CN112574283A - Human MUC1 specific binding polypeptide and extraction method thereof - Google Patents
Human MUC1 specific binding polypeptide and extraction method thereof Download PDFInfo
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Abstract
The invention discloses a specific binding polypeptide of human MUC1, and the amino acid sequence of the specific binding polypeptide is HKFIPHRDTSIA. The polypeptide is verified by methods such as biological panning, positive phage amplification, DNA sequence determination of polypeptide presented by coded phage, ELISA determination of screened positive phage and the like. The polypeptide has small molecular weight, easy chemical synthesis and low immunogenicity, can be specifically combined with human MUC1 protein, and has good application value in the field of tumor diagnosis and treatment with human MUC1 protein as a target spot.
Description
Technical Field
The invention belongs to the technical field of protein and polypeptide extraction and separation, and particularly relates to an extraction technology of human MUC1 specific binding polypeptide.
Background
Tumor is one of diseases seriously threatening human health, and with the deep understanding of people on tumor and the continuous development of scientific technology, the clinical drugs for tumor diagnosis and treatment are continuously appeared, but how to effectively improve the specificity of tumor diagnosis and treatment drugs and reduce the combination of tumor diagnosis and treatment drugs and non-tumor tissues is still a difficult problem restricting the tumor diagnosis to advance to the accurate stage. The targeting drug strategy is an effective method for solving the problem, and the method utilizes molecules (such as receptors and other molecules) which are highly expressed compared with normal tissues of tumor tissues as targets, and combines substances (such as antibodies, ligands or small molecular polypeptides and the like) which specifically recognize the molecules with the tumor diagnosis and treatment drug, so as to realize the specific enrichment of the tumor diagnosis and treatment drug in the tumor tissues.
The design strategy of the polypeptide-mediated targeting drug is an effective method for improving the enrichment degree of the drug in lesion tissues, increasing the curative effect of the drug and reducing the dosage and toxic and side effects of the drug in recent years. The discovery of a good polypeptide-directed drug requires two conditions, the first being a specific drug target. Mucin 1(mucin antigen 1, MUC1) is just such a drug target. Mucin 1(MUC 1) is the earliest protein found in the mucin family, and in normal physiological environment, MUC1 is expressed in the apical expression and polarity distribution in the near-luminal or glandular luminal surface of epithelial cells in many tissues and organs. In tumor cells, MUC1 shows the characteristics of up-regulation of expression, abnormal glycosylation, polarity disappearance and the like, plays an important role in the occurrence and development of tumors by regulating and controlling the processes of proliferation, epithelial-mesenchymal transition, epigenetics, immune escape and the like of the tumor cells, and is an ideal drug target in accurate diagnosis and treatment of the tumors.
The second condition of polypeptide-targeted drug design is that a molecule capable of specifically recognizing the target is required to be used as a targeted molecule. Phage display peptide library technology, which has become an effective tool for finding targeting molecules, has developed rapidly in the last 20 years. The inventors of the technology shared the nobel chemical prize in 2018.
Experiments such as phage ELISA and the like prove that the 1 polypeptide can specifically identify MUC1 by using MUC1 as a target molecule and obtaining a small molecular polypeptide from a phage display random 12 peptide library, and has a good application prospect in the research and development of tumor diagnosis and treatment medicines taking MUC1 as a target spot.
In the prior art, the screening of MUC1 mucin antigen mimic epitope in lung cancer T7 phage library (Qiu et al, 2016.05), the screening of tumor MUC1/Y mucin binding peptide by using phage random peptide library (Zhang Li Xin, 2001.09), the cloning, expression analysis and screening research of binding peptide of MUC1/Y mucin (Zhang Li Xin, 2000.06), and the Chinese patent application CN110511269A all studied the screening technology of MUC1 or MUC4 polypeptide. However, these documents and patent applications differ in the strains, buffers and eluents selected, in the conditions for reaction and treatment, and in the amino acid sequence of the selected polypeptide, and therefore in the specificity of binding to MUC 1.
