CN111100209A

CN111100209A - Recombinant protein G3P20-31 and preparation method and application thereof

Info

Publication number: CN111100209A
Application number: CN202010053979.4A
Authority: CN
Inventors: 潘鹏涛; 王选年; 邹凡雨; 李文明
Original assignee: Xinxiang University
Current assignee: Xinxiang University
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2020-05-05

Abstract

The invention discloses a recombinant protein G3P20-31 and a preparation method and application thereof, belonging to the technical field of DNA recombination in bioengineering. The invention utilizes the phage display technology to clone the gene fragment of the N-terminal 20-31 peptide segment of the encoded P53 protein into the PIII protein gene of a filamentous phage vector, prepares a recombinant protein for displaying the P53 protein epitope, displays the recombinant protein on the surface of a phage, and is further used for detecting the serum P53 antibody of a tumor patient. The invention also discloses application of the recombinant protein G3P20-31 in preparation of a biological product for detecting a serum P53 antibody of a tumor patient, and the biological product has the advantages of strong specificity, high sensitivity, simple preparation, low cost and the like.

Description

Recombinant protein G3P20-31 and preparation method and application thereof

Technical Field

The invention relates to the technical field of DNA recombination in bioengineering, in particular to a recombinant protein G3P20-31 and a preparation method and application thereof.

Background

Cancer is a main killer threatening human health, and the number of cancer diseases is about 260 ten thousand and about 180 ten thousand of deaths each year in China. The reason for this phenomenon is many, and one important reason is that human beings cannot effectively diagnose the occurrence and development process of tumors, especially early diagnosis.

p53 is a cancer suppressor gene, the protein of which plays an important regulatory role in maintaining normal cell division and growth; however, once the gene is mutated, the encoded mutant protein has prolonged half-life, loses the cancer inhibition effect, accumulates in cells and stimulates the immune system to produce the P53 antibody. Research shows that the serum P53 antibody can be used as a broad-spectrum tumor marker for early detection of tumors and screening of high risk groups of tumors.

At present, the detection of serum p53 antibody is mainly carried out based on recombinant p53 protein, and the preparation and purification of protein have the disadvantages of complex operation, time consumption, relatively high price, low sensitivity and the like. Therefore, the problem to be solved by those skilled in the art is to provide a recombinant protein G3P20-31 and a preparation method and application thereof.

Disclosure of Invention

In view of the above, the invention provides the recombinant protein G3P20-31 and the preparation method and application thereof, the preparation method is simple, the cost is low, the sensitivity is high, and the recombinant protein G3P20-31 can be used for detecting the serum P53 antibody of the tumor patient.

In order to achieve the purpose, the invention adopts the following technical scheme:

the epitope recognized by serum P53 antibody in tumor patients is mainly located in the N segment of P53 protein, wherein the N segment 20-31 amino acids are a key epitope recognized by the antibody.

A recombinant protein G3P20-31 comprises P53 protein epitope (SDLWKLLPENNV) and filamentous phage minor coat protein G3P, specifically, N segment of P53 protein 20-31 amino acid SDLWKLLPENNV is displayed on filamentous phage minor coat protein PIII; the amino acid sequence of the recombinant protein G3P20-31 is shown in SEQ ID NO. 1. The black bold part is P53 protein epitope (SDLWKLLPENNV).

MKYLLPTAAAGLLLLAAQPAMASDLWKLLPENNVGPGGLSLEAETV ESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQ CYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYI NPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGT VTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEY QGQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGS GSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDG FIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQ SVECRPYVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILR NKES；SEQ ID NO.1。

Further, the nucleotide sequence for coding the recombinant protein G3P20-31 is shown in SEQ ID NO. 2.

The black bold part (nucleotide sequence: 67-102) is the nucleotide sequence encoding epitope (SDLWKLLPENNV) of P53 protein; the nucleotide sequence before black bold (nucleotide sequence: 1-66) is a signal peptide encoding the PIII protein; the black bold underlined part (nucleotide sequence 103-126) is Linker, which is connected with the PIII protein of the target peptide multi-phage, and aims to enable the displayed exogenous peptide to better exert the function; the nucleotide sequence (127-1344) following the black bold underlined portion is the gene for the PIII protein.

