Background
Bacillus thuringiensis (Bt) Protein is a parasporal Crystal inclusion secreted by gram-positive bacteria widely existing in soil, has Insecticidal activity on various target insects, is called Insecticidal Crystal Protein (ICP), is also called endotoxin or Bt toxic Protein, and is a main component of Bt insecticides in the market at present. After it is ingested in the form of protoxin by the target insect, it is hydrolyzed by specific protease in the alkaline environment of insect midgut to produce a protein which can tolerate the action of protease. The activated protein first binds to a specific receptor on the midgut cell membrane and then inserts into the cell membrane, forming a solubility hole that causes the cell to release its contents, eventually leading to insect death. In addition, insect-resistant transgenic crops of Bt proteins have also been promoted worldwide and have achieved very good insecticidal effects. There are many varieties of Bt proteins, and Bt proteins can be divided into two major classes based on the insecticidal spectrum of the encoded protein and the homology of the encoded gene sequence: the Crystal protein family (Cry) and the Cytolytic protein family (Cyt), respectively. The Cry genes are divided into 4 groups, namely Cry1, Cry2, Cry3 and Cry 4; according to the homology size of amino acids, the Cry1 gene is further classified into: cry1A, Cry1B, Cry1C and Cry 1D. Along with the globalization of transgenic crops, people begin to have a concern about the safety of transgenic crops, and whether Bt protein with the highest market share of pesticides has a threat to human beings or not is also receiving more and more attention from countries around the world. Therefore, the method has important significance for detecting the Bt protein in the transgenic food.
Currently, Bt protein detection mainly depends on immunological analysis, and Enzyme-Linked Immunosorbent Assay (ELISA) is the most common method, especially double-antibody sandwich ELISA. In a double antibody sandwich ELISA, the antigen is specifically selected by two antibodies (capture antibody and detection antibody), making this method very specific and sensitive.
The core of the immunological analysis method is the specific recognition and binding between antigen and antibody, so that the preparation of antibody specifically binding to antigen is a prerequisite and core for the development of immunological analysis method. The research of antibodies has been rapidly developed in recent years, and has been developed from conventional polyclonal and monoclonal antibodies to the field of genetically engineered antibodies represented by single-chain antibodies, phage display antibodies, single-domain heavy-chain antibodies, anti-idiotypic antibodies, and the like. Compared with the traditional antibody preparation which must undergo complex procedures such as animal immunization, cell fusion, positive clone screening and the like, the genetic engineering antibody has become a hotspot of the current research with the advantages of orientable modification, convenient panning and the like. In addition, in recent years, many scholars including China have started to pay attention to research on novel antibodies which do not mainly comprise immunoglobulins, for example, nucleic acid aptamers based on SELEX technology, artificial antibodies based on molecularly imprinted polymer technology, antibody analogs based on Lipocalin protein family, and the like all have made favorable progress, and provide a solid theoretical and methodological basis for the alternative research of traditional antibodies. By observing the development dynamics of the antibody, the novel antibody has the characteristics of small molecular weight, simple structure, self evolution, quick preparation and development trend. For example, monoclonal antibodies, single-chain antibodies, and single-domain heavy-chain antibodies have decreasing molecular weights of 150 kDa, 30 kDa to 50 kDa, and 15 kDa to 20kDa, and antibody analogs with Lipocalin as the backbone protein, whereas aptamers have only a small nucleic acid sequence.
In recent years there has been intense research and development of peptidomimetics, one of the goals being to gradually modify, substitute, engineer and finally compress complex molecules with biological activity into small molecules containing only functional units, referred to as rational design of peptidomimetics. Thus, polypeptide molecules can become potential novel antibody molecules. With the rapid development of the phage display peptide library technology, through panning of the phage display peptide library, polypeptide molecules specifically bound to specific target molecules can be rapidly and simply obtained without the need of an animal immunization process. The phage display peptide library technology has the main characteristic that phage display polypeptides specifically bound with a target body can be effectively screened, and the technology is widely applied to the aspects of exploring the interaction binding sites between receptors and ligands, seeking ligand molecules with high affinity bioactivity, exploring epitopes of unknown protein spatial structures, developing novel vaccines and the like.