Disclosure of Invention
In view of the above disadvantages of the prior art, the present invention provides a method for extracting a polypeptide capable of binding to human MUC1 with high specificity, which is specifically achieved by the following techniques.
A human MUC1 specific binding polypeptide, wherein the amino acid sequence of the human MUC1 specific binding polypeptide is HKFIPHRDTSIA.
The invention mainly aims at the problem of poor specificity of the existing tumor diagnosis and treatment medicine, takes a human MUC1 protein as a classic tumor marker as a target molecule, and obtains 1 polypeptide sequence by performing biological elutriation on a phage display random 12 peptide library. Experiments such as phage ELISA and the like show that the peptide can specifically identify human MUC1 protein, and has good application prospect in the research and development of tumor diagnosis and treatment medicines taking human MUC1 protein as a target spot.
The invention also provides an extraction method of the human MUC1 specific binding polypeptide, which is characterized by comprising the following steps
S1, biological elutriation: adding MUC1 solution into the pore plate, inoculating Escherichia coli strain and tetracycline-containing culture medium, and culturing; pouring out residual liquid, adding TBS sealing liquid and washing for a plurality of times; adding a phage solution for amplification, purification and elution to obtain eluted phage; adding the eluted phage solution into an escherichia coli culture for amplification, and separating to obtain amplified eluent; repeating the second and third rounds of screening to obtain screened positive plaques;
s2, amplification of positive phage: adding the overnight culture of the escherichia coli into a culture medium for dilution, dipping a plurality of well-separated blue plaques for inoculation, culture and amplification; centrifuging and taking the supernatant to obtain amplified positive phage liquid;
s3, DNA sequencing of encoded phage-displayed polypeptides: extracting phage single-chain DNA, sequencing by using a sequencer by taking the phage single-chain DNA as a template to obtain a phage single-chain DNA sequence, and deducing an amino acid sequence of polypeptide presented by the phage;
s4, performing ELISA determination verification on the screened positive phage.
Preferably, in the method for extracting the human MUC 1-specific binding polypeptide, the step S1 specifically comprises:
s11, adding 200ul of NaHCO with concentration of 100ug/mL into each well of 96-well plate, and adding 0.1mol/L3Incubating the diluted human MUC1 protein solution overnight at 4 ℃ with shaking; inoculating 10mL of Escherichia coli in 20mLLB culture medium containing tetracycline, and culturing at 37 deg.C; taking out the 96-well plate, and removing residual liquid; adding 200uL of TBS blocking solution, and incubating for 2h at 4 ℃; washing with TBST buffer solution for several times to remove residual liquid;
s12, diluting 200ul of the mixture with TBST buffer solution to contain 2X 1011pfu phage solution, the TBST buffer solution is TBS solution containing 0.1% Tween; adding the protein into the sealed human MUC1 coated well of the 96-well plate obtained in the step S12, and incubating for 1h at room temperature; pouring out the liquid, washing for a plurality of times by using TBST buffer solution, and removing the residual liquid in each washing; then eluting with 100 μ L of eluent, and rapidly adding neutralizing solution for neutralization to obtain eluted phage solution;
s13, taking 1 mu L of the eluted phage solution of the step S12 to measure titer, adding the rest eluted phage solution into 20mL of escherichia coli culture at early logarithmic growth stage, and carrying out shake culture at 37 ℃ for 4.5 h; centrifuging the mixed solution at 4 deg.C and 10000rpm for 10min, and centrifuging the supernatant for the second time; adding 80% of the supernatant obtained by the second centrifugation into 1/6 PEG8000/NaCl solution in the volume of the supernatant, and standing overnight at 4 ℃;