ATGAAATACCTATTGCCTACGGCGGCCGCTGGATTGTTATTACTCG CGGCCCAGCCGGCCATGGCATCAGACCTATGGAAACTACTTCCTGAA AACAACGTTGGCCCGGGAGGCCTGTCTCTAGAAGCCGAAACTGTTG AAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTG GAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCT GTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGT TACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTG GTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCG GTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTA TATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCC GCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCAT GTTTCAGAATAATAGGTTCCGAAATAGGCAGGGTGCATTAACTGTTTAT ACGGGCACTGTTACTCAAGGCACTGACCCCGTTAAAACTTATTACCAGT ACACTCCTGTATCATCAAAAGCCATGTATGACGCTTACTGGAACGGTAA ATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATGAGGATCCATTCGTTT GTGAATATCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGC TGGCGGCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGCGG CTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGTGGCGGTTC CGGTGGCGGCTCCGGTTCCGGTGATTTTGATTATGAAAAAATGGCAAA CGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACA GTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCT GCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATG GTGCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGG TGACGGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTTACCTT CTTTGCCTCAGTCGGTTGAATGTCGCCCTTATGTCTTTGGCGCTGGTAA ACCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGT GTCTTTGCGTTTCTTTTATATGTTGCCACCTTTATGTATGTATTTTCGACGTTTGCTAACATACTGCGTAATAAGGAGTCT；SEQ ID NO.2。

Further, a preparation method of the recombinant protein G3P20-31 comprises the following specific steps:

(1) carrying out Bgl I enzyme digestion on the vector fADL-le, and recovering the enzyme-digested vector fADL-le;

(2) synthetic phage display epitope SDLWKLLPENNV:

synthesizing two complementary DNA fragments which code the 20 th to 31 th amino acids MEEPQSDPSVEP at the N end of the P53 protein;

5’-CGGCCATGGCATCAGACCTATGGAAACTACTTCCTGAAAACAAC GTTGGCCCGGG-3’；SEQ IDNO.3；

5’-GGGCCAACGTTGTTTTCAGGAAGTAGTTTCCATAGGTCTGATG CCATGGCCGGCT-3’；SEQ IDNO.4；

dissolving the two fragments, mixing in an equimolar way, denaturing at 94 ℃ for 5min, and then renaturing at 58 ℃ for 4min to ensure that the two fragments are complementarily combined into a double chain to synthesize a target fragment;

(3) connecting the enzyme-digested vector fADL-le obtained in the step (1) with the target fragment synthesized in the step (2), and reacting overnight at 16 ℃ to obtain a recombinant plasmid;

(4) transforming the recombinant plasmid into escherichia coli JM109 competent cells, selecting positive clones, and performing PCR identification;

(5) phage displaying the recombinant protein G3P20-31 were prepared using positive clones identified as correct.

Further, the recombinant protein G3P20-31 is applied to the preparation of biological products for detecting tumor patient serum P53 antibodies.

According to the technical scheme, compared with the prior art, the invention discloses and provides the recombinant protein G3P20-31 and the preparation method and application thereof, the gene fragment of the N-terminal 20-31 peptide segment of the encoded P53 protein is cloned into the PIII protein gene of the filamentous phage vector by utilizing the phage display technology, the recombinant protein displaying the P53 protein epitope is prepared, and the recombinant protein is displayed on the surface of the phage, so that the recombinant protein is used for detecting the serum P53 antibody of a tumor patient. The invention also discloses application of the recombinant protein G3P20-31 in preparation of a biological product for detecting a serum P53 antibody of a tumor patient, and the biological product has the advantages of strong specificity, high sensitivity, simple preparation, low cost and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 shows the result of Bgl I digestion in fADL-le vector of the present invention;

wherein, 1, Marker; 2-3, fADL-le vector; 4, carrying out enzyme digestion on the fADL-le vector by Bgl I;

FIG. 2 shows PCR-verified positive clones of recombinant fADL-le vector according to the present invention;

wherein, 1, Marker; 2-6, positive cloning;

FIG. 3 is a partial sequencing peak diagram of the recombinant phage vector of the present invention;

wherein, the 884-919 is a nucleotide sequence for coding a target polypeptide;

FIG. 4 shows a Westernblot analysis of recombinant protein G3P20-31 according to the present invention;

wherein, 1, 3 and 5 are Marker; 2, the recombinant protein G3P20-31 is hybridized with a monoclonal antibody of the anti-PIII protein (positive control); 4, 6 are the recombinant protein G3P20-31 which is respectively hybridized with the serum of a P53 antibody positive tumor patient and a healthy human (negative control);

