CN110283232A - Peptide molecule and its application in conjunction with Cry1Da albumen - Google Patents

Peptide molecule and its application in conjunction with Cry1Da albumen Download PDF

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CN110283232A
CN110283232A CN201910508465.0A CN201910508465A CN110283232A CN 110283232 A CN110283232 A CN 110283232A CN 201910508465 A CN201910508465 A CN 201910508465A CN 110283232 A CN110283232 A CN 110283232A
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cry1da
peptide molecule
molecule
albumen
peptide
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CN110283232B (en
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何庆华
游凯豪
危泰龙
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Jiangxi Zhongzao Biotechnology Co ltd
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Nanchang University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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Abstract

The present invention relates to field of biotechnology, more particularly to the peptide molecule and its application for combining Cry1Da albumen, by using Cry1Da albumen as target molecule, put into phage random displayed polypeptide library, pass through the affine panning conditions such as unique negative sieve, buffer, coating time, competitor elution, obtain a kind of peptide molecule that can specifically bind Cry1Da albumen, amino acid sequence are as follows: PTAVGGS.The alternative traditional Cry1Da antibody molecule of peptide molecule mentioned by the present invention, binding molecule as Cry1Da directly applies to immunology detection analysis, the peptide molecule high specificity, binding performance height, structural stability, acid and alkali-resistance, prepare the features such as simple and quick.

Description

Peptide molecule and its application in conjunction with Cry1Da albumen
Technical field
The invention belongs to field of biotechnology, and in particular to peptide molecule and its application in conjunction with Cry1Da albumen.
Background technique
Bacillus thuringiensis (Bacillus thuringiensis, Bt) albumen is a kind of be widely present in soil A kind of parasporal crystal content secreted by gram-positive bacterium has insecticidal activity to all kinds of target insects, referred to as kills Worm crystalline protein (ICP, Insecticidal Crystal Protein), also known as delta-endotoxin or Bt toxalbumin, are current The main component of Bt insecticide on the market.After it is taken in the form of parent toxin by targeted insect, in insect midgut alkaline environment By differential protein enzyme hydrolysis, the albumen for being resistant to albumen enzyme effect is generated.Albumen after activation is first and on midgut epithelial cells film Specific receptor combines, and is then inserted into cell membrane, forms dissolubility hole, causes cell to discharge its content, finally lead Cause insect death.In addition, the anti-pest GM crop of Bt albumen has also extended to the whole world and has obtained good insecticidal effect.Bt There are many kinds of the types of albumen, according to the homology of the insecticidal spectrum of coding albumen and coding gene sequence, can be divided into Bt albumen Two major classes: being crystalline protein family (Crystal protein, Cry) and cytolytic proteins family (Cytolytic respectively Protein, Cyt).Cry gene therein is divided into 4 monoids, i.e. Cry1, Cry2, Cry3, Cry4 again；According to amino acid Homology size is categorized further, as: Cry1A, Cry1B, Cry1C, Cry1D on the basis of Cry1 gene.With transgenosis The globalization of crop, people start the safety to genetically modified crops and generate misgivings, and as biocides market occupation rate highest Bt albumen, if to the mankind exist threaten the also concern increasingly by countries in the world.Therefore, to Bt egg in GM food White detection is of great significance.
Immune analysis is relied primarily on for the detection of Bt albumen at present, wherein with enzyme linked immunosorbent assay (Enzyme- Linked Immunosorbent Assay, ELISA) especially double crush syndrome is most commonly seen.In double crush syndrome In, antigen makes the specific and sensitive of this method by the specific selection of two kinds of antibody (capture antibody and detection antibody) It spends all very high.
