CN117964704A - Peptide aptamer capable of specifically recognizing thyroxine as well as screening method and application thereof - Google Patents

Peptide aptamer capable of specifically recognizing thyroxine as well as screening method and application thereof Download PDF

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Publication number
CN117964704A
CN117964704A CN202410080495.7A CN202410080495A CN117964704A CN 117964704 A CN117964704 A CN 117964704A CN 202410080495 A CN202410080495 A CN 202410080495A CN 117964704 A CN117964704 A CN 117964704A
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Prior art keywords
peptide aptamer
thyroxine
preferred embodiments
amino acid
seq
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卢晓玲
魏铮
王梦杰
黄杰
李凌昌
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Changsha Yixin Biotechnology Co ltd
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Changsha Yixin Biotechnology Co ltd
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Abstract

The invention discloses a peptide aptamer specifically recognizing thyroxine, a screening method and application thereof, wherein the sequence of the peptide aptamer comprises the following components: an amino acid sequence shown in any one of SEQ ID NO. 1-11; or the amino acid sequence shown in any one of SEQ ID NO. 1-11 is subjected to substitution and/or deletion and/or addition of one or more amino acids, and has the same function as the protein shown in any one of SEQ ID NO. 1-11; or an amino acid sequence having the same function as the amino acid sequence shown in any one of SEQ ID NO.1 to 11. The peptide aptamer provided by the invention has the advantages of low immunogenicity, easiness in preparation, good stability, high in vivo affinity, high tissue permeability, small batch-to-batch difference and the like, has the potential of replacing monoclonal antibodies, and can be used for developing thyroxine rapid detection reagents.

Description

Peptide aptamer capable of specifically recognizing thyroxine as well as screening method and application thereof
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a peptide aptamer capable of specifically recognizing thyroxine, and a screening method and application thereof.
Background
Thyroxine, one of the indispensable hormones of the body, has the effects of maintaining normal growth and development, promoting metabolism and maintaining normal functions of the cardiovascular and nervous systems. Lack of thyroxine and excessive thyroxine secretion can cause the body to produce symptoms of hyponychium, hyperthyroidism and the like. The current methods for detecting thyroxine include enzyme-linked immunoassay, electrochemiluminescence, radioimmunoassay, liquid chromatography-mass spectrometry (LC-MS) and fluorescence immunoassay. In the immunological method, the antibody is expensive, the preparation is complicated, and the problem of cross reaction exists.
While an emerging molecular recognition element, peptide aptamer, is exhibiting its endless potential to replace antibodies in molecular recognition. Compared with antibodies, the peptide aptamer has the advantages of low immunogenicity, easy preparation, good stability, small batch-to-batch difference and the like. Peptide aptamers have become a new choice for replacing monoclonal antibodies due to their better stability, in vivo affinity and tissue permeability than antibodies, coupled with lower immunogenicity. Thus, there is a need to provide thyroxine-specific peptide aptamers for use in place of thyroxine antibodies.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a peptide aptamer specifically recognizing thyroxine, which has the potential of replacing thyroxine antibodies and can be used for developing a thyroxine rapid detection method.
The invention also provides a nucleic acid molecule.
The invention also provides a biological material related to the nucleic acid molecules.
The invention also provides a product.
The invention also provides a conjugate.
The invention also provides a screening method of the peptide aptamer.
The invention also provides a characterization method of the peptide aptamer.
The invention also provides application of the peptide aptamer.
According to a first aspect of the present invention there is provided a peptide aptamer that specifically recognizes thyroxine, the sequence of the peptide aptamer comprising:
(1) An amino acid sequence shown in any one of SEQ ID NO. 1-11; or (b)
(2) Amino acid sequences shown in any one of SEQ ID NO. 1-11 are subjected to substitution and/or deletion and/or addition of one or more amino acids, and have the same functions as the proteins shown in any one of SEQ ID NO. 1-11; or (b)
(3) An amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% homology with the amino acid sequence shown in any one of SEQ ID NO.1 to 11 and having the same function as the amino acid sequence shown in any one of SEQ ID NO.1 to 11.
According to a second aspect of the present invention there is provided a nucleic acid molecule encoding a peptide aptamer according to the first aspect of the present invention.
According to a third aspect of the present invention, there is provided a biological material associated with a nucleic acid molecule, the biological material comprising at least one of (1) to (7):
(1) An expression cassette comprising a nucleic acid molecule according to the second aspect of the invention;
(2) A vector comprising a nucleic acid molecule according to the second aspect of the invention;
(3) A vector comprising the expression cassette of (1);
(4) A transgenic cell line comprising a nucleic acid molecule according to the second aspect of the invention;
(5) A transgenic cell line comprising the expression cassette of (1);
(6) A transgenic cell line comprising the vector of (2);
(7) A transgenic cell line comprising the vector of (3).