FIG. 5 shows the result of detecting serum P53 antibody of breast cancer patients with the recombinant protein G3P20-31 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious 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 construction of recombinant phage

(1) Extraction of phage vector fADL-le

The plasmid-commercialized phage vector fADL-le (purchased from Antibody Design laboratories, Catalog number: PD020) was extracted using Axygen's plasmid miniprep kit, and the specific steps were as follows:

1) taking 6ml of bacterial liquid JM109 (preserved in a laboratory and transformed with a vector fADL-le) cultured in an LB culture medium overnight, centrifuging at 12000 Xg for 1min, and discarding the supernatant;

2) adding 250 mul of Buffer S1, and suspending the bacteria for precipitation, wherein the suspension needs to be uniform and small bacteria blocks are not left;

3) adding 250 μ l Buffer S2, gently and fully turning over for 4-6 times, mixing well, and fully cracking the thallus until a clear solution is formed;

4) adding 350 μ l Buffer S3, gently and turning over for 6-8 times, centrifuging at 12000 × g for 10 min;

5) sucking the supernatant centrifuged in the step 4), transferring the supernatant into a preparation tube, centrifuging at 12000 Xg for 1min, and removing the filtrate;

6) the prepared tube is put back into a centrifuge tube, 500 mu l of BufferW1 is added, 12000 Xg is added, centrifugation is carried out for 1min, and the filtrate is discarded;

7) the prepared tube is put back into a centrifuge tube, 700 mul of BufferW2 is added, 12000 Xg is added, centrifugation is carried out for 1min, and the filtrate is discarded; the mixture was washed once more with 700. mu.l of BufferW2 in the same manner, and the filtrate was discarded;

8) placing the prepared tube back into a 2ml centrifuge tube, and centrifuging for 1min at 12000 Xg;

9) the preparation tube was transferred to a new 1.5ml centrifuge tube, 50. mu.l of Eluent solution was added to the center of the membrane of the preparation tube, left to stand at room temperature for 1min, and centrifuged at 12000 Xg for 1 min.

(2) Enzyme digestion of fADL-le

Bgl I enzyme digestion vector fADL-le, the specific enzyme digestion reaction system is as follows:

(3) recovery of enzyme digestion vectors

The vector fADL-le and the cleaved vector fADL-le were subjected to agarose gel electrophoresis, and the results are shown in FIG. 1. The fADL-le vector mainly exists in a circular form and a linear form, is changed into a linear form after enzyme digestion (only cut off about 10 bp), has faster electrophoresis rate than a linear no-load conformation, and preliminarily proves that the enzyme digestion is successful.

And (3) carrying out gel cutting recovery on the vector which is completely cut by enzyme and proved by agarose gel electrophoresis, and operating according to the instruction of the DNA gel recovery kit, wherein the operation is as follows:

1) cutting the agarose gel containing the target DNA under an ultraviolet lamp, completely absorbing the liquid on the surface of the gel by using a paper towel, cutting the gel, and calculating the weight of the gel, wherein the weight is used as the volume of the gel;

2) adding 3 volumes of Buffer DE-A, mixing uniformly, heating at 75 ℃, and mixing discontinuously until the gel is completely melted;

3) adding 0.5 Buffer DE-B with the volume of the Buffer DE-A, and uniformly mixing;

4) sucking the mixed solution in the step 3), transferring the mixed solution into a DNA preparation tube, centrifuging at 12000 Xg for 1min, and removing the filtrate;

5) placing the prepared tube back into a 2ml centrifuge tube, adding 500 μ l of BufferW1, centrifuging at 12000 Xg for 1min, and removing the filtrate;

6) the prepared tube is put back into a centrifuge tube, 700 mul of BufferW2 is added, 12000 Xg is added, centrifugation is carried out for 1min, and the filtrate is discarded; the mixture was washed once more with 700. mu.l of BufferW2 in the same manner, and the filtrate was discarded;

7) placing the prepared tube back into a 2ml centrifuge tube, and centrifuging for 1min at 12000 Xg;

8) the preparation tube was transferred to a new 1.5ml centrifuge tube, 10. mu.l of Eluent solution was added to the center of the membrane of the preparation tube, left to stand at room temperature for 1min, and centrifuged at 12000 Xg for 1 min.