s14, taking out the liquid obtained in the step S12, centrifuging for 15min at 4 ℃ and 10000rpm, and taking out the precipitate; centrifuging once again, and sucking up residual supernatant; resuspending with 1mL TBS buffer solution, centrifuging at 10000rpm for 5min under the condition of resuspension of 4 ℃, taking the supernatant, adding a PEG/NaCl solution with the volume of 1/6 supernatant, and carrying out ice bath for 1 h; centrifuging at 4 deg.C and 10000rpm for 15min, and removing supernatant; centrifuging again, and sucking up residual supernatant;
s15, using 200uL containing 0.01% NaN3Centrifuging the TBS solution at 4 ℃ and 10000rpm to resuspend the precipitate obtained in the step S13 for 1min, and taking the supernatant to obtain an amplified eluent, namely completing the first round of screening; 1uL of the amplified eluent is used for measuring the titer, and the residual liquid is stored at 4 ℃ for standby; the titer of the phage was determined by observing the results of the titer measurement in step S14, and the titer calculated from the titer was equivalent to a phage content of 2X 1011Volume of phage solution of pfu;
s16, newly coating 1 hole of a 96-hole plate with 200uL of human MUC1 protein solution, and performing a second round of screening by adopting the method of S11-S15, wherein the concentration of the target protein is 50mg/L, and the concentration of Tween in the TBST buffer solution in the second round of screening is 0.5%;
s17, newly coating one hole of a 96-hole plate with 200ul of human MUC1 protein solution with the concentration of 100ug/mL, and performing the third screening by adopting the method of the steps S11-S15, wherein the concentration of the target protein is 20 mg/L; performing titer determination on 1ul of the third eluate, and storing the rest eluate at 4 deg.C; in the titer assay, 50 well-separated positive plaques were randomly picked from plates with a plaque number of less than 100.
Preferably, in the method for extracting the human MUC 1-specific binding polypeptide, the step S2 specifically comprises:
s21, diluting an overnight culture of ER2738 with LB culture medium according to a ratio of 1:100, and subpackaging the diluted culture in a plurality of 50mL culture tubes according to 1 mL/tube;
s22, dipping 50 well-separated blue plaques, respectively putting the blue plaques into the culture tube in the step S21, and carrying out shake culture at 37 ℃ for 4.5h to complete amplification;
s23, centrifuging the amplified phage liquid obtained in the step S22 for 30S; taking the supernatant and centrifuging again; and taking 80% of the supernatant obtained by the second centrifugation to obtain the amplified phage liquid.
Preferably, in the method for extracting the human MUC 1-specific binding polypeptide, the step S3 specifically comprises:
s31, extracting the single-stranded DNA of the positive phage obtained in the step S2;
s32, automatically sequencing on an automatic sequencer by using the single-stranded DNA as a template and a sequencing primer with the sequence of 5 '-CCCTCATAGTTAGCGTAACG-3';
and S33, deducing the amino acid sequence of the peptide presented by the phage according to the DNA sequencing result.
Preferably, in the method for extracting the human MUC 1-specific binding polypeptide, the step S4 specifically comprises:
s41, adding 200ul of NaHCO with concentration of 100ug/mL into each well of 96-well plate, and adding 0.1mol/L3Setting a coated well of 1% BSA solution as a negative control for each well of a human MUC1 protein solution diluted by the solution, and incubating overnight at 4 ℃;
s42, pouring out the coating solution, adding the sealing solution, and sealing for 2h at 4 ℃; then pouring off the confining liquid, washing for a plurality of times by using TBST buffer solution, and removing residual liquid in each washing;
s44, combining phages with the same single-stranded DNA sequence according to the sequencing result of the step S3, respectively adding a plurality of remaining purified phages into human MUC1 protein coated wells, and incubating for 2h at room temperature;
s45, washing for several times by using TBST buffer solution, adding 100 mu L of HRP-labeled mouse anti-M13 phage mAb with the dilution of 1:5000 into each hole, and incubating for 1h at room temperature; the absorbance at A490nm was measured after several additional washes with TBST buffer and OPD development.
More preferably, in the method for extracting the human MUC 1-specific binding polypeptide, in step S42, the blocking solution is pH 8.6, 5mg/L BSA and 0.1mol/L NaHCO3The mixed solution of (1).