According to a fourth aspect of the present invention, there is provided a product comprising at least one of a peptide aptamer according to the first aspect of the present invention, a peptide aptamer encoded by a nucleic acid molecule according to the second aspect of the present invention and a biological material according to the third aspect of the present invention.
In some embodiments of the invention, the product comprises at least one of a drug, a reagent, a test plate, a kit, a test chip.
According to a fifth aspect of the present invention there is provided a conjugate comprising: a peptide aptamer according to the first aspect of the invention and a coupling moiety; the conjugate moiety comprises at least one of a detectable label, a drug, a toxin, an electron dense label, biotin/avidin, a spin label, an antibody Fc fragment, an antibody scFv fragment, a radioisotope, an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, and a viral coat protein.
According to a sixth aspect of the present invention, there is provided a method of screening a peptide aptamer according to the first aspect of the present invention, the method comprising the steps of:
s1: designing and synthesizing a random DNA library;
S2: integrating the random DNA obtained in the step S1 onto a phagemid vector to obtain a recombinant phagemid; transferring the recombinant phagemid into escherichia coli, then carrying out super-infection by using auxiliary phage to obtain super-infected escherichia coli, and culturing the super-infected escherichia coli overnight;
s3: collecting the bacterial liquid cultured overnight to obtain a phage display library;
s4: incubating the phage display library obtained in the step S3 with thyroxine, and collecting the eluate of the first round;
s5: repeatedly screening the eluate obtained in the step S4 to obtain a peptide aptamer library with strong binding force with thyroxine;
s6: and (3) sequencing the peptide aptamer library obtained in the step (S5) to obtain the amino acid sequence of the peptide aptamer specifically recognizing thyroxine.
In some embodiments of the invention, the sequence of the random DNA library of step S1 is: 5'-CACCGG CGCACCTGCGGCCAG-MNNMNNMNNMNNMNNMNNMNNMNNMNNMNNMNNM NNMNNMNNMNN-TAGCTGGGCCGCATAGAAAGG-3', wherein N represents any one of A, T, G and C, and M represents A or C.
In some embodiments of the invention, the phagemid vector of step S2 comprises pcatab 5E.
In some embodiments of the invention, the step S2 employs restriction enzymes SfiI, notI to integrate the random DNA onto the phagemid.
In some embodiments of the invention, the escherichia coli of step S2 is TG 1 escherichia coli.
In some embodiments of the invention, the helper phage of step S2 comprises any one of M13KO7 and VCSM 13.
In some embodiments of the invention, the incubation time for the superinfection of step S2 is 20-40 min.
In some preferred embodiments of the invention, the incubation time for the superinfection of step S2 is 30min.
In some embodiments of the invention, the step S3 further comprises centrifugation and collection of supernatant after collection of the overnight cultured bacterial liquid.
In some preferred embodiments of the invention, the centrifugation conditions are 4000g centrifugation at 4℃for 15min.
In some preferred embodiments of the present invention, the step S3 further comprises mixing the collected supernatant with a PEG-NaCl solution, incubating and centrifuging to collect a first precipitate.
In some more preferred embodiments of the invention, the mixing volume ratio of the supernatant to the PEG-NaCl solution is (4-6): 1.
In some more preferred embodiments of the invention, the mixing volume ratio of the supernatant to PEG-NaCl solution is 5:1.
In some more preferred embodiments of the invention, the PEG-NaCl solution is a 2.5mol/L NaCl solution containing 20% PEG 8000.
In some more preferred embodiments of the invention, the incubation time is 0.5 to 2 hours.
In some more preferred embodiments of the invention, the incubation time is 1h.
In some more preferred embodiments of the invention, the centrifugation conditions are 4000g centrifugation at 4℃for 15min.
In some more preferred embodiments of the present invention, the step S3 further comprises re-suspending the collected first precipitate as a suspension and adding a PEG-NaCl solution, mixing and incubating and centrifugally collecting a second precipitate.
In some more preferred embodiments of the invention, the buffer used for the resuspension is PBS.
In some more preferred embodiments of the invention, the mixing volume ratio of the suspension to PEG-NaCl solution is (4-6): 1.
In some more preferred embodiments of the invention, the mixing volume ratio of the suspension to PEG-NaCl solution is 5:1.
In some more preferred embodiments of the invention, the incubation time is 0.5 to 2 hours.
In some more preferred embodiments of the invention, the incubation time is 1h.
In some more preferred embodiments of the invention, the centrifugation conditions are 4000g centrifugation at 4℃for 15min.
In some more preferred embodiments of the present invention, the step S3 further comprises re-suspending the collected second precipitate and then centrifuging and collecting the supernatant.
In some more preferred embodiments of the invention, the buffer used for the resuspension is PBS.
In some more preferred embodiments of the invention, the centrifugation conditions are 12000rpm centrifugation at 4℃for 1min.
In some embodiments of the invention, the thyroxine of step S4 comprises bovine serum albumin-coupled thyroxine (T4-BSA).