(4) Synthesis of phage display epitopes

dissolving the two fragments, mixing in equimolar mode, denaturing at 94 ℃ for 5min, and renaturing at 58 ℃ for 4min to allow the two fragments to be complementarily combined into a double chain.

(5) Connection of target fragment and fADL-le enzyme digestion vector

Connecting the target fragment synthesized in the step (4) with the vector after the enzyme digestion of fADL-le, reacting overnight at 16 ℃, wherein the connection reaction system is as follows:

(6) recombinant plasmid transformed Escherichia coli JM109 competent cells

1) Adding 10 μ l of the ligation product into competent cells of Escherichia coli JM109, mixing, and ice-cooling for 30 min;

2) carrying out heat shock for 90s at 42 ℃;

3) immediately placing on ice, and carrying out ice bath for 10 min;

4) adding 800 μ l LB culture medium, 37 deg.C, 100rpm, 45 min;

5) uniformly coating the transformation product on an LB solid culture medium containing kanamycin, after complete absorption, inversely placing the transformation product in an incubator at 37 ℃, and culturing for 12 hours overnight;

6) and (4) selecting positive clones, and carrying out PCR identification.

(7) PCR identification of bacterial liquid

Selecting a single clone, transferring the single clone into 5ml of LB liquid culture medium containing kanamycin resistance, at 37 ℃, 200rpm and 8h, respectively sucking 1 mul of bacterial liquid as a template to carry out bacterial liquid PCR identification, wherein primers and amplification conditions are as follows:

upstream Primer (PF): 5'-CCGTGCATCTGTCCTCGTTCAA-3', respectively; SEQ ID No. 5;

downstream Primer (PR): 5'-GTTTTCAGGAAGTAGTTTCCATAGGTC-3', respectively; SEQ ID No. 6;

an upstream primer PF is designed at about 700bp upstream of the insertion site of the pIII gene of the vector, and PCR verification is performed by using the antisense strand of the inserted fragment as a downstream primer.

The PCR reaction system is as follows:

the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 8 min; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min.

The amplified products were subjected to agarose gel electrophoresis, and the results of PCR verification are shown in FIG. 2. After PCR amplification, the positive clone has specific target band at 700bp, and the negative clone has no specific band after PCR amplification. The result of FIG. 2 shows that a specific target band appears at 700bp, which initially indicates that the coding fragment of the foreign peptide (SDLWKLLPENNV) is successfully inserted into the pIII gene of the phage, and the recombinant phage vector is successfully constructed.

After the reaction is finished, the screened positive clones are sent to Shanghai Biotechnology service company for sequencing.

The sequencing result of the recombinant vector is as follows, and the target fragment (the black bold line part and the figure 3) is successfully cloned into the PIII gene of the phage vector fADL-le and is completely consistent with the connected original sequence, which indicates that the construction of the recombinant vector is successful and can be used for preparing the recombinant protein G3P20-31 in the next step.

TCCTGACTGGTATAATGAGCCAGTTCTTAAAATCGCATAAGGTAAT TCAAAATGATTAAAGTTGAAATTAAACCATCTCAAGCGCAATTTACTAC CCGTTCTGGTGTTTCTCGTCAGGGCAAGCCTTATTCACTGAATGAGCAG CTTTGTTACGTTGATTTGGGTAATGAATATCCGGTGCTTGTCAAGATTA CTCTCGACGAAGGTCAGCCAGCGTATGCGCCTGGTCTGTACACCGTGCA TCTGTCCTCGTTCAAAGTTGGTCAGTTCGGTTCTCTTATGATTGACCGTCTGCGCCTCGTTCCGGCTAAGTAACATGGAGCAGGTCGCGGATTTCGAC ACAATTTATCAGGCGATGATACAAATCTCCGTTGTACTTTGTTTCGCGC TTGGTATAATCGCTGGGGGTCAAAGATGAGTGTTTTAGTGTATTCTTTC GCCTCTTTCGTTTTAGGTTGGTGCCTTCGTAGTGGCATTACGTATTTTAC CCGTTTAATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCT CCGTAGCCGTTGCTACCCTCGTTCCGATGCTGTCTTTCGCTGCTGAGGG TGACGATCCCGCAAAAGCGGCCTTTAACTCCCTGCAAGCCTCAGCGAC CGAATATATCGGTTATGCGTGGGCGATGGTTGTTGTCATTGTCGGCGCA ACTATCGGTATCAAGCTGTTTAAGAAATTCACCTCGAAAGCAAGCTGAT AAACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTGTCGACTA ACGAGGGCAAATCATGAAATACCTATTGCCTACGGCGGCCGCTGGATT GTTATTACTCGCGGCCCAGCCGGCCATGGCATCAGACCTATGGAAACTACTTCCTGAAAA CAACGTTGGCCCGGGAGGCCTGTCTCTAGAAGCCG AAACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATC；SEQ ID NO.7。