Preferably, in the method for extracting the human MUC1 specific binding polypeptide, the eluent is a mixed solution of BSA (bovine serum albumin) with the pH of 2.2 and 10g/L and Glycine-HCl with the pH of 0.2 mol/L; the neutralization solution is Tris-HCl solution with the pH value of 9.1 and 1 mol/L.
Compared with the prior art, the invention has the advantages that: the polypeptide capable of specifically recognizing the human MUC1 protein provided by the invention has the advantages of small molecular weight, easiness in chemical synthesis and low immunogenicity, and has good application value in the field of tumor diagnosis and treatment with MUC1 as a target spot.
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FIG. 1 is the absorbance at A490nm of a polypeptide specifically recognizing human MUC1 protein, prepared in example 1;
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The specific binding polypeptide of human MUC1 provided by the application is extracted by the following method:
s1, biological elutriation: adding MUC1 solution into the pore plate, inoculating Escherichia coli strain and tetracycline-containing culture medium, and culturing; pouring out residual liquid, and adding TBS sealing liquid for washing; adding a phage solution for amplification, purification and elution to obtain eluted phage; adding the eluted phage solution into an escherichia coli culture for amplification, and separating to obtain amplified eluent; repeating the second and third rounds of screening to obtain screened positive plaques;
the specific steps of step S1 are as follows:
s11, adding 200ul of NaHCO with concentration of 100ug/mL into each well of 96-well plate, and adding 0.1mol/L3Placing the diluted human MUC1 protein solution in a wet box, slightly shaking at 4 ℃, and incubating overnight; inoculating 10mL of Escherichia coli ER2738 into 20mL of LB medium (for amplifying phage) containing tetracycline, and culturing at 37 ℃;taking out the 96-well plate, pouring out the liquid, then reversely buckling the 96-well plate on a clean paper towel, and slightly deducting the residual liquid; adding 200uL of TBS blocking solution, and incubating for 2h at 4 ℃; washing with TBST buffer solution for 6 times, and removing residual liquid in each washing;
s12, diluting 200ul of the mixture with TBST buffer solution to contain 2X 1011pfu phage solution, the TBST buffer solution is TBS solution containing 0.1% Tween; adding the protein into the sealed human MUC1 coated well of the 96-well plate obtained in the step S12, and incubating for 1h at room temperature; pouring out the liquid, washing for 10 times by using TBST buffer solution, and reversely buckling the 96-well plate on a clean paper towel for each washing to slightly subtract the residual liquid; then eluting with 100 μ L of eluent, and rapidly adding neutralizing solution for neutralization to obtain eluted phage solution;
s13, taking 1 mu L of the eluted phage solution of the step S12 to measure the titer, adding the rest of the eluted phage solution into 20mL of escherichia coli R2738 culture in the early logarithmic growth phase, and carrying out vigorous shaking culture at 37 ℃ for 4.5 h; centrifuging the mixed solution at 4 deg.C and 10000rpm for 10min, and centrifuging the supernatant for the second time; adding 80% of the supernatant obtained by the second centrifugation into 1/6 PEG8000/NaCl solution in the volume of the supernatant, and standing overnight at 4 ℃;
s14, taking out the liquid obtained in the step S12, centrifuging for 15min at 4 ℃ and 10000rpm, and taking out the precipitate; centrifuging once again, and sucking up residual supernatant by using a micropipette; resuspending with 1mL TBS buffer solution, precipitating the residual cells, and centrifuging at 10000rpm for 5min under the condition of resuspension at 4 ℃; adding the supernatant into a PEG/NaCl solution with the volume of 1/6 supernatant, and carrying out ice bath for 1 h; centrifuging at 4 deg.C and 10000rpm for 15min, and carefully decanting the supernatant; centrifuging again, and sucking up residual supernatant with a micropipette;
s15, using 200uL containing 0.01% NaN3Centrifuging the TBS solution at 4 ℃ and 10000rpm to resuspend the precipitate obtained in the step S13 for 1min, taking the supernatant and transferring the supernatant into a new centrifugal tube to obtain an amplified eluent, namely completing the first round of screening; 1uL of the amplified eluent is used for measuring the titer, and the residual liquid is stored at 4 ℃ for standby; the titer of the phage was determined by observing the results of the titer measurement in step S14, and the titer calculated from the titer was equivalent to a phage content of 2X 1011Volume of phage solution of pfu;
s16, newly coating 1 hole of a 96-hole plate with 200uL of human MUC1 protein solution, and performing a second round of screening by adopting the method of S11-S15, wherein the concentration of the target protein is 50mg/L, and the concentration of Tween in the TBST buffer solution in the second round of screening is 0.5%;
s17, newly coating one hole of a 96-hole plate with 200ul of human MUC1 protein solution with the concentration of 100ug/mL, and performing the third screening by adopting the method of the steps S11-S15, wherein the concentration of the target protein is 20 mg/L; the eluted product of the third round is not required to be amplified, 1ul of the eluted product of the third round is taken for titer determination, and the rest of the eluted product is stored at 4 ℃; in the titer assay, 50 well-separated positive plaques were randomly picked from plates with a plaque number of less than 100.