In some embodiments of the invention, the co-incubation of step S4 is performed in a microplate.
In some preferred embodiments of the invention, the T4-BSA is coated on the bottom of a microplate prior to co-incubation.
In some more preferred embodiments of the invention, the coating is used at a T4-BSA concentration of 100 μg/mL.
In some more preferred embodiments of the present invention, the solvent for T4-BSA is a carbonate buffer (pH 9.6).
In some more preferred embodiments of the invention, the coating temperature is 35 to 40 ℃.
In some more preferred embodiments of the invention, the coating temperature is 37 ℃.
In some more preferred embodiments of the invention, the coating is for a period of time ranging from 0.5 to 2 hours.
In some more preferred embodiments of the invention, the time of coating is 1h.
In some more preferred embodiments of the invention, the step of blocking with a blocking buffer is also carried out after the coating and before the co-incubation.
In some more preferred embodiments of the invention, the blocking buffer is PBS buffer containing 5mg/mL BSA.
In some more preferred embodiments of the invention, the temperature of the blocking is between 35 and 40 ℃.
In some more preferred embodiments of the invention, the temperature of the seal is 37 ℃.
In some more preferred embodiments of the invention, the blocking time is 20 to 50 minutes.
In some more preferred embodiments of the invention, the blocking time is 30 minutes.
In some embodiments of the invention, the phage display library of step S4 is mixed with T4-BSA at a ratio of (1X 10 8～1×1010): 1 cfu/. Mu.g.
In some preferred embodiments of the invention, the phage display library of step S4 is mixed with T4-BSA at a ratio of 1X 10 9 when co-incubated: 1 cfu/. Mu.g.
In some embodiments of the invention, the temperature of the co-incubation in step S4 is room temperature (20-30 ℃).
In some embodiments of the invention, the co-incubation time of step S4 is 0.5 to 2 hours,
In some preferred embodiments of the invention, the co-incubation time of step S4 is 1h.
In some embodiments of the invention, the co-incubation of step S4 is performed by slow shaking on a shaker.
In some preferred embodiments of the invention, the slow shaking incubation is at a rotational speed of 30 to 70rpm.
In some more preferred embodiments of the invention, the slow shaking incubation is at a rotational speed of 50rpm.
In some embodiments of the present invention, the collecting the first round of eluate in step S4 includes discarding the liquid in the microplate, washing the microplate with a washing solution, adding an elution buffer for elution, collecting the eluate, and adding a neutralization buffer to obtain the first round of eluate.
In some preferred embodiments of the invention, the wash solution is a PBS buffer containing 0.05-0.4 v/v% Tween 20.
In some more preferred embodiments of the invention, the wash solution is PBS buffer containing 0.1v/v% Tween 20.
In some preferred embodiments of the invention, the elution buffer is an acidic buffer.
In some more preferred embodiments of the invention, the acidic buffer is glycine-hydrochloric acid buffer (pH 2.2).
In some preferred embodiments of the invention, the conditions for the elution are shaking at 30-70 rpm for 10-20 min on a shaker at room temperature (20-30 ℃).
In some more preferred embodiments of the invention, the conditions for the elution are shaking at 50rpm for 15min on a shaker at room temperature (20-30 ℃).
In some preferred embodiments of the invention, the neutralization buffer is an alkaline buffer.
In some more preferred embodiments of the invention, the alkaline buffer is Tris-HCl buffer (pH 9.2).
In some preferred embodiments of the invention, the volume ratio of elution buffer to neutralization buffer is 20: (1-5).
In some preferred embodiments of the invention, the volume ratio of elution buffer to neutralization buffer is 20:3.
In some embodiments of the invention, the repeated screening of step S5 comprises 3 rounds of screening, a second round of screening, a third round of screening, and a fourth round of screening, respectively.
In some preferred embodiments of the invention, the step of the second round of screening differs from step S4 only in that the second round of screening is coated with a concentration of T4-BSA of 75. Mu.g/mL and a concentration of Tween 20 in the wash of 0.2v/v%.
In some preferred embodiments of the invention, the step of the third round of screening differs from step S4 only in that the concentration of T4-BSA used in the second round of screening coating is 50. Mu.g/mL and the concentration of Tween 20 in the wash solution is 0.3v/v%.
In some preferred embodiments of the invention, the fourth round of screening does not need T4-BSA coating, the eluate obtained by the third round of screening is added for incubation after blocking, and the supernatant and the cleaning solution after incubation are collected, thus obtaining the fourth round of eluate.
In some more preferred embodiments of the invention, the blocking buffer is PBS buffer containing 5mg/mL BSA.
In some more preferred embodiments of the invention, the temperature of the blocking is between 35 and 40 ℃.
In some more preferred embodiments of the invention, the temperature of the seal is 37 ℃.
In some more preferred embodiments of the invention, the blocking time is 20 to 50 minutes.
