 10 competent cells of Escherichia coli Top, select monoclonal at random after overnight growth row overnight
Bacterium colony carries out PCR identifications, and the positive colony for identifying gained is sent to Bo Shang Bioisystech Co., Ltd and carries out sequence, and profit
Obtained positive colony gene order is compared, and then to constructed with NCBI and V-BASE DNAPLOT databases
The integrality of ribosomal display gene library is analyzed.
Embodiment 2：Ribosomal display and affine screening
1. in-vitro transcription is translated：Using constructed ribosomal display gene library as template, using TNT Quick
Coupled Transcription/Translation Systems in-vitro transcription translation kits, the standard for preparing 50 μ L are anti-
System is answered, each component is added as shown in table 2, is positioned in 30 DEG C of thermostat water baths and is reacted 60 min, prepares antibody-core
Sugared body-mRNA triplet complexs（ARM）.
2. affine screening：Envelope antigen CIT-OVA and carrier protein OVA are configured to end respectively using buffer solution is coated with
A concentration of 0.1 mg/mL, is added separately in ELISA Plate, and per 100 μ L of hole, 4 DEG C overnight.Liquid in hole is discarded, is washed with PBST
Wash 5 times, every time 3 min.It is closed using 5 % skimmed milks, per hole 100 μ L, 37 DEG C of 2 h of closing.Liquid in hole is discarded, is used
PBSTM is washed 5 times, and 3 min, 300 μ L PBST cleaning solutions are finally added in ELISA Plate, 20 min are pre-chilled on ice every time.
The product that in-vitro transcription is translated is added in the coated enzyme mark holes of carrier protein OVA, it, will after 4 DEG C of 1 h of effect
Liquid is transferred in the coated enzyme mark holes of envelope antigen CIT-OVA in hole, and 4 DEG C of 2 h of effect discard liquid in hole, use PBSTM
Washing 3 times, every time 3 min, then the DEPC water quick wash 2 times with precooling, discard liquid in hole, ELISA Plate are placed on ice in advance
Cold 20 min adds 11 μ L DEPC water and carries out original position RT-PCR recycling.The second affine Select to use comlete antigen CIT-BSA of wheel
It is screened with carrier protein BSA as envelope antigen, two groups of antigens carry out affine screening alternately as envelope antigen.
3. original position RT-PCR is recycled：RT-PCR primer in situ, primer sequence such as 3 institute of table are designed according to spacer region C κ segments
Show, the enzyme mark hole after affine screening carries out reverse transcription recycling, is carried out according to kit specification RT-PCR：65 DEG C of effects 5
Min is placed in and acts on 30 s on ice, and 30 DEG C of 10 min of effect, 42 DEG C of 30 min of effect, 85 DEG C of 5 min of effect, 4 DEG C act on 5
min.Reaction product is the screened cDNA of affine screening.
4. the structure of PCR amplification and new round displaying template：Design one short-movie section primer KZ of synthesis, primer sequence
For（5’- GAACAGACCACCATGSAGG-3’）, using KZ as sense primer, reverse transcription primer is as downstream primer, to being obtained
The cDNA obtained carries out PCR amplification, and reaction condition is：
95 ℃ 3 min；95 DEG C of 30 s, 60 DEG C of 1 min, 72 DEG C of 1 min, totally 30 recycle；72 ℃ 10
min；
Reaction product is the acquired antibody gene of affine screening.After reaction, by all PCR products through 1 %
Agarose gel electrophoresis identifies that observation gained purpose band, utilizes Wizard SV Gel and PCR Clean- under ultraviolet lamp
Up System gel reclaims kits recycle purpose band.So far, the affine screening of a wheel terminates, and PCR recovery products can
For building the template of next round ribosomal display.
Using primer T7R as sense primer, reverse transcription primer expands PCR recovery products, reacts as downstream primer
Condition is：
95 ℃ 3 min；95 DEG C of 30 s, 50 DEG C of 1 min, 72 DEG C of 1 min, totally 30 recycle；72 ℃ 10 min；
Reaction product is the template of new round ribosomal display.Outer transcription and translation-affine screening-original position the RT- of repeat body
PCR recycles this process, after six wheel screenings, as shown in figure 3, with the progress of screening round, the genetic fragment obtained
It is gradually shortened, the abundance of genetic fragment gradually increases.Wherein, primer D2 is for the first round and the reversion after the affine screening of fourth round
Record recycling and PCR amplification, primer D3 is for the reverse transcription recycling after the second wheel and the 5th affine screening of wheel and PCR amplification, primer
D4 is for the reverse transcription recycling after third round and the 6th affine screening of wheel and PCR amplification.