S2, amplification of positive phage: adding the overnight culture of Escherichia coli ER2738 into the culture medium, diluting, dipping 50 well-separated blue plaques, inoculating, culturing and amplifying; centrifuging and taking the supernatant to obtain amplified positive phage liquid;
the specific steps of the above-mentioned step S2 are,
s21, diluting an overnight culture of ER2738 with LB culture medium according to a ratio of 1:100, and subpackaging the diluted culture in 1 mL/tube into 50mL culture tubes;
s22, dipping 50 well-separated blue plaques, respectively putting the blue plaques into the culture tube in the step S21, and carrying out shake culture at 37 ℃ for 4.5h to complete amplification;
s23, centrifuging the amplified phage liquid obtained in the step S22 for 30S; taking the supernatant and centrifuging again; and taking 80% of the supernatant obtained by the second centrifugation to obtain the amplified phage liquid. The amplified phage liquid can be stored for several weeks at 4 ℃, and if the amplified phage liquid is stored for a long time, sterile glycerol with the final concentration of 50 percent can be added to the amplified phage liquid for storage at the temperature of-20 ℃.
S3, DNA sequencing of encoded phage-displayed polypeptides: extracting phage single-chain DNA, sequencing by using a sequencer by taking the phage single-chain DNA as a template to obtain a phage single-chain DNA sequence, and deducing an amino acid sequence of polypeptide presented by the phage;
the specific steps of the above-mentioned step S3 are,
s31, extracting the single-stranded DNA of the positive phage obtained in the step S2;
s32, taking the single-stranded DNA as a template, and automatically sequencing on an ABI 310DNA automatic sequencer by using a sequencing primer provided in the phage random peptide library kit and a sequencing primer with a sequence of 5 '-CCCTCATAGTTAGCGTAACG-3';
s33, deducing that the amino acid sequence of the peptide presented by the phage is HKFIPHRDTSIA according to the DNA sequencing result.
S4, performing ELISA determination verification on the screened positive phage, and specifically comprising the following steps:
s41, adding 200ul of NaHCO with concentration of 100ug/mL into each well of 96-well plate, and adding 0.1mol/L3Setting a coated well of 1% BSA solution as a negative control for each well of a human MUC1 protein solution diluted by the solution, and incubating overnight at 4 ℃;
s42, pouring out the coating solution, adding the sealing solution, and sealing for 2h at 4 ℃; then pouring off the confining liquid, washing for a plurality of times by using TBST buffer solution, and removing residual liquid in each washing;
s44, merging phages with the same single-stranded DNA sequence according to the sequencing result of the step S3, respectively adding 36 purified phages with different DNA sequence numbers into human MUC1 protein coated wells, and incubating for 2h at room temperature;
s45, washing with TBST buffer solution for several times, adding 100 mu L of HRP-labeled mouse anti-M13 phage mAb (1:5000) with the dilution of 1:5000 into each well, and incubating for 1h at room temperature; the absorbance at A490nm was measured after several additional washes with TBST buffer and OPD development. The result of absorbance detection is shown in figure 1, and it can be seen that the polypeptide of the above sequence has very good effect of specifically binding to human MUC1 protein.