In some more preferred embodiments of the invention, the blocking time is 30 minutes.
In some more preferred embodiments of the invention, the incubation temperature is room temperature (20-30 ℃).
In some more preferred embodiments of the invention, the incubation time is 0.5 to 2 hours.
In some more preferred embodiments of the invention, the incubation time is 1h.
In some more preferred embodiments of the invention, the incubation is a slow shaking incubation on a shaker.
In some more preferred embodiments of the invention, the slow shaking incubation is at a rotational speed of 30 to 70rpm.
In some more preferred embodiments of the invention, the slow shaking incubation is at a rotational speed of 50rpm.
In some more preferred embodiments of the invention, the wash solution is PBS buffer containing 0.1v/v% Tween 20.
According to a seventh aspect of the present invention, there is provided a method of characterising a peptide aptamer according to the first aspect of the invention, the characterisation method comprising at least one of a phage capture assay and a molecular docking assay.
In some embodiments of the invention, the phage capture assay comprises the steps of:
A1: incubating the peptide aptamer and thyroxine on a shaker, and collecting eluent;
a2: and (3) carrying out gradient dilution on the eluent in the step (A1), coating a plate after continuous gradient infection of host bacteria, counting colony numbers the next day, and calculating the recovery rate.
In some preferred embodiments of the invention, the thyroxine of step A1 comprises bovine serum albumin-coupled thyroxine (T4-BSA).
In some preferred embodiments of the invention, the co-incubation described in step A1 is performed in a microplate.
In some preferred embodiments of the invention, the T4-BSA is coated on the bottom of a microplate prior to co-incubation.
In some more preferred embodiments of the invention, the coating is used at a T4-BSA concentration of 10 μg/mL.
In some more preferred embodiments of the present invention, the solvent for T4-BSA is a carbonate buffer (pH 9.6).
In some more preferred embodiments of the invention, the coating temperature is 35 to 40 ℃.
In some more preferred embodiments of the invention, the coating temperature is 37 ℃.
In some more preferred embodiments of the invention, the coating is for a period of time ranging from 0.5 to 2 hours.
In some more preferred embodiments of the invention, the time of coating is 1h.
In some more preferred embodiments of the invention, the step of blocking with a blocking buffer is also carried out after the coating and before the co-incubation.
In some more preferred embodiments of the invention, the blocking buffer is PBS buffer containing 5mg/mL BSA.
In some more preferred embodiments of the invention, the temperature of the blocking is between 35 and 40 ℃.
In some more preferred embodiments of the invention, the temperature of the seal is 37 ℃.
In some more preferred embodiments of the invention, the blocking time is 20 to 50 minutes.
In some more preferred embodiments of the invention, the blocking time is 30 minutes.
In some embodiments of the invention, the phage display library of step A1 is mixed with T4-BSA at a ratio of (1X 10 8～1×1010): 1 cfu/. Mu.g.
In some preferred embodiments of the invention, the phage display library of step A1 is mixed with T4-BSA at a ratio of 1X 10 9: 1 cfu/. Mu.g.
In some embodiments of the invention, the temperature of the co-incubation described in step A1 is room temperature (20-30 ℃).
In some embodiments of the invention, the co-incubation time described in step A1 is from 0.5 to 2 hours,
In some preferred embodiments of the invention, the co-incubation time described in step A1 is 1h.
In some embodiments of the invention, the rotation speed of the co-incubation on the shaker as described in step A1 is between 30 and 70rpm.
In some preferred embodiments of the invention, the rotation speed of the co-incubation on the shaker as described in step A1 is 50rpm.
In some embodiments of the invention, the step A1 of collecting the eluate comprises the steps of discarding the liquid in the microplate, washing the microplate with a washing solution, adding an elution buffer for elution, collecting the eluate, and adding a neutralization buffer.
In some preferred embodiments of the invention, the wash solution is a PBS buffer containing 0.05-0.4 v/v% Tween 20.
In some more preferred embodiments of the invention, the wash solution is PBS buffer containing 0.1v/v% Tween 20.
In some preferred embodiments of the invention, the elution buffer is an acidic buffer.
In some more preferred embodiments of the invention, the acidic buffer is glycine-hydrochloric acid buffer (pH 2.2).
In some preferred embodiments of the invention, the conditions for the elution are shaking at 30-70 rpm for 10-20 min on a shaker at room temperature (20-30 ℃).
In some more preferred embodiments of the invention, the conditions for the elution are shaking at 50rpm for 15min on a shaker at room temperature (20-30 ℃).
In some preferred embodiments of the invention, the neutralization buffer is an alkaline buffer.
In some more preferred embodiments of the invention, the alkaline buffer is Tris-HCl buffer (pH 9.2).
In some preferred embodiments of the invention, the volume ratio of elution buffer to neutralization buffer is 20: (1-5).
In some preferred embodiments of the invention, the volume ratio of elution buffer to neutralization buffer is 20:3.