The core of immunological analysis method is specific recognition and combination between Ag-Ab, therefore prepares specificity Just become the premise and core of development immunological analysis method in conjunction with the antibody of antigen.The research of antibody develops rapidly in recent years, From traditional polyclonal, monoclonal antibody, it is developed to single-chain antibody, phage displaying antibody, single domain heavy chain antibody, resists Idiotype antibody etc. is the genetic engineering antibody field of representative.Animal immune, cell are subjected to compared to conventional antibodies preparation The advantages that complicated processes such as fusion, positive colony screening, genetic engineering antibody is facilitated with its orientable transformation, elutriation, has become mesh The hot spot of preceding research.In addition to this, numerous scholars in recent years including China, which have begun, is conceived to not with immune globulin Based on white composed structure novel antibodies research, such as the aptamer based on SELEX technology, based on molecular engram polymerize The artificial antibody of body technique and the antibody analog based on Lipocalin protein family etc. achieve gratifying progress, are The substitution Journal of Sex Research of conventional antibodies provides sturdy theory and method basis.Make a general survey of the development trend of antibody, it can be seen that Novel antibodies have the characteristics that molecular weight is small, structure is simple, self can evolve, prepare quick and development trend.For example, monoclonal Antibody, single-chain antibody, single domain heavy chain antibody molecular weight gradually decrease, respectively 150,30-50,15-20kDa, with Lipocalin is that the antibody analog of skelemin is 18-20kDa, and aptamer is then only a bit of nucleic acid sequence.
People are keen to research and development peptide mimics in recent years, the first purpose be by biologically active complicated molecule by It walks modification, replace, transformation, the final small molecule being compressed to only comprising functional unit, the referred to as rational design of peptide mimics.Cause This, peptide molecule can become potential novel antibody molecules.With the rapid development of phage-displayed polypeptides library technology, by biting The elutriation in phage display peptide library can be obtained quickly, simply and specific target body molecular specificity knot without animal immune process The peptide molecule of conjunction.Phage display peptide library technology is mainly characterized by effectively filtering out and target target body specific bond Phage-displayed polypeptides, the technology are exploring the binding site that interacts between receptor and ligand, are seeking the work of high-affinity biology Property ligand molecular, explore agnoprotein matter space structure epitope, the development of new generation vaccine etc. is widely used.
The present invention is more from phage display using unique affine elutriation mode by phage-displayed polypeptides library technology Quickly screened in peptide library can with the peptide molecule in conjunction with Cry1Da protein-specific, the peptide molecule have with it is traditional Cry1Da is polyclonal, the similar immunology detection characteristic of monoclonal antibody molecule, can be used as the substitute of traditional Cry1Da antibody Immune analysis system applied to Cry1Da.This method avoid prepare animal required for traditional monoclonal antibody molecule The processes such as immune, cell fusion, monoclonal screening have without immunologic process, screen convenient, the period is short, preparation cost is low etc. Feature.
Summary of the invention
The purpose of the present invention is a kind of peptide molecule of combination Cry1Da albumen and its applications
To achieve the above object, the technical solution adopted by the present invention is that:
The present invention puts into phage random displayed polypeptide library, passes through unique negative sieve, buffering using Cry1Da albumen as target molecule The affine panning conditions such as liquid, coating time, competitor elution, obtain a kind of polypeptide that can specifically bind Cry1Da albumen point Son, amino acid sequence are as follows: PTAVGGS.
In aforementioned polypeptides molecular structure, capitalization English letter respectively represents known natural L-form amino acid residue or its D- type One kind of isomers, i.e. S represent serine residue, and P represents proline residue, and H represents histidine residues, and it is residual that G represents glycine Base, T represent threonine residues, and V represents valine residue, and A represents alanine residue.
The invention further relates to the nucleotide of encoding such polypeptides molecule amino acid sequence (PTAVGGS), sequences are as follows:
CCT ACT GCT TAT GGT GGT TCT
The present invention refer to peptide molecule can Phage amplification, chemical synthesis or genetic engineering recombinantly express by way of into The a large amount of preparations of row.Phage amplification, which refers to, to show the bacteriophage for having peptide molecule, by way of biology amplification, mass propagation Production shows the bacteriophage particles for having peptide molecule.Chemical synthesis refers to the amino acid sequence according to the mimic epitope announced, and leads to The mode for crossing chemically synthesized polypeptide carries out Peptide systhesis.The mode of genetic engineering recombinant expression, which refers to, will encode the base of peptide molecule Cause carries out a large amount of preparations of peptide molecule by being cloned into expression vector in the form of polypeptide-fusion protein.