In the step of extracting the polypeptide, the eluent used is a mixed solution of BSA (bovine serum albumin) with the pH value of 2.2 and 10g/L and Glycine-HCl with the concentration of 0.2 mol/L; the neutralization solution is Tris-HCl solution with pH 9.1 and 1 mol/L; the blocking solution used was pH 8.6, 5mg/L BSA and 0.1mol/L NaHCO3The mixed solution of (1).
Sequence listing
<110> Hubei academy of culture and management
<120> human MUC1 specific binding polypeptide and extraction method thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
His Lys Phe Ile Pro His Arg Asp Thr Ser Ile Ala
1 5 10
Claims (8)
1. A human MUC 1-specific binding polypeptide, wherein the amino acid sequence of the human MUC 1-specific binding polypeptide is HKFIPHRDTSIA.
2. The method of claim 1, comprising the step of extracting the human MUC 1-specific binding polypeptide
S1, biological elutriation: adding MUC1 solution into the pore plate, inoculating Escherichia coli strain and tetracycline-containing culture medium, and culturing; pouring out residual liquid, adding TBS sealing liquid and washing for a plurality of times; adding a phage solution for amplification, purification and elution to obtain eluted phage; adding the eluted phage solution into an escherichia coli culture for amplification, and separating to obtain amplified eluent; repeating the second and third rounds of screening to obtain screened positive plaques;
s2, amplification of positive phage: adding the overnight culture of the escherichia coli into a culture medium for dilution, dipping a plurality of well-separated blue plaques for inoculation, culture and amplification; centrifuging and taking the supernatant to obtain amplified positive phage;
s3, DNA sequencing of encoded phage-displayed polypeptides: extracting phage single-chain DNA, sequencing by using a sequencer by taking the phage single-chain DNA as a template to obtain a phage single-chain DNA sequence, and deducing an amino acid sequence of polypeptide presented by the phage;
s4, performing ELISA determination verification on the screened positive phage.
3. The method of claim 2, wherein step S1 is specifically comprised of:
s11, adding 200ul of NaHCO with concentration of 100ug/mL into each well of 96-well plate, and adding 0.1mol/L3Incubating the diluted human MUC1 protein solution overnight at 4 ℃ with shaking; inoculating 10mL of Escherichia coli into 20mL of LB culture medium containing tetracycline, and culturing at 37 ℃; taking out the 96-well plate, and removing residual liquid; adding 200uL of TBS blocking solution, and incubating for 2h at 4 ℃; washing with TBST buffer solution for several times to remove residual liquid;
s12, diluting 200ul of the mixture with TBST buffer solution to contain 2X 1011pfu phage solution, the TBST buffer solution is TBS solution containing 0.1% Tween; adding the protein into the sealed human MUC1 coated well of the 96-well plate obtained in the step S12, and incubating for 1h at room temperature; pouring out the liquid, washing for a plurality of times by using TBST buffer solution, and removing the residual liquid in each washing; then eluting with 100 μ L of eluent, and rapidly adding neutralizing solution for neutralization to obtain eluted phage solution;
s13, taking 1 mu L of the eluted phage solution of the step S12 to measure titer, adding the rest eluted phage solution into 20mL of escherichia coli culture at early logarithmic growth stage, and carrying out shake culture at 37 ℃ for 4.5 h; centrifuging the mixed solution at 4 deg.C and 10000rpm for 10min, and centrifuging the supernatant for the second time; adding 80% of the supernatant obtained by the second centrifugation into 1/6 PEG8000/NaCl solution in the volume of the supernatant, and standing overnight at 4 ℃;
s14, taking out the liquid obtained in the step S12, centrifuging for 15min at 4 ℃ and 10000rpm, and taking out the precipitate; centrifuging once again, and sucking up residual supernatant; resuspending with 1mL TBS buffer solution, centrifuging at 10000rpm for 5min under the condition of resuspension of 4 ℃, taking the supernatant, adding a PEG/NaCl solution with the volume of 1/6 supernatant, and carrying out ice bath for 1 h; centrifuging at 4 deg.C and 10000rpm for 15min, and removing supernatant; centrifuging again, and sucking up residual supernatant;