The invention rapidly screens polypeptide molecules capable of being specifically combined with Cry1Da protein from a phage display polypeptide library by a phage display polypeptide library technology and a unique affinity panning mode, the polypeptide molecules have immunological detection characteristics similar to that of the traditional Cry1Da polyclonal and monoclonal antibody molecules, and can be used as a substitute of the traditional Cry1Da antibody in a Cry1Da immunological analysis system. The method avoids the processes of animal immunization, cell fusion, monoclonal screening and the like required by the preparation of the traditional monoclonal antibody molecules, and has the characteristics of no need of an immunization process, convenience in screening, short period, low preparation cost and the like.
Disclosure of Invention
The invention aims to provide a polypeptide molecule combined with Cry1Da protein and application thereof
In order to achieve the purpose, the invention adopts the technical scheme that:
the Cry1Da protein is used as a target molecule, a phage random display peptide library is put into the target molecule, and a polypeptide molecule capable of specifically binding the Cry1Da protein is obtained through the unique conditions of negative sieve, buffer solution, coating time, competitor elution and the like of affinity panning, and the amino acid sequence of the polypeptide molecule is as follows: PTAGGS.
In the molecular structure of the polypeptide, capital English letters respectively represent known natural L-type amino acid residues or one of D-type isomers thereof, namely S represents serine residues, P represents proline residues, H represents histidine residues, G represents glycine residues, T represents threonine residues, V represents valine residues, and A represents alanine residues.
The invention also relates to a nucleotide for coding the polypeptide molecule amino acid sequence (PTAGGS), which has the following sequence:
CCT ACT GCT TAT GGT GGT TCT
the polypeptide molecule can be prepared in large quantity by means of phage amplification, chemical synthesis or genetic engineering recombinant expression. Phage amplification refers to the mass propagation and production of phage particles displaying polypeptide molecules by using phage displaying polypeptide molecules through a biological amplification mode. Chemical synthesis refers to polypeptide synthesis by chemically synthesizing polypeptides according to the published amino acid sequences of mimotopes. The mode of genetic engineering recombinant expression refers to the mass production of polypeptide molecules in the form of polypeptide-fusion proteins by cloning genes encoding the polypeptide molecules into expression vectors.
The invention also relates to the application of the polypeptide molecule in immunological detection and analysis. The type of immunological detection includes an immunological assay detection type based on an antigen-antibody specific reaction, such as enzyme-linked immunosorbent assay.
Compared with the prior art, the invention has the beneficial effects that:
the polypeptide molecule provided by the invention can replace the traditional Cry1Da antibody molecule, can be used as a binding molecule of Cry1Da to be directly applied to immunological detection and analysis, and has the characteristics of strong specificity, high binding performance, structural stability, acid and alkali resistance, simple and rapid preparation and the like.
Detailed Description
Example 1 affinity panning and identification of polypeptide molecules that specifically bind to Cry1Da proteins
1) The specific method for affinity panning and specific binding of Cry1Da protein to polypeptide molecules comprises the following steps: placing the ELISA plate on an ultra-clean workbench, sterilizing for half an hour by ultraviolet irradiation, coating Cry1Da protein of 2 μ g/mL on the ELISA plate hole (100 μ L/hole), sealing with sterilized lunch box, and storing in a refrigerator at 4 deg.C overnight. Removed from 4 ℃, washed 8 times in a clean bench with PBST (10mM PBS, pH 7.4, containing 0.8% Tween-20(v/v)), patted dry on sterile absorbent paper; 5% gelatin blocking solution was added and incubated at 37 ℃ for 5 hours. To obtain polypeptide molecules that specifically bind Cry1Da and reduce non-specific binding, the present invention provides for random phage display of a polypeptide library (phage display heptapeptide library, purchased from NEB corporation, diluted phage amplification medium with PBS, approximately 4.0X 10) prior to affinity panning11pfu) are respectively put into an enzyme-labeled plate hole coated with BSA protein (10 mug/mL), OVA protein (10 mug/mL) and skimmed milk (10 mug/mL) for negative screening, and supernatant in the hole is collected for standby. Taking an enzyme-labeled plate hole coated with Cry1Da protein, washing with PBST for 4 times, adding 100 mu L of the supernatant of the hole passing through the negative sieve into each hole, and carrying out shaking reaction at 37 ℃ for 1 hour. Unbound phage were discarded, washed 6 times with PBST, and bound phage were competitively eluted with anti-Cry 1Da monoclonal antibody for 8min before neutralization with 1M Tris-HCl (pH 9.0). 10 μ L of eluted phage was titered and the remainder was used to infect 30mL of E.coli ER2738 strain grown to early log. The next day, phages were purified by PEG/NaCl precipitation and the titer of the amplified phages was determined.