In some embodiments of the invention, the gradient dilution described in step A2 is a 10-fold gradient dilution.
In some embodiments of the invention, the continuous gradient of step A2 is 10 2 -fold dilution, 10 3 -fold dilution, and 10 4 -fold dilution.
In some embodiments of the present invention, the recovery rate in step A2 is calculated by the formula: recovery = (phage recovery titer/phage input titer) ×100%.
In some embodiments of the invention, the molecular docking assay comprises the steps of:
B1: predicting the three-dimensional structure of the peptide aptamer by Alphafold < 2 >, so as to obtain a predicted three-dimensional structure;
b2: and (3) docking the predicted three-dimensional structure in the step (B1) with a thyroxine T4 molecular structure by using MOE molecular docking software, and predicting the combination energy of the three-dimensional structure and the thyroxine T4 molecular structure.
In some preferred embodiments of the invention, the thyroxine T4 molecular structure of step B2 was downloaded from PubChem website, pubChem CID was 5819.
According to an eighth aspect of the present invention, there is provided the use of a peptide aptamer according to the first aspect of the present invention and/or a peptide aptamer obtained by screening according to the screening method of the sixth aspect of the present invention in the preparation of a thyroxine-detecting product.
In some embodiments of the invention, the product comprises at least one of a drug, a reagent, a test plate, a kit, a test chip.
According to a preferred (specific) embodiment of the invention, there is at least the following advantageous effect:
Compared with thyroxine antibodies, the peptide aptamer capable of specifically recognizing thyroxine provided by the invention has the advantages of low immunogenicity, easiness in preparation, good stability, high in vivo affinity, high tissue permeability, small batch-to-batch difference and the like, has the potential of replacing monoclonal antibodies, and can be used for developing thyroxine rapid detection reagents.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a schematic diagram of the screening principle and flow chart of embodiment 1 of the present invention;
FIG. 2 is a graph showing the recovery of the Seq1-11 peptide aptamer in the phage capture assay of example 2 of the invention;
FIG. 3 is a graph showing the results of recovery contribution rates of the Seq5, 7, 8, 10 peptide aptamers in the phage capture assay of example 2 of the present invention;
FIG. 4 is a predicted graph of the secondary structure of the peptide aptamers Seq1 to Seq11 of example 2 of the present invention;
FIG. 5 is a graph showing the results of scoring of the docking of the Seq1-11 peptide aptamer of example 2 of the invention with a thyroxine molecule.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Example 1
The embodiment provides a peptide aptamer specifically recognizing thyroxine, the principle and flow of a specific screening method are shown in figure 1, the peptide aptamer with the sequence shown as SEQ ID NO. 1-11 is obtained through screening, and the screening method specifically comprises the following steps:
1) Preparing a phage display library:
① The Shanghai biological engineering company is entrusted to synthesize a random single-stranded DNA library, and the library sequence is 5'-CACCGGCGCACCTGCGGCCAG-MNNMNNMNNMNNMNNMNNMNNMNNMNN MNNMNNMNNMNNMNNMNN-TAGCTGGGCCGCATAGAAAGG-3'; wherein N represents any one of A, T, G and C, M represents A or C;
② Annealing and extending the single-stranded DNA library in the step ① by using Klenow fragment (purchased from Bao Ri doctor materials technology (Beijing)) to synthesize a double-stranded DNA library, cloning the double-stranded DNA onto pCANTAB 5E phagemid vector by using restriction enzymes SfiI and NotI, and constructing to obtain recombinant phagemid;
③ Transferring the recombinant phagemid obtained in the step ② into escherichia coli TG 1, placing the escherichia coli TG 1 in a shaking table at 37 ℃, shaking at 200rpm for 40min, and then coating a plate, and performing total transformation for 10 times; after culturing for 24 hours, washing with 2YT liquid culture medium and collecting bacteria on a plate to obtain bacteria with recombinant phagemid, which can be used immediately or added with equal volume of 50% glycerol (equal volume of physiological saline and glycerol) for mixing and freezing at-80deg.C;
④ Inoculating 10 μl of the recombinant phagemid-carrying bacteria obtained in step ③ into 30mL 2YT liquid medium (containing 100 μg/mL ampicillin), and culturing at 37deg.C at 200rpm to logarithmic phase; adding helper phage M13KO7, mixing gently, standing at 37deg.C, and incubating for 30min to obtain super-infected cells;