s15, using 200uL containing 0.01% NaN3Centrifuging the TBS solution at 4 ℃ and 10000rpm to resuspend the precipitate obtained in the step S13 for 1min, and taking the supernatant to obtain an amplified eluent, namely completing the first round of screening; 1uL of the amplified eluent is used for measuring the titer, and the residual liquid is stored at 4 ℃ for standby; watch withThe titer of the phage was determined by examining the titer measured in step S14, and the titer corresponding to the phage content of 2X 10 was calculated11Volume of phage solution of pfu;
s16, newly coating 1 hole of a 96-hole plate with 200uL of human MUC1 protein solution, and performing a second round of screening by adopting the method of S11-S15, wherein the concentration of the target protein is 50mg/L, and the concentration of Tween in the TBST buffer solution in the second round of screening is 0.5%;
s17, newly coating one hole of a 96-hole plate with 200ul of human MUC1 protein solution with the concentration of 100ug/mL, and performing the third screening by adopting the method of the steps S11-S15, wherein the concentration of the target protein is 20 mg/L; performing titer determination on 1ul of the third eluate, and storing the rest eluate at 4 deg.C; in the titer assay, 50 well-separated positive plaques were randomly picked from plates with a plaque number of less than 100.
4. The method of claim 2, wherein step S2 is specifically comprised of:
s21, diluting an overnight culture of ER2738 with LB culture medium according to a ratio of 1:100, and subpackaging the diluted culture in a plurality of 50mL culture tubes according to 1 mL/tube;
s22, dipping 50 well-separated blue plaques, respectively putting the blue plaques into the culture tube in the step S21, and carrying out shake culture at 37 ℃ for 4.5h to complete amplification;
s23, centrifuging the amplified phage liquid obtained in the step S22 for 30S; taking the supernatant and centrifuging again; and taking 80% of the supernatant obtained by the second centrifugation to obtain the amplified phage liquid.
5. The method of claim 2, wherein step S3 is specifically comprised of:
s31, extracting the single-stranded DNA of the positive phage obtained in the step S2;
s32, automatically sequencing on an automatic sequencer by using the single-stranded DNA as a template and a sequencing primer with the sequence of 5 '-CCCTCATAGTTAGCGTAACG-3';
and S33, deducing the amino acid sequence of the peptide presented by the phage according to the DNA sequencing result.
6. The method of claim 2, wherein step S4 is specifically comprised of:
s41, adding 200ul of NaHCO with concentration of 100ug/mL into each well of 96-well plate, and adding 0.1mol/L3Setting a coated well of 1% BSA solution as a negative control for each well of a human MUC1 protein solution diluted by the solution, and incubating overnight at 4 ℃;
s42, pouring out the coating solution, adding the sealing solution, and sealing for 2h at 4 ℃; then pouring off the confining liquid, washing for a plurality of times by using TBST buffer solution, and removing residual liquid in each washing;
s44, combining phages with the same single-stranded DNA sequence according to the sequencing result of the step S3, respectively adding a plurality of remaining purified phages into human MUC1 protein coated wells, and incubating for 2h at room temperature;
s45, washing for several times by using TBST buffer solution, adding 100 mu L of HRP-labeled mouse anti-M13 phage mAb with the dilution of 1:5000 into each hole, and incubating for 1h at room temperature; the absorbance at A490nm was measured after several additional washes with TBST buffer and OPD development.
7. The method of claim 6, wherein the blocking solution is pH 8.6, 5mg/L BSA and 0.1mol/L NaHCO in step S423The mixed solution of (1).
8. The method of claim 3 or 6, wherein the eluent is a mixture of BSA (2.2 pH, 10 g/L) and Glycine-HCl (0.2 mol/L); the neutralization solution is Tris-HCl solution with the pH value of 9.1 and 1 mol/L.
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