The invention further relates to application of the peptide molecule in immunology detection analysis.The type of immunology detection includes The immune analysis detection type based on Ag-Ab specific reaction such as MBP enzyme linked immuno-adsorbent assay.
Compared with prior art, the beneficial effects of the present invention are:
The alternative traditional Cry1Da antibody molecule of peptide molecule mentioned by the present invention, the binding molecule as Cry1Da Directly apply to immunology detection analysis, the peptide molecule high specificity, binding performance height, structural stability, acid and alkali-resistance, preparation The features such as simple and quick.
Specific embodiment
The affine elutriation and its identification of the peptide molecule in conjunction with Cry1Da protein-specific of embodiment 1.
1) affine elutriation peptide molecule in conjunction with Cry1Da protein-specific method particularly includes: be placed on ELISA Plate ultra-clean After workbench ultraviolet irradiation sterilizes half an hour, 2 μ g/mL Cry1Da albumen are coated in ELISA Plate hole (100 hole μ L/), with sterilizing Lunch box be sealed in 4 DEG C of refrigerator overnights.It is taken out from 4 DEG C, with PBST, (10mM PBS, pH 7.4 include in super-clean bench 0.8%Tween-20 (v/v)) washing 8 times, it is patted dry on sterilizing blotting paper；37 DEG C of 5% gelatin confining liquid are added to be incubated for 5 hours. For the peptide molecule for obtaining specific binding Cry1Da, non-specific binding is reduced, the present invention, will before carrying out affine elutriation Phage random displayed polypeptides library (phage display heptapeptide library, be purchased from NEB company, with PBS dilute Phage amplification liquid, about 4.0 ×1011Pfu) investment is coated with BSA albumen (10 μ g/mL), OVA albumen (10 μ g/mL), skim milk (10 μ g/mL) respectively Negative sieve is carried out in ELISA Plate hole, and it is stand-by to collect supernatant in hole.The ELISA Plate hole for being coated with Cry1Da albumen is taken, is washed with PBST 4 times, every hole is added the hole supernatant that 100 μ L pass through above-mentioned negative sieve, 37 DEG C oscillating reactions 1 hour.Unbonded bacteriophage is discarded, is used PBST is washed 6 times, is eluted 8min with anti-being at war with property of Cry1Da monoclonal antibody in conjunction with upper bacteriophage, is used 1M Tris- afterwards HCl (pH 9.0) is neutralized.10 μ L wash-out bacteriophages are taken to survey titre, remaining is used to infect 30mL and grows to logarithm early period E.coli ER2738 bacterial strain is expanded.With PEG/NaCl deposition and purification bacteriophage, and bacteriophage after amplification is measured within second day Titre.
Then the 2nd wheel and the 3rd wheel elutriation are successively carried out, panning step is substantially the same with the first round, but each twice later The phagocytosis scale of construction of addition is 2 × 1011Pfu, the 2nd, 3 wheel Cry1Da protein standard substances peridium concentration be respectively 1 μ g/mL and 0.5 μ g/mL, the phage-displayed polypeptides of screening and the lath incubation time for being coated with Cry1Da albumen be respectively 15min, 10min, cleaning solution concentration are respectively 1.0%PBST and 1.25%PBST.
2) identification of positive phage clones: the random picking 50 from the plate for measuring phage titre after third round elutriation A bacteriophage spot, carries out the amplification of bacteriophage, and direct coated ELISA method and sandwich ELISA detection side is respectively adopted The identification of method progress positive phage clones.
Direct coated ELISA's method particularly includes: coating Cry1Da albumen is in ELISA Plate, 4 DEG C of overnight incubations.Second day use After PBST (10mM PBS, 0.05%Tween-20 (v/v)) is washed 3 times, closed with the PBS containing 5% skimmed milk power, 37 DEG C be incubated for 1 hour, investment 100 μ L obtain positive bacteriophage expand liquid, be incubated for 30min.1:5000 times of diluted HRP mark is added Remember anti-M13 bacteriophage secondary antibody 100 μ L, 37 DEG C of incubation 45min.100 μ L tmb substrate liquid are added, are protected from light colour developing 6min, microplate reader Read the absorption value at 450nm.BSA, OVA antigen are coated with as negative control, remaining ELISA operating method is identical.