⑤ The super-infected cells of step ④ were centrifuged at 4000g for 10min at 4℃and the supernatant carefully discarded, and the pellet was resuspended in 30mL 2YT-AK broth (containing 100. Mu.g/mL ampicillin and 50. Mu.g/mL kanamycin) and shake-cultured overnight on a shaker at 37℃at 200 rpm;
⑥ Collecting the bacterial liquid cultured overnight in the step ⑤, centrifuging 4000g at 4 ℃ for 15min to precipitate bacterial bodies, collecting supernatant, adding pre-cooled 2.5M NaCl solution containing 20% PEG8000 (the mixing volume ratio of the supernatant to the PEG-NaCl solution is 5:1), fully and uniformly mixing, and then placing the mixture on ice for incubation for 1h to precipitate phage; centrifuging at 4 ℃ for 15min at 4000g, and discarding supernatant; re-suspending the precipitate with 1mL PBS, adding the pre-cooled PEG-NaCl solution (the mixing volume ratio of the suspension to the PEG-NaCl solution is 5:1), reversing and mixing for a plurality of times, and incubating on ice for 1h; centrifuging at 4 ℃ for 15min at 4000g, and discarding supernatant; adding 200 mu L PBS to the precipitate for resuspension, centrifuging at 12000rpm for 1min at 4 ℃, and carefully collecting the supernatant to obtain a phage display library;
⑦ Absorbing 10 mu L of the phage display library in the step ⑥, carrying out 3 times of serial gradient dilution (10 2、103 and 10 4 times dilution respectively), respectively infecting host bacteria TG 1, then plating and counting, calculating the titer of the phage library according to the colony count of the next day, and detecting and calculating to obtain the titer of the phage display library reaching 1.06 multiplied by 10 13 cfu/mL;
2) Binding force screening of phage display library to thyroxine:
① T4-BSA (thyroxine coupled bovine serum albumin) was diluted to 100. Mu.g/mL with a carbonate coating buffer at pH 9.6 and added to the microplate, 100. Mu.L per well, coated for 1h at 37 ℃; taking out the coated microporous plate, pouring out the coating buffer solution in the hole, and immediately beating on sterile paper;
② Setting a control hole (namely coating is not carried out, only sealing treatment is carried out) and a screening hole (coating and sealing treatment) respectively, and adding 300 mu L of sealing buffer solution (namely PBS containing 5mg/mL BSA) into each of the control hole and the screening hole to enable the sealing buffer solution to fill the small holes, and sealing at 37 ℃ for 30min;
③ Aspirate blocking buffer and beat as much as possible the residual liquid as described in step ①, wash the microplate 3 times with 300 μl of 0.1% PBST buffer (i.e. PBS containing 0.1v/v% tween 20) and beat dry the liquid;
④ After washing, 100. Mu.L of phage library prepared in the step 1) is added into each well (diluted to about 1X 10 10 cfu in advance), mixed uniformly, rotated carefully so that the liquid fully infiltrates the bottom of the well, and placed on a shaking table at 50rpm and slowly shaken for 1 hour at room temperature;
⑤ Pouring out the liquid in the microwell plate and beating the liquid as dry as possible, and rapidly washing the microwell plate with 300 mu L of 0.1% PBST buffer for 6 times as described in step ①; adding 100 μl of elution buffer (Gly-HCl, pH 2.2), and eluting at 50rpm on a shaker at room temperature for 15min; collecting eluent, immediately adding 15 mu L of neutralization buffer (Tris-HCl, pH 9.2), and uniformly mixing to obtain a first round of screening;
⑥ Repeatedly screening the first round of screening material in the step ⑤ according to the step ①～⑤, only adjusting the coating concentration of the T4-BSA to 75 mug/mL, adjusting the concentration of Tween in PBST to 0.2v/v%, and obtaining a second round of screening material by screening under the same conditions;
⑦ Repeatedly screening the second round of screening material in the step ⑥ according to the step ①～⑤, only adjusting the coating concentration of the T4-BSA to 50 mug/mL, adjusting the concentration of Tween in PBST to 0.3v/v%, and obtaining a third round of screening material by screening under the same conditions;
⑧ Adding the third round of screening described in step ⑦ to wells without T4-BSA coating (still blocking step) for incubation, blocking and incubation performed according to step ②～④; after incubation, the supernatant was collected, the microwell plates were rapidly washed 6 times with 300 μl of 0.1% PBST buffer as described in step ① and all wash solutions were collected; combining the collected supernatant and the cleaning liquid to obtain a fourth-round screening object;
⑨ Sequencing the fourth round of screening in the step ⑧, and identifying and screening to obtain 11 peptide aptamer sequences, wherein 5 repeated sequences are obtained, and the sequence information and the sequencing frequency of the sequences are shown in the table 1.