Embodiment 3：The expression and identification of single-chain antibody
1. the cloning and expression of single-chain antibody gene segment：The pcr amplification product for taking abundance higher and pMD18-T carriers
Conversion is attached, all monoclonal bacterium colonies are selected after overnight growth and carry out PCR identifications, identification gained positive colony is through sequencing
Analysis.Select gene order it is complete, without terminator codon, without 32 positive colonies and expression vector of lethal mutation
PTIG-TRX carries out prokaryotic expression.
2. the identification of single-chain antibody prokaryotic expression product：Positive recombinant plasmid is pressed 1:100 ratio is inoculated into 100 mL
LB liquid medium（Containing ampicillin, Amp）In, it is placed in 37 DEG C, carries out to bacterium solution in the constant-temperature table of 250 rpm
OD600 nmWhen being 0.7（About 3.5 h）, add in the IPTG derivants of final concentration of 1 mM, be replaced in 25 DEG C, 130 rpm
Induced expression is carried out in constant-temperature table, bacterium solution is recycled in 50 mL centrifuge tubes after inducing 6 h, 8000 rpm centrifuge 10 min,
Supernatant is discarded, will be precipitated uniformly using the PBS solution of 10 mL, 50 mM, ultrasound cracking thalline, ultrasound are utilized under condition of ice bath
Cracking condition is set as 4 s of processing, is spaced 4 s, super to split 30 min.It is super to split, 8000 rpms centrifugation 10 mins limpid to bacterium solution.It takes
It is super split after supernatant precipitation carry out SDS-PAGE and Western blot identifications, as shown in figure 4, expression product surpass it is upper after splitting
Cleer and peaceful precipitation has apparent band at about 32.0 kDa, and it is consistent to be expected size with target fragment.And purpose in supernatant
The total amount of albumen is higher than the amount in precipitation, and it is to be present in cell membrane and thin with soluble activated state to illustrate prokaryotic expression product
In pericentral siphon chamber between cell wall.
3. the combination activity of indirect ELISA detection single-chain antibody：With coating buffer solution respectively by coupled antigen CIT-BSA
1000 times are diluted with carrier protein BSA, is added in ELISA Plate, per 100 μ L of hole, 4 DEG C overnight.Liquid in hole is discarded, uses PBST
Washing 5 times, 3 min, pats dry every time.It is closed using 5 % skimmed milks, per hole 100 μ L, 37 DEG C of 1 h of closing.It discards in hole
Liquid is washed 5 times with PBST, and 3 min, pats dry every time.Expression product is surpassed to the supernatant after splitting and is separately added into CIT-BSA and BSA
In coated hole, per hole 100 μ L, 37 DEG C of 2 h of closing.Liquid in hole is discarded, is washed 5 times with PBST, 3 min, pats dry every time.
The anti-His tag antibodies of mouse are diluted to suitable working concentration using confining liquid, in adding hole, per 100 μ L of hole, 37 DEG C of incubations
1 h.Liquid in hole is discarded, is washed 5 times with PBST, 3 min, pats dry every time.By the sheep anti-mouse igg of horseradish peroxidase-labeled
Secondary antibody is diluted to appropriate working concentration, in adding hole, per hole 100 μ L, 37 DEG C of 1 h of incubation.Liquid in hole is discarded, uses PBST
Washing 5 times, 3 min, pats dry every time.Add the OPD developing solutions of 100 μ L Fresh, 37 DEG C of 15 min of incubation per hole.Often
Hole respectively adds 50 μ L, 2 M H2SO4Terminate liquid terminates reaction.The light absorption value at 492 nm wavelength is measured using microplate reader, such as Fig. 5 institutes
Show, have in 32 recombinant plasmids 13 can with envelope antigen CIT-BSA occur association reaction, and not with carrier protein BSA
Association reaction occurs, wherein the single-chain antibody that number is 23,68 and 109 has higher OD492 nmValue.