Sandwich ELISA detection method method particularly includes: firstly, being diluted with 10mM PBS (pH 7.4) anti- Cry1Da monoclonal antibody (number 1a) to 1 μ g/mL, 100 holes μ L/ are coated with 96 hole elisa Plates, 4 DEG C of overnight incubations.Second day use After PBST (10mM PBS, 0.05%Tween-20 (v/v)) is washed 3 times, closed with the PBS containing 5% skimmed milk power, 37 DEG C be incubated for 1 hour；Cry1Da protein standard substance 50ng/mL, 100 37 DEG C of the hole μ L/ incubation 45min is added.Put into that 100 μ L are to be measured to be bitten Thallus spot expands liquid (1.0 × 1012Pfu), using the PBS solution without Cry1Da protein standard substance as blank control, with It is positive right that Cry1Da protein-specific, which combines and marks the anti-Cry1Da monoclonal antibody (number 2b) of horseradish peroxidase, According to, using naive phage display peptide library dilution as negative control, 37 DEG C of incubation 45min.Remaining hole adds in addition to Positive control wells Enter 1: 5000 times of diluted HRP and marks anti-M13 bacteriophage secondary antibody 100 μ L, 37 DEG C of incubation 45min.100 μ L tmb substrates are added Liquid, is protected from light colour developing 6min, and microplate reader reads the absorption value at 450nm.Choose OD450Phage clone of 2 times greater than negative control For positive colony.
3) it specifically binds the binding performance identification of Cry1Da peptide molecule: specific knot is carried out using the method for ELISA Close Cry1Da peptide molecule binding performance identification, method particularly includes: be coated with various concentration Cry1Da albumen (0.01, 0.25,0.5,1,5,10,20,40,100ng/mL) in ELISA Plate, 4 DEG C of overnight incubations.Second day with PBST (10mM PBS, 0.05%Tween-20 (v/v)) washing 3 times after, closed with the PBS containing 5% skimmed milk power, 37 DEG C be incubated for 1 hour, throw The positive bacteriophage amplification liquid for entering 100 μ L acquisition, is incubated for 30min.1: 5000 times of diluted HRP is added and marks anti-M13 bacteriophage Secondary antibody 100 μ L, 37 DEG C of incubation 45min.100 μ L tmb substrate liquid are added, are protected from light colour developing 6min, microplate reader is read at 450nm Absorption value.It has been coated with 0.01 as the result is shown, 0.25,0.5,1,5,10,20,40, the OD value in the hole of 100ng/mL CrylDa albumen Respectively 0.2,0.3,0.38,0.52,1.0,1.2,1.8,2.0,2.6, are presented good binding performance, and negative hole is colourless, show Show unbonded.
4) it identification of the specific binding Cry1Da peptide molecule for Cry1Da specificity: is carried out using the method for ELISA The cross reaction of peptide molecule and Cry1Ac, Cry1Ab homologous protein identifies, method particularly includes: be coated with respectively Cry1Ac, Cry1Ab albumen is in ELISA Plate, 4 DEG C of overnight incubations.It is washed with PBST (10mM PBS, 0.05%Tween-20 (v/v)) within second day It after 3 times, is closed with the PBS containing 5% skimmed milk power, 37 DEG C are incubated for 1 hour, put into the 100 identified specific bindings of μ L The phage clone (1.0 × 10 of Cry1Da11Pfu) it is incubated for 30min.1: 5000 times of diluted HRP is added and marks anti-M13 bacteriophage Secondary antibody 100 μ L, 37 DEG C of incubation 45min.100 μ L tmb substrate liquid are added, are protected from light colour developing 6min, microplate reader is read at 450nm Absorption value.If the polypeptide of Cry1Da can be specifically bound to Cry1Ac, Cry1Ab no cross reaction, be coated with Cry1Ac, Kong Yingwu absorbance value (OD) value of Cry1Ab albumen.It has put into the ELISA Plate hole of Cry1Ac, Cry1Ab and has had no as the result is shown Color shows that the phage clone of the anti-Cry1Da of acquisition is not combined with Cry1Ac, Cry1Ab, has excellent specificity.