Then, the 2 nd and 3 rd rounds of panning were performed in sequence, and the panning procedure was substantially the same as in the first round, but the amount of phage added in each of the two subsequent rounds was 2X 1011pfu, cycle 2, and 3 Cry1Da protein standards were coated at 1. mu.g/mL and 0.5. mu.g/mL, respectively, the incubation times of the selected phage-displayed polypeptides with Cry1Da protein-coated strips were 15min and 10min, respectively, and the wash concentrations were 1.0% PBST and 1.25% PBST, respectively.
2) Identification of positive phage clones: randomly picking 50 phage spots from the plate for determining the phage titer after the third round of panning, amplifying the phage, and respectively adopting a direct coating ELISA method and a sandwich enzyme-linked immunosorbent assay method to identify the positive phage clone.
The specific method of the direct coating ELISA is as follows: cry1Da protein was coated on an enzyme plate and incubated overnight at 4 ℃. The following day, the cells were washed 3 times with PBST (10mM PBS, 0.05% Tween-20(v/v)), blocked with PBS containing 5% skim milk powder, incubated at 37 ℃ for 1 hour, charged with 100. mu.L of the obtained positive phage amplification solution, and incubated for 30 min. Adding 1: a5000-fold dilution of HRP-labeled anti-M13 phage secondary antibody was incubated at 100. mu.L for 45min at 37 ℃. Adding 100 μ L TMB substrate solution, developing for 6min in dark, and reading the absorption value at 450nm with enzyme-labeling instrument. BSA and OVA antigens are coated as negative controls, and the rest ELISA operation methods are the same.
The sandwich enzyme-linked immunosorbent assay method comprises the following specific steps: first, the anti-Cry 1Da monoclonal antibody (number 1a) was diluted to 1. mu.g/mL, 100. mu.L/well with 10mM PBS (pH 7.4), coated with a 96-well microplate, and incubated overnight at 4 ℃. The following day after 3 washes with PBST (10mM PBS, 0.05% Tween-20(v/v)), blocked with PBS containing 5% skim milk powder, and incubated at 37 ℃ for 1 hour; cry1Da protein standard 50ng/mL was added and incubated at 37 ℃ for 45min at 100. mu.L/well. 100 μ L of phage plaque amplification solution (1.0X 10) to be tested was added12pfu), using PBS solution without Cry1Da protein standard as blank control, anti-Cry 1Da monoclonal antibody (code 2b) which specifically binds to Cry1Da protein and is labeled with horseradish peroxidase as positive control, using original phage display peptide library diluent as negative control, and incubating for 45min at 37 ℃. 100 μ L of HRP-labeled anti-M13 phage secondary antibody diluted 1: 5000 fold was added to the wells except for the positive control wells and incubated at 37 ℃ for 45 min. Adding 100 μ L TMB substrate solution, developing for 6min in dark, and reading the absorption value at 450nm with enzyme-labeling instrument. Selection of OD450Phage clones 2 times larger than the negative control were positive clones.
3) Identification of binding properties of specifically binding Cry1Da polypeptide molecules: the identification of the binding performance of the specific binding Cry1Da polypeptide molecule is carried out by adopting an ELISA method, which comprises the following steps: different concentrations of Cry1Da protein (0.01, 0.25, 0.5, 1, 5, 10, 20, 40, 100ng/mL) were coated on the microplate and incubated overnight at 4 ℃. The following day, the cells were washed 3 times with PBST (10mM PBS, 0.05% Tween-20(v/v)), blocked with PBS containing 5% skim milk powder, incubated at 37 ℃ for 1 hour, charged with 100. mu.L of the obtained positive phage amplification solution, and incubated for 30 min. A1: 5000 fold dilution of a secondary HRP-labeled anti-M13 phage antibody at 100. mu.L was added and incubated at 37 ℃ for 45 min. Adding 100 μ L TMB substrate solution, developing for 6min in dark, and reading the absorption value at 450nm with enzyme-labeling instrument. The results show that the OD values of wells coated with 0.01, 0.25, 0.5, 1, 5, 10, 20, 40, 100ng/mL CrylDa protein are 0.2, 0.3, 0.38, 0.52, 1.0, 1.2, 1.8, 2.0, 2.6, respectively, showing good binding performance, the negative wells are colorless, showing no binding.