4. competitive ELISA detects the inhibition of single-chain antibody：With the carbonate buffer solution of pH 9.6 by envelope antigen
Penicillium citrinum toxin-BSA is diluted to 5 μ g/mL, and ELISA Plate adds in 100 μ L per hole, and 4 DEG C overnight, and coating buffer of inclining is washed with PBST
Liquid washs 5 times per hole, and each 3min is patted dry；100 μ L, 5% skimmed milk powers are added in per hole, 37 DEG C of closing 1h, incline deblocking liquid, often
Hole is washed 5 times, and each 3min is patted dry；The single-chain antibody of 50 μ L and the Penicillium citrinum of the different extension rates of 50 μ L are respectively added in per hole
Toxin standard items, and control wells are arranged in parallel, using negative serum as negative control, blank control is made with PBST, 37 DEG C are incubated 1h,
Washing 5 times, each 3min is patted dry；The anti-His tag antibodies of mouse are diluted to suitable working concentration, adding hole using confining liquid
It is interior, per hole 100 μ L, 37 DEG C of 1 h of incubation.Liquid in hole is discarded, is washed 5 times with PBST, 3 min, pats dry every time.By horseradish mistake
The sheep anti-mouse igg secondary antibody of oxide enzyme label is diluted to appropriate working concentration, in adding hole, per 100 μ L of hole, 37 DEG C of incubations
1 h.Liquid in hole is discarded, is washed 5 times with PBST, 3 min, pats dry every time.The OPD of 100 μ L Fresh is added to show per hole
Color liquid, 37 DEG C of 15 min of incubation.Add 50 μ L, 2 M H per hole2SO4Terminate liquid terminates reaction.It is measured using microplate reader
492nm light absorption values, OD when no CIT inhibits492 nmNumerical value is as B0, corresponding OD when various concentration CIT inhibits492 nmNumerical value is made
For B, with B/B0For ordinate, a concentration of abscissas of CIT are added with every hole, standard curve are drawn, as shown in fig. 6, with CIT
The increase of concentration, OD492nmValue continuously decreases, and scFv 23 is to the IC of CIT50It is worth for 3.2705 μ g/mL, minimum detection limit
LOD:IC10For 0.0111 μ g/mL；The IC of scFv 6850It is worth for 1.2422 μ g/mL, minimum detection limit LOD:IC10It is 0.0031
μg/mL；The IC of scFv 10950It is worth for 2.7626 μ g/mL, minimum detection limit LOD:IC10For 0.0061 μ g/mL.
5. the identification of cross reaction：Using racing ELISA detecting method, single-chain antibody and Penicillium patulum are detected respectively
Toxin（PAT）, aflatoxin B1（AFB1）, fumonisin B1（FB1）And ochratoxin A（OTA）Between cross reaction
Property, measure corresponding OD492 using microplate readernm, the half inhibiting rate of calculating antibody（IC50）, and then calculate cross reacting rate：CR
（%）=100×IC50（CIT）/ IC50（Other small molecule toxins）, cross reaction qualification result such as the following table 4 of single-chain antibody.
4 single-chain antibody cross reaction qualification result of table shows that the single-chain antibody that the present invention obtains is small to aflatoxin etc.
Molecular antigen cross reaction is extremely low, illustrates that the method for the present invention obtains the specific single-chain antibody for Penicillium citrinum toxin.
Present invention combination most preferred embodiment is described, however after the above for having read the present invention, ability
Field technique personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application appended claims
Book limited range.