The sequencing of embodiment 2. and the peptide molecule encoding gene of Cry1Da specific binding and its amino acid sequence are really It is fixed
Identification being shown to, the bacteriophage for having specific binding Cry1Da peptide molecule expands, extracts the DNA of bacteriophage Sequencing template.Simplified process is as follows: carrying out Phage amplification, after first step centrifugation, 800 μ L are transferred to one containing bacteriophage supernatant New centrifuge tube.200 μ L PEG/NaCl precipitating phages are added.Precipitating is resuspended in 100 μ L iodide buffers after centrifugation (10mM Tris-HCl (pH 8.0), 1mM EDTA, 4M NaI) is added 250 μ L dehydrated alcohols and precipitates DNA, use again after centrifugation 70% ethanol washing precipitates (DNA sequencing template).Precipitating is finally resuspended in 20 μ L aqua sterilisas, and 2 μ L is taken to carry out Ago-Gel electricity Swimming analysis；5 μ L bacteriophage templates are taken to carry out DNA sequencing, -96gIII sequencing primer are as follows: 5'-HOCCC TCA TAG TTA GCG TAA CG-3'.It can get the amino acid sequence of peptide molecule: PTAVGGS according to DNA sequencing result and password sublist.
A large amount of preparations of embodiment 3. and the peptide molecule of Cry1Da specific binding
1) in a manner of Phage amplification
It will be added to 30ml in the culture for being inoculated with E.coli ER2738 with the bacteriophage that Cry1Da is specifically bound, 37 DEG C of 220rpm, shaken cultivation 4.5h.Culture is transferred in another centrifuge tube, 4 DEG C of 8000rpm are centrifuged 30min, by supernatant Top 80% is transferred in a fresh tube, and the PEG/NaCl of 1/6 volume is added, stands overnight at 4 DEG C.4 DEG C of 8000rpm are centrifuged PEG/ NaCL stands solution 30min.Supernatant is abandoned, precipitating is resuspended in 1mL PBS, and 200 μ L PEG/NaCl are added, stand 2h at 4 DEG C, Residual supernatant is sucked after 12000rpm centrifugation 10min, then of short duration centrifugation.200 μ L PBS are added to be resuspended, as bacteriophage Expand liquid.
2) it is prepared in a manner of polypeptide-fusion protein
The external source encoding gene of A.PCR amplification peptide molecule
PCR reaction system: (50 μ L)
PCR reaction condition:
Using PCR product QIAquick Gel Extraction Kit purified pcr product, trace dna quantitative instrument is quantitative.The coding base of peptide molecule Because of sequence are as follows: CCT ACT GCT TAT GGT GGT TCT
B. the double digestion of external source encoding gene and expression vector
The external source code gene of ACC65I and Eag I enzyme and expression vector is respectively adopted, and (pMAl-pIII, NEB company, can Express MBP fusion protein) carry out double digestion.
C. after digestion product connection and conversion
Plasmid pMal-PIII and target fragment are mixed with 1: 15 (molar ratio), 12h is connected in 20 DEG C of water-baths, takes 15 μ L Connection product adds in 100 μ L competent cell DH5a, mixes well.After ice bath 35min, 42 DEG C of water-bath heat shock 60s, ice immediately 800 μ L LB liquid mediums are added after bath 3min, 37 DEG C, 200rpm cultivates 1h, and 8000rpm is centrifuged 5min, sucks supernatant and leave and take About 300 μ L are coated in LB-A solid (Amp) culture medium, and 37 DEG C are incubated overnight, and obtain subclone positive colony.
D. Cloning Transformation
The subclone that above-mentioned steps are obtained extracts purpose plasmid with Tiangeng kit, takes 10 μ L thin to 100 μ L competence In born of the same parents TB1, mix well.After ice bath 20min, 45 DEG C of water-bath heat shock 60s add the training of 800 μ LLB liquid immediately after ice bath 5min Nutrient solution, 37 DEG C, 200rpm cultivates 45min, and 7000rpm is centrifuged 6min, sucks supernatant and leave and take about 300 μ L, be coated on LB-A solid (Amp) in culture medium, 37 DEG C are incubated overnight, and obtain positive colony.