4) Identification of specific binding to Cry1Da polypeptide molecules specific for Cry1 Da: the cross reaction identification of polypeptide molecules and Cry1Ac and Cry1Ab homologous proteins is carried out by adopting an ELISA method, and the specific method comprises the following steps: cry1Ac and Cry1Ab proteins are coated on the ELISA plate respectively, and incubated overnight at 4 ℃. The following day, after 3 washes with PBST (10mM PBS, 0.05% Tween-20(v/v)), blocked with PBS containing 5% skim milk powder, incubated at 37 ℃ for 1 hour, 100. mu.L of phage clones identified as specifically binding to Cry1Da (1.0X 10)11pfu) for 30 min. A1: 5000 fold dilution of a secondary HRP-labeled anti-M13 phage antibody at 100. mu.L was added and incubated at 37 ℃ for 45 min. Adding 100 μ L TMB substrate solution, developing for 6min in dark, and reading the absorption value at 450nm with enzyme-labeling instrument. If the polypeptide capable of specifically binding to Cry1Da has no cross reaction to Cry1Ac and Cry1Ab, the wells coated with Cry1Ac and Cry1Ab proteins should have no light absorption (OD) value. The results show that no color exists in the plate wells of the enzyme labeled plate which is thrown into Cry1Ac and Cry1Ab, and that the obtained phage clone which resists Cry1Da is not combined with Cry1Ac and Cry1Ab, so that the phage clone has excellent specificity.
Example 2 sequencing of the Gene encoding a polypeptide molecule that specifically binds to Cry1Da and determination of its amino acid sequence
And amplifying the phage which is identified and displayed with the specific binding Cry1Da polypeptide molecule, and extracting a DNA sequencing template of the phage. The brief procedure is as follows: phage amplification was performed and after the first centrifugation step 800. mu.L of phage-containing supernatant was transferred to a new centrifuge tube. Add 200 u L PEG/NaCl precipitation phage. After centrifugation, the pellet was resuspended in 100. mu.L of iodide buffer (10mM Tris-HCl (pH 8.0), 1mM EDTA, 4M NaI), 250. mu.L of absolute ethanol was added to precipitate the DNA, and after centrifugation, 70% was addedThe pellet (DNA sequencing template) was washed with ethanol. Finally, the sediment is resuspended in 20 mu L of sterilized water, and 2 mu L of the sediment is taken for agarose gel electrophoresis analysis; taking 5 mu L of phage template for DNA sequencing, wherein-96 gIII sequencing primers are as follows: 5' -HOCCC TCA TAG TTA GCG TAA CG-3'. The amino acid sequence of the polypeptide molecule can be obtained according to the DNA sequencing result and the codon table: PTAGGS.
Example 3 Mass preparation of polypeptide molecules specifically binding to Cry1Da
1) By means of phage amplification
The phage specifically binding to Cry1Da were added to 30ml of E.coli ER2738 inoculated culture at 37 ℃ at 220rpm for 4.5h with shaking. The culture was transferred to another centrifuge tube, centrifuged at 8000rpm at 4 ℃ for 30min, the upper 80% of the supernatant was transferred to a fresh tube, added with 1/6 volumes of PEG/NaCl and allowed to stand overnight at 4 ℃. The PEG/NaCL solution was centrifuged at 8000rpm at 4 ℃ for 30 min. Discard the supernatant, resuspend the pellet in 1mL PBS, add 200. mu.L PEG/NaCl, stand at 4 ℃ for 2h, centrifuge at 12000rpm for 10min, centrifuge briefly, and aspirate the residual supernatant. Adding 200 mu L PBS for re-suspending to obtain phage amplification solution.