<110>Zhejiang University
<120>Anti- Penicillium citrinum toxin single-chain antibody and preparation method and application
<160>15
<210>1
<211>243
<212>PRT
<213>Artificial sequence
<220>
<223>scFv 23
<400>1
Gln Val His Leu Glu Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Phe Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr val Asp Lys Ser Ser Ser Thr Ala His
65 70 75 80
Met Glu Leu Leu Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Gly Thr Met Ile Ala Tyr Trp Gly Gln Gly Thr Thr Val Ser Ser Gly
100 105 110
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile
115 120 125
Val Leu Thr Gln Ser Pro Ala Ser Leu Ser Met Ala Val Arg Lys Lys
130 135 140
Val Thr Phe Arg Phe Met Pro Asn Thr Asp Ile Asp Asp Asp Val Asn
145 150 155 160
Trp Phe Arg Gln Lys Pro Gly Ala Pro Pro Asp Leu Leu Ile Ser Glu
165 170 175
Val Asn Ser Pro Arg Pro Gly Val Pro Ser Arg Phe Ser Ser Ser Val
180 185 190
Tyr Gly Thr Asp Phe Val Phe Val Ile Glu Asn Met Leu Ser Glu Asp
195 200 205
Val Ala Asp Cys Tyr Cys Leu Gln Gly Ala Asn Leu Pro Leu His Val
210 215 220
Arg Leu Gly Asp Gln Ala Gly Asn Pro Asn Val Lys Leu Asn Arg Arg
225 230 235 240
Pro Ala Gly
<210>2
<211>244
<212>PRT
<213>Artificial sequence
<220>
<223>scFv 68
<400>2
Gln Val Gln Leu Glu Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Gln Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Phe
20 25 30
Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu
50 55 60
Lys Asp Arg Val Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Arg Asn Trp Asp Phe Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly
100 105 110
Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser His Lys Phe
130 135 140
Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser
145 150 155 160
Gln Asp Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln
165 170 175
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val
180 185 190
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
195 200 205
Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
210 215 220
Tyr Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
225 230 235 240
Lys Thr Ser Lys
<210>3
<211>250
<212>PRT
<213>Artificial sequence
<220>
<223>scFv 109
<400>3
Gln Val His Leu Glu Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Val Ser Leu Thr Cys Thr Val Thr Gly Ile Ser Ile Thr Thr Gly
20 25 30
Asn Tyr Arg Trp Ser Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu
35 40 45
Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Thr Ile Thr Tyr Asn Pro Ser
50 55 60
Leu Thr Gly Arg Thr Thr Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe
65 70 75 80
Phe Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Thr Tyr Cys
85 90 95
Ala Arg Asp Arg Gly Asn Gly Asn Phe Gly Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser His Lys Phe Thr
130 135 140
Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln
145 150 155 160
Asp Val Gly Ala Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser
165 170 175
Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro
180 185 190
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
195 200 205
Ser Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr
210 215 220
Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys
225 230 235 240
Thr Ser Lys Leu Asn Arg Arg Pro Ala Arg
245 250
<210>4
<211>64
<212>DNA
<213>Artificial sequence
<220>
<223>T7-RBS
<400>4
gcagctaata cgactcacta taggaagaac agaccaccat gsaggtscas ctcgagsagt 60
ctgg 64
<210>5
<211>23
<212>DNA
<213>Mouse（Mus musculus）
<220>
<221>V region
<400>5
saggtscasc tcgagsagtc tgg 23
<210>6
<211>32
<212>DNA
<213>Mouse（Mus musculus）
<220>
<221>V region
<400>6
tgaggagacg gtgaccgtgg tcccttggcc cc 32
<210>7
<211>20
<212>DNA
<213>Mouse（Mus musculus）
<220>
<221>V region
<400>7
gayattgtgy tracacagtc 20
<210>8
<211>21
<212>DNA
<213>Mouse（Mus musculus）
<220>
<221>V region
<400>8
acgtttkgat ttccagcttg g 21
<210>9
<211>35
<212>DNA
<213>Mouse（Mus musculus）
<220>
<221>C region
<400>9
ccaagctgga aatcmaaacg tgctgatgct gcacc 35
<210>10
<211>24
<212>DNA
<213>Mouse（Mus musculus）
<220>
<221>C region
<400>10
cactcattcc tgttgaagct cttg 24
<210>11
<211>19
<212>DNA
<213>Mouse（Mus musculus）
<220>
<221>C region
<400>11
cgtgagggtg ctgctcatg 19
<210>12
<211>22
<212>DNA
<213>Mouse（Mus musculus）
<220>
<221>C region
<400>12
ggggtagaag ttgttcaaga ag 22
<210>13
<211>19
<212>DNA
<213>Mouse（Mus musculus）
<220>
<221>C region
<400>13
ctggatggtg ggaagatgg 19
<210>14
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>Linker-R
<400>14
ccacggtcac cgtctcctca ggcggcggcg gctc 34
<210>15
<211>29
<212>DNA
<213>Artificial sequence
<220>
<223>Linker-F
<400>15
gactgtgtya rcacaatrtc ggagcctcc 29
Claims (3)
1. a kind of single-chain antibody of anti-Penicillium citrinum toxin, which is characterized in that the amino acid sequence of the antibody is respectively such as SEQ ID
No：1、SEQ ID No：2 and SEQ ID No：Shown in 3.