TABLE 1 amino acid sequence information and sequencing frequency of peptide aptamers
Example 2 characterization method of peptide aptamer specifically recognizing thyroxine
The present embodiment provides a characterization method for specifically recognizing thyroxine peptide aptamer and characterizes peptide aptamers with sequences shown in SEQ ID No. 1-11 obtained by screening in embodiment 1, specifically including the following steps:
1) Preparing a phage display library: the specific procedure for the preparation of PT-1 to PT-11 was as follows, using the phage display library (original library) prepared in example 1 as a control group, and respectively synthesizing monoclonal phages (PT-1 to PT-11) capable of displaying peptide aptamers having sequences as shown in SEQ ID No.1 to 11 as an experimental group:
① Taking 10 mu L of host bacteria with PT-1-PT-11 gene phagemid (the host bacteria are monoclonal strains which are screened and protected after the sequencing of the example 1), inoculating the host bacteria into 30mL of 2YT liquid culture medium (containing 100 mu g/mL ampicillin), and culturing the host bacteria to logarithmic phase at the temperature of 37 ℃ at the speed of 200 rpm; adding helper phage M13KO7, mixing gently, standing at 37deg.C, and incubating for 30min to obtain super-infected cells;
② The super-infected cells of step ① were centrifuged at 4000g for 10min at 4℃and the supernatant carefully discarded, and the pellet was resuspended in 30mL 2YT-AK broth (containing 100. Mu.g/mL ampicillin and 50. Mu.g/mL kanamycin) and shake-cultured overnight on a shaker at 37℃at 200 rpm;
③ Collecting the bacterial liquid cultured overnight in the step ②, centrifuging 4000g at 4 ℃ for 15min to precipitate bacterial bodies, collecting supernatant, adding a precooled 2.5M NaCl solution containing 20% PEG8000 (the mixing volume ratio of the supernatant to the PEG-NaCl solution is 5:1), fully and uniformly mixing, and then placing the mixture on ice for incubation for 1h to precipitate phage; centrifuging at 4 ℃ for 15min at 4000g, and discarding supernatant; re-suspending the precipitate with 1mL PBS, adding the pre-cooled PEG-NaCl solution (the mixing volume ratio of the suspension to the PEG-NaCl solution is 5:1), reversing and mixing for a plurality of times, and incubating on ice for 1h; centrifuging at 4 ℃ for 15min at 4000g, and discarding supernatant; adding 200 mu L PBS to the precipitate for resuspension, centrifuging at 12000rpm for 1min at 4 ℃, and carefully collecting the supernatant to obtain PT-1-PT-11 monoclonal phage;
④ And (3) respectively sucking 10 mu L of the monoclonal phage in the step ③, carrying out 3 times of serial gradient dilution (10 2、103 and 10 4 times of dilution respectively), respectively infecting host strain TG 1, then plating and counting, calculating the titer of the phage library according to the colony number of the next day, and detecting and calculating to obtain the monoclonal phage titer reaching 10 12 cfu/mL.
2) Phage capture assay:
① Coating: T4-BSA (thyroxine coupled bovine serum albumin) was diluted to 10. Mu.g/mL using a carbonate coating buffer pH 9.6 and added to the microplate, 100. Mu.L per well, 12 wells total (i.e., experimental wells); meanwhile, a control hole coated with 100 mu L of 5mg/mL BSA is also arranged; coating for 1h at 37 ℃;
② Closing: taking out the coated microporous plate, pouring out the coating buffer solution in the hole, and immediately beating on sterile paper; the experimental and control wells were blocked by adding 300. Mu.L of blocking buffer (i.e., PBS containing 5mg/mL BSA);
③ Incubation: aspirate blocking buffer and beat as much as possible the residual liquid as described in step ②, wash the microplate 3 times with 300 μl of 0.1% pbst buffer (i.e. PBS containing 0.1v/v% tween 20) and beat dry the liquid; adding 11 monoclonal antibodies of PT-1 to PT-11 and an original library control, 100 mu L/hole, incubating L h;
④ Eluting: PBST is used for washing the plate for 6 times, 100 mu L of elution buffer (Gly-HCl, pH 2.2) is added, the elution is immediately recovered after 15min of elution, and 15 mu L of neutralization buffer (Tris-HCl, pH 9.2) is added for uniform mixing;
⑤ Titer determination: performing gradient dilution on each group of eluents, coating a plate after three continuous gradients (10 2、103 and 10 4 times dilution) are adopted to infect host bacteria, and calculating the recovery rate by counting colony numbers in the next day; recovery = (phage recovery titer/phage input titer) ×100%; the results are shown in FIGS. 2 to 3.
3) Molecular docking test:
① Firstly, predicting three-dimensional structures of 11 candidate peptide aptamers by using Alphafold online websites (https:// colab. Research. Google. Com/github/sokrypton/ColabFold/blob/main/alphafold2. Ipynb), wherein the prediction results are shown in figure 4;
② Downloading thyroxine T4 molecular files at PubChem website (https:// pubchem. Ncbi. Nl. Gov /), pubChem CID being 5819;
③ Docking of candidate polypeptides with thyroxine T4 was performed using MOE software, and the binding energy of both was predicted, with the predicted results shown in figure 5.