2) Preparation by means of polypeptide-fusion proteins
PCR amplification of exogenous coding genes of polypeptide molecules
And (3) PCR reaction system: (50. mu.L)
And (3) PCR reaction conditions:
and purifying the PCR product by using a PCR product recovery kit, and quantifying by using a trace nucleic acid quantifier. The coding gene sequence of the polypeptide molecule is as follows: CCT ACT GCT TAT GGT GGT TCT
B. Double enzyme digestion of exogenous coding gene and expression vector
ACC65I and Eag I enzymes are respectively adopted to carry out double enzyme digestion on the exogenous coding gene and an expression vector (pMAl-pIII, NEB company can express MBP fusion protein).
C. Ligation and transformation of products after enzyme digestion
Plasmid pMal-PIII and the target fragment were mixed at a molar ratio of 1: 15, ligated in a water bath at 20 ℃ for 12 hours, 15. mu.L of the ligation product was added to 100. mu.L of competent cell DH5a, and mixed well. After ice bath for 35min, performing water bath heat shock at 42 ℃ for 60s, immediately performing ice bath for 3min, supplementing 800 mu L of LB liquid culture solution, culturing at 37 ℃ and 200rpm for 1h, centrifuging at 8000rpm for 5min, sucking supernatant, taking about 300 mu L, coating the supernatant in an LB-A solid (Amp) culture medium, and performing overnight culture at 37 ℃ to obtain a subclone positive clone.
D. Clonal transformation
The subclone obtained by the above steps is extracted with Tiangen kit to obtain the objective plasmid, and 10. mu.L to 100. mu.L of competent cell TB1 is taken and mixed well. After ice-bath for 20min, performing heat shock in a water bath at 45 ℃ for 60s, immediately performing ice-bath for 5min, supplementing 800 mu LLB liquid culture solution, culturing at 37 ℃ for 45min at 200rpm, centrifuging at 7000rpm for 6min, sucking out supernatant, taking about 300 mu L, coating the supernatant in an LB-A solid (Amp) culture medium, and performing overnight culture at 37 ℃ to obtain positive clones.
E. Expression of polypeptide-MBP fusion proteins
And selecting a single colony from the plate of the positive clone obtained above, inoculating the single colony in 5mL of LB-A and 0.5% of cane sugar at 37 ℃ for 220r/min, carrying out shake culture overnight, inoculating the overnight culture in 50mL of LB-A and 0.2% of cane sugar culture media according to the inoculation amount (v/v) of 1%, respectively inoculating the culture in 3 bottles, carrying out shake culture at 37 ℃ for 220r/min, adding IPTG (isopropyl-beta-thiogalactoside) to the three bottles of culture until the final concentration is 0.3mmol/L when the bacterial concentration OD600 of the culture reaches 0.6, carrying out shake culture at 120r/min, centrifuging the three bottles of culture at 4 ℃ for 15min by using an inducer (IPEG solution) to collect thallus precipitates, and discarding the supernatant. Resuspending the cells in 400mL of 30mM Tris-HCl, 25% sucrose, pH 8.0(80mL/g of wet cell weight), adding EDTA to 1mM, shaking for 5-10min at room temperature, centrifuging for 15min at 8000g and 4 ℃, discarding the supernatant, resuspending the precipitate in 400mL of precooled 5mM MgS04, shaking for 15min on ice, shaking for 8000g and 4 ℃, centrifuging for 20min, retaining the supernatant, adding 8mL of 1M Tris-HCl to the supernatant, and centrifuging for pH 7.4 to obtain polypeptide-MBP fusion protein, wherein the fusion protein can be directly used for detection of Cry1Da in an ELISA (enzyme-linked immunosorbent assay) based on antigen-antibody.
Example 4 application of Cry1 Da-specific binding polypeptide molecules in ELISA
(1) Sample extraction
Weighing 5g of a sample to be detected, adding 25mL of PBS-methanol solution, and oscillating at 200rpm for 5 minutes; filtering the extractive solution with whatman No. 1 filter paper, adding 1mL of PBS (phosphate buffer solution, pH 7.2) into 1mL of filtrate, and mixing to obtain sample extractive solution.
(2) Coating and sealing
1. Direct detection method
The extract was directly coated on an ELISA plate and incubated overnight at 4 ℃. The following day, after 3 washes with PBST (10mM PBS, 0.05% Tween-20(v/v)), blocked with PBS containing 5% nonfat dry milk, and incubated at 37 ℃ for 1 hour.