2. a kind of preparation method of anti-Penicillium citrinum toxin single-chain antibody according to claim 1, which is characterized in that by with
Lower step prepares：
（1）Mouse spleen total serum IgE is taken, after reverse transcription synthesizes cDNA, PCR expands VH, Linker, VL and C kappa gene piece respectively
Section；
（2）VH, Linker and VL genetic fragment using purifying recycling is template, is assembled by Overlapping PCR PCR method
Single-chain antibody scFv gene libraries；
（3）It is template using the single-chain antibody scFv gene libraries and C kappa genes of purifying recycling, passes through Overlapping PCR PCR side
Method builds ribosomal display gene library；
（4）Constructed ribosomal display gene library is as template, using TNT Quick Coupled
Transcription/Translation Systems in-vitro transcription translation kits, prepare antibody-ribosomal-mRNA three
Composite；
（5）Using artificial coupled antigen Penicillium citrinum toxin-BSA/BSA and Penicillium citrinum toxin-OVA/OVA to antibody-ribosomal-
MRNA triplets complex carries out affine screening；
（6）Affine screening product is recycled using RT-PCR method in situ, products therefrom is the DNA gene pieces after screening
Section, the outer transcription and translation-affine screening-original position RT-PCR of repeat body recycle this process, after six wheel screenings, obtain high abundance
Genetic fragment；
（7）The genetic fragment obtained finds that 32 sequences are complete through sequencing and after comparing analysis, without lethal mutation,
All genetic fragments are connected with expression vector pTIG-TRX and forms recombinant plasmid prokaryotic expression is carried out in e. coli bl21,
Expression product is taken to surpass the precipitation of the supernatant after splitting and carries out SDS-PAGE and WB identifications respectively；
（8）Artificial coupled antigen Penicillium citrinum toxin-BSA and carrier protein BSA are used respectively as envelope antigen, empty carrier bacterium colony
Induced product as blank control, expression product is surpassed using indirect ELISA detection method split the combination activity of rear supernatant into
Row identification identifies the super Competitive assays effect for splitting rear supernatant using racing ELISA detecting method, obtains 3 altogether
The single-chain antibody of a anti-Penicillium citrinum toxin, is scFv 23, scFv 68 and scFv 109 respectively, and amino acid sequence is respectively such as
SEQ ID No：1、SEQ ID No：2 and SEQ ID No：Shown in 3.
3. a kind of purposes of the anti-Penicillium citrinum toxin single-chain antibody according to claim 1 in detection kit is prepared,
It is characterized in that, the application in measure agricultural product and food samples are prepared in the remaining detection kit of Penicillium citrinum endotoxin contamination.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101289760A (en) * | 2008-06-24 | 2008-10-22 | 江苏省农业科学院 | Antibody library of bacteriophages and applications in immunoassay of pesticide residue |
| CN101845094A (en) * | 2010-02-08 | 2010-09-29 | 福州大学 | Double-cross-linking chemical synthesis method of citrinin-protein-coupled antigen and preparation method of anti-citrinin monoclonal antibody |
| CN103965358A (en) * | 2014-04-22 | 2014-08-06 | 南昌大学 | Nano antibody for simulating citrinin (CIT) antigen and application thereof |
-
2015
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101289760A (en) * | 2008-06-24 | 2008-10-22 | 江苏省农业科学院 | Antibody library of bacteriophages and applications in immunoassay of pesticide residue |
| CN101845094A (en) * | 2010-02-08 | 2010-09-29 | 福州大学 | Double-cross-linking chemical synthesis method of citrinin-protein-coupled antigen and preparation method of anti-citrinin monoclonal antibody |
| CN103965358A (en) * | 2014-04-22 | 2014-08-06 | 南昌大学 | Nano antibody for simulating citrinin (CIT) antigen and application thereof |
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