From FIGS. 2 and 3, it can be seen that the recovery rates of Seq5, seq7, seq8, and Seq10 are significantly improved over the library, and significantly stronger than other clones, and can be used as subjects for further analysis; since thyroxine T4 has too small a molecular weight, the BSA coupling method was used for immobilization during coating. Therefore, wells coated with BSA alone were also required as controls in evaluating affinity. Here, the recovery rate of the coated T4-BSA wells was N, the recovery rate of the coated BSA wells was N0, the contribution rate of T4 and BSA to the recovery rate was calculated from the ratio of the difference between N and N0 to N, and the candidate peptide sequences were evaluated by combining the recovery rate and the recovery contribution rate, and the recovery contribution rates were calculated for Seq5, seq7, seq8, and Seq10, whereby it was found that the recovery rate of Seq5 was the highest, but the recovery contribution rate of T4 was not the same as that of the other three candidate sequences, and the recovery rate of Seq7 was the third one.
As can be seen from FIGS. 4 and 5, the molecular predicted docking results are consistent with phage capture assay results, and Seq7 and Seq8 perform best.
In summary, the present protocol provides 11 peptide aptamer sequences from a co-screen, and the peptide aptamers have improved affinity compared to the original library, as compared to recovery from phage capture assays. According to the mutual verification results of the phage capture test and the molecular docking test, the Seq7 and the Seq8 perform optimally, and are the optimal candidate sequences of thyroxine peptide aptamer.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims (10)

1. A peptide aptamer that specifically recognizes thyroxine, wherein the sequence of the peptide aptamer comprises:
(1) An amino acid sequence shown in any one of SEQ ID NO. 1-11; or (b)
(2) Amino acid sequences shown in any one of SEQ ID NO. 1-11 are subjected to substitution and/or deletion and/or addition of one or more amino acids, and have the same functions as the proteins shown in any one of SEQ ID NO. 1-11; or (b)
(3) An amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% homology with the amino acid sequence shown in any one of SEQ ID NO.1 to 11 and having the same function as the amino acid sequence shown in any one of SEQ ID NO.1 to 11.
2. A nucleic acid molecule encoding the peptide aptamer of claim 1.
3. A biological material associated with a nucleic acid molecule, wherein the biological material comprises at least one of (1) to (7):
(1) An expression cassette comprising the nucleic acid molecule of claim 2;
(2) A vector comprising the nucleic acid molecule of claim 2;
(3) A vector comprising the expression cassette of (1);
(4) A transgenic cell line comprising the nucleic acid molecule of claim 2;
(5) A transgenic cell line comprising the expression cassette of (1);
(6) A transgenic cell line comprising the vector of (2);
(7) A transgenic cell line comprising the vector of (3).
4. A product comprising at least one of the peptide aptamer of claim 1, the peptide aptamer encoded by the nucleic acid molecule of claim 2, and the biological material of claim 3.
5. The product of claim 4, wherein the product comprises at least one of a drug, a reagent, a test plate, a kit, and a test chip.
6. A conjugate, the conjugate comprising: the peptide aptamer of claim 1 and a coupling moiety; the conjugate moiety comprises at least one of a detectable label, a drug, a toxin, an electron dense label, biotin/avidin, a spin label, an antibody Fc fragment, an antibody scFv fragment, a radioisotope, an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, and a viral coat protein.
7. A method of screening for a peptide aptamer according to claim 1 or encoded by a nucleic acid molecule according to claim 2, comprising the steps of:
s1: designing and synthesizing a random DNA library;
S2: integrating the random DNA library obtained in the step S1 onto a phagemid vector to obtain a recombinant phagemid; transferring the recombinant phagemid into escherichia coli, then carrying out super-infection by using auxiliary phage to obtain super-infected escherichia coli, and culturing the super-infected escherichia coli overnight;
s3: collecting the bacterial liquid cultured overnight to obtain a phage display library;
s4: incubating the phage display library obtained in the step S3 with thyroxine, and collecting the eluate of the first round;
s5: repeatedly screening the eluate obtained in the step S4 to obtain a peptide aptamer library with strong binding force with thyroxine;
s6: and (3) sequencing the peptide aptamer library obtained in the step (S5) to obtain the amino acid sequence of the peptide aptamer specifically recognizing thyroxine.
8. The method according to claim 7, wherein the random DNA library of step S1 has the sequence: 5'-CACCGGCGCACCTGCGGCCAG-MNNMNNMNNMNNMNNMNNMNNMNN MNNMNNMNNMNNMNNMNNMNN-TAGCTGGGCCGCATAGAAAGG-3', wherein N represents any one of A, T, G and C, and M represents A or C.
9. Use of a peptide aptamer according to claim 1 and/or a peptide aptamer screened by a screening method according to any one of claims 7 to 8 in the preparation of a thyroxine detection product.
10. The use of claim 9, wherein the product comprises at least one of a drug, a reagent, a test plate, a kit, a test chip.
CN202410080495.7A 2024-01-19 2024-01-19 Peptide aptamer capable of specifically recognizing thyroxine as well as screening method and application thereof Pending CN117964704A (en)

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