2. Sandwich ELISA method
anti-Cry 1Da monoclonal antibody (1. mu.g/ml) diluted in 10mM PBS (pH 7.4) was coated on the microplate and incubated overnight at 4 ℃. The following day, after washing 3 times with PBST (10mM PBS, 0.05% Tween-20(v/v)), blocking with PBS containing 5% skim milk powder, incubating at 37 ℃ for 1 hour, washing the plate with PBST for 6 times, ultra-clean bench drying, and storing at 4 ℃ for later use.
(3) Establishment of a Standard Curve
A. Based on direct detection method
The strips were removed and 100. mu.L of a double dilution of Cry1Da protein standard (concentration range 0-2000ng/mL), 100. mu.L/well, incubated at 37 ℃ for 45min, per well. mu.L of phage displaying a molecule that specifically binds to the Cry1Da polypeptide (1.0X 10)11pfu) or expressed polypeptide-MBP fusion protein. Adding 1: a5000-fold dilution of either HRP-labeled anti-M13 phage secondary antibody at 100. mu.L or enzyme-labeled anti-MBP antibody was incubated at 37 ℃ for 45 min. Then developed with TMB substrate and OD read450An ELISA standard curve was established. The results show that: of the standard curveThe linear detection range is 0.1-400 ng/mL.
B. Sandwich ELISA method
Taking out the strips treated in the step (2), putting 100 mu L of the Cry1Da protein standard substance (the concentration range is 0-2000ng/mL) diluted by times into each hole, and incubating for 45min at 37 ℃ at 100 mu L/hole. mu.L of phage displaying a molecule that specifically binds to the Cry1Da polypeptide (1.0X 10)11pfu) or expressed polypeptide-MBP fusion protein. A1: 5000 fold dilution of either HRP-labeled anti-M13 phage secondary antibody at 100. mu.L or enzyme-labeled anti-MBP antibody was added and incubated at 37 ℃ for 45 min. Then developed with TMB substrate and OD read450A sandwich ELISA standard curve was established. The results show that: the linear detection range of the standard curve is 0.01-180 ng/mL.
(4) Detection of samples
And (3) taking out the lath processed in the step (2), respectively operating according to a direct or sandwich ELISA method, and reversely deducing the content of Cry1Da in the sample according to a standard curve.
Example 5 immunological assay stability assay for Cry1 Da-specific binding polypeptide molecules
Acid and alkali resistance test
The phage display polypeptide molecule/polypeptide-MBP fusion protein capable of being specifically combined with Cry1Da and the Cry1Da monoclonal antibody are respectively diluted into working solutions by buffers with pH values of 4.0, 5.0, 6.0, 7.4 and 8.0, and ELISA determination is respectively carried out on the working solutions, wherein the specific method comprises the following steps: cry1Da protein was coated on an enzyme plate and incubated overnight at 4 ℃. The following day, after washing 3 times with PBST (10mM PBS, 0.05% Tween-20(v/v)), blocking with PBS containing 5% skim milk powder, incubating at 37 ℃ for 1 hour, adding 100 μ L of CrylDa-specific binding polypeptide molecule or Cry1Da monoclonal antibody diluted to working solution with buffer solution at pH 4.0, 5.0, 6.0, 7.4, 8.0, respectively, and incubating for 30 min. A1: 5000 fold dilution of HRP-labeled secondary anti-M13 phage or anti-IgG antibody was added at 100. mu.L and incubated at 37 ℃ for 45 min. Adding 100 μ L TMB substrate solution, developing for 6min in dark, and reading the absorption value at 450nm with enzyme-labeling instrument.
The experimental results show that: the polypeptide molecule is used as a sandwich ELISA of Cry1Da recognition element, and the OD value is kept constant when the pH value is 5.0, 6.0, 7.4 and 8.0; the ELISA using the Cry1Da monoclonal antibody as a recognition element shows that the OD value changes irregularly when the pH value is 5.0, 6.0 and 8.0, and the immunological analysis performance of the antibody is obviously influenced, which shows that the polypeptide molecule used as the substitute of the Cry1Da antibody has better acid-base resistance.
Sequence listing
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