E. the expression of polypeptide-MBP fusion protein
By the positive clone molecule of above-mentioned acquisition, a single colonie is chosen from plate and is inoculated in 5mL LB-A, in 0.5% sucrose, 37 DEG C, 220r/min, shaken cultivation is overnight, and overnight culture is inoculated in the LB-A of 50mL by 1% inoculum concentration (v/v), and 0.2% In sucrose culture medium, 3 bottles of inoculation respectively, 37 DEG C, 220r/min shaken cultivation, when culture bacterial concentration OD600 reaches 0.6 When, IPTG to final concentration of 0.3mmol/L is added into three bottles of cultures, 120r/min shaken cultivation, (IPEG is molten by inducer Liquid) in 4 DEG C, 5000g, centrifugation 15min collects bacterial sediment, abandons supernatant.Cell is resuspended in 400mL 30mM Tris-HCl, 25% sucrose, pH 8.0 (80mL/g wet cell weight) are added EDTA to 1mM, shake 5-10min, 8000g, 4 DEG C of centrifugations at room temperature 15min abandons supernatant, and precipitating is resuspended in the 5mM MgS04 of 400ml pre-cooling, shakes 15min, 8000g, 4 DEG C on ice, is centrifuged 20min retains supernatant, and 8mL 1M Tris-HCl, pH 7.4 is added into supernatant, obtains polypeptide-MBP fusion protein, can incite somebody to action The fusion protein is directly used in ELISA etc. based on the inspection for being used for Cry1Da in the immune detecting system of antigen-antibody as antibody It surveys.
Embodiment 4. specifically binds application of the Cry1Da peptide molecule in ELISA
(1) sample extraction
5g sample to be tested is weighed, 25mL PBS- methanol solution is added, 200rpm vibrates 5 minutes；Extracting solution is used After No. 1 filter paper of whatman is filtered, after taking 1mL filtrate that 1mL PBS (phosphate buffer, pH=7.2) mixing is added, i.e., For sample extracting solution, for use.
(2) it is coated with and closes
1. direct detecting method
By extracting solution direct coated in ELISA Plate, 4 DEG C of overnight incubations.Second day with PBST (10mM PBS, 0.05% Tween-20 (v/v)) washing 3 times after, closed with the PBS containing 5% skimmed milk power, 37 DEG C be incubated for 1 hour.
2. sandwich ELISA method
With the diluted anti-Cry1Da monoclonal antibody (1 μ g/ml) of 10mM PBS (pH 7.4), coated elisa plate, 4 DEG C of incubations Overnight.After second day is washed 3 times with PBST (10mM PBS, 0.05%Tween-20 (v/v)), with containing 5% skimmed milk power PBS is closed, and after 37 DEG C are incubated for 1 hour, are dried up 4 DEG C with 6 super-clean benches of PBST board-washing and is saved for use.
(3) foundation of standard curve
A. it is based on direct detecting method
Lath is taken out, every hole puts into Cry1Da protein standard substance (the concentration range 0-2000ng/ of 100 μ L doubling dilutions ML), 100 hole μ L/, 37 DEG C of incubation 45min.It puts into 100 μ L and shows the bacteriophage for having specific binding Cry1Da peptide molecule (1.0×1011Pfu) or expression polypeptide-MBP fusion protein.1:5000 times of diluted HRP is added and marks anti-M13 bacteriophage two Anti- 100 μ L or enzyme mark anti-MBP antibody, 37 DEG C of incubation 45min.Then it is developed the color with tmb substrate, reads OD450Establish ELISA mark Directrix curve.As the result is shown: the linear detection range of the standard curve is 0.1-400ng/mL.
B. sandwich ELISA method
The lath handled well through step (2) is taken out, the Cry1Da protein standard substance that every hole puts into 100 μ L doubling dilutions is (dense Degree range is 0-2000ng/mL), 100 holes μ L/, 37 DEG C of incubation 45min.Putting into 100 μ L displaying has specific binding Cry1Da more The bacteriophage (1.0 × 10 of peptide molecule11Pfu) or expression polypeptide-MBP fusion protein.1: 5000 times of diluted HRP label is added Anti- 100 μ L of M13 bacteriophage secondary antibody or enzyme mark anti-MBP antibody, 37 DEG C of incubation 45min.Then it is developed the color, is read with tmb substrate OD450Establish sandwich ELISA standard curve.As the result is shown: the linear detection range of the standard curve is 0.01-180ng/mL.
(4) detection of sample
The lath handled well through step (2) is taken out, is operated respectively according to direct or sandwich ELISA method, according to mark Directrix curve retrodicts out the content of Cry1Da in sample.
The immunology detection stability experiment of embodiment 5. and Cry1Da specific binding polypeptide molecule
Resistance to acid and alkali experiment
By can with Cry1Da specifically bind phage-displayed polypeptides molecule/polypeptide-MBP fusion protein and Cry1Da it is mono- Clonal antibody uses pH=4.0 respectively, and 5.0,6.0,7.4,8.0 buffer is diluted to working solution, carries out ELISA measurement respectively, Method particularly includes: coating Cry1Da albumen is in ELISA Plate, 4 DEG C of overnight incubations.Second day with PBST (10mM PBS, 0.05% Tween-20 (v/v)) washing 3 times after, closed with the PBS containing 5% skimmed milk power, 37 DEG C be incubated for 1 hour, put into 100 μ L uses pH=4.0 respectively, and 5.0,6.0,7.4,8.0 buffer is diluted to the polypeptide point of the CrylDa specific binding of working solution Son or Cry1Da monoclonal antibody are incubated for 30min.1: 5000 times of diluted HRP is added and marks anti-M13 bacteriophage secondary antibody or anti- IgG secondary antibody 100 μ L, 37 DEG C of incubation 45min.100 μ L tmb substrate liquid are added, are protected from light colour developing 6min, microplate reader is read at 450nm Absorption value.
Experimental result is shown: using peptide molecule as the sandwich ELISA of Cry1Da recognition component, in pH=5.0, and 6.0, When 7.4,8.0, OD value is kept constant；Using Cry1Da monoclonal antibody as the ELISA of recognition component, in pH=5.0,6.0, Irregularities variation is presented in OD value when 8.0, and the immune analysis performance of antibody is significantly affected, shows as Cry1Da antibody The peptide molecule of substitute has better soda acid patience.
Sequence table
<110>University Of Nanchang
<120>in conjunction with the peptide molecule of Cry1Da albumen and its application
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 7
<212> PRT
<213>artificial sequence (Artificial Sequence)
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Pro Thr Ala Val Gly Gly Ser
1 5
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<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cctactgctt atggtggttc t 21

Claims (6)

1. combining the peptide molecule of Cry1Da albumen, it is characterised in that its amino acid sequence are as follows: PTAVGGS.
2. encoding the nucleotide of peptide molecule amino acid sequence described in claim 1.
3. nucleotide sequence as claimed in claim 2 is corresponding are as follows: CCT ACT GCT TAT GGT GGT TCT.
4. peptide molecule preparation method as described in claim 1, it is characterised in that pass through Phage amplification, chemical synthesis or gene The mode of engineering recombinant expression is largely prepared；The Phage amplification refers to the bacteriophage of displayed polypeptides molecule, passes through The mode of biology amplification, mass propagation production show there is the polypeptide that can specifically bind bacillus thuringiensis Cry1Da albumen point The bacteriophage particles of son；The chemical synthesis refers to the amino acid sequence according to peptide molecule, by way of chemically synthesized polypeptide Carry out Peptide systhesis；The mode of the genetic engineering recombinant expression refers to and will encode the gene of peptide molecule, by being cloned into table Up to carrier, largely prepared in the form of polypeptide-fusion protein.
5. application of the peptide molecule described in claim 1 in immunology detection analysis.
6. application as claimed in claim 5, it is characterised in that peptide molecule substitutes traditional Cry1Da protein polyclone, monoclonal Application of the antibody in immunology detection analysis.
CN201910508465.0A 2019-06-12 2019-06-12 Polypeptide molecule combined with Cry1Da protein and application thereof Active CN110283232B (en)

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