Example 2 preparation of phage displaying recombinant protein G3P20-31

1) 200 μ l of sequencing-correct JM109 was inoculated into a medium containing 100ml of LB liquid medium (100 μ g/ml Kar)⁺) In the test tube, shaking vigorously for 10h at 37 ℃;

2)8000rpm, 10min, 4 ℃, and reserving supernatant;

4) adding one sixth volume of PEG/NaCl solution, vortexing, and standing overnight at 4 deg.C;

5) centrifuging at 12000rpm for 15min, and dissolving phage precipitate with 1ml TBS;

6) transferring the solution into 1.5ml EP tubes, centrifuging at 14000rpm for 1min, carefully transferring the supernatant into 1.5ml EP tubes, adding 150 μ l PEG/NaCl into each EP tube, mixing uniformly, and standing overnight at 4 ℃;

7) centrifuge at 14000rpm for 15min, dissolve the phage pellet with 100. mu.l TBS, and store in a refrigerator at 4 ℃.

Example 3Western-blot analysis of phage displaying recombinant protein G3P20-31

In order to verify that the P53 protein N-terminal 20-31 polypeptide and PIII are expressed in a fusion mode and displayed on the surfaces of phage successfully, the prepared phage are respectively hybridized with anti-PIII monoclonal antibody and P53 antibody positive tumor patient serum, and the specific steps are as follows:

1) after SDS-PAGE, cutting the separating gel and placing the separating gel in a transfer buffer solution;

2) cutting a PVDF membrane and filter paper according to the size of glue, soaking the filter paper in a transfer buffer solution, soaking the PVDF membrane in methanol for 1min, and then placing the PVDF membrane in the transfer buffer solution;

3) converting the voltage of 80V to 2 h;

4) cutting the membrane into small strips after ponceau red dyeing to separate the proteins on the membrane from each other;

5) putting the membrane into sealing liquid for sealing for 1 h;

6) PBST washing three times, each time interval is 5 min;

7) adding the components according to the proportion of 1: commercial murine anti-PIII monoclonal antibody (NEB) at 1000 dilution or p53 antibody positive cancer patient serum at 1:100 dilution or 1:100 diluting healthy human serum at 37 ℃ for 1 h;

8) PBST washing three times, each time interval is 5 min;

9) diluting goat anti-mouse IgG or goat anti-human IgG to working concentration, placing the membrane strip in the diluted goat anti-mouse IgG or goat anti-human IgG, and keeping the temperature at 37 ℃ for 45 min;

10) PBST washing three times, each time interval is 5 min;

11) the ECL luminescence solution was diluted to the working concentration, dropped on the PVDF membrane, and exposed for 30 seconds.

The result is shown in fig. 4, the recombinant protein G3P20-31 can simultaneously generate specific reaction with the anti-PIII monoclonal antibody and the tumor patient serum P53 antibody, and the target band positions are the same, and the recombinant protein has no cross reaction with the healthy human serum, which indicates that the recombinant protein G3P20-31 is successfully expressed and can specifically recognize the tumor patient serum P53 antibody.

Example 4G3P20-31 phage detection of serum P53 antibody ELISA results for breast cancer patients

(1)G3P20-31-ELISA

The method comprises the following steps of taking phage displaying recombinant protein G3P20-31 as a detection antigen, and carrying out detection on serum P53 antibodies of 60 breast cancer patients and 60 healthy people (negative control) by an ELISA method:

1) coating a 96-hole enzyme label plate by using a phage displaying recombinant protein G3P20-31 as a coating antigen (G3P20-31-ELISA for short), wherein the concentration is 30 mu G/ml, each hole is 50 mu l, and the plate is placed in a wet box for overnight at 4 ℃;

2) on the next day, wash 3 times with PBST buffer solution, 3min each time, wash twice with PBS solution, 3min each time;

3) adding 200 mul of sealing liquid into each hole, sealing at 37 ℃ for 1 h;

4) washing, adding 50 μ l of human serum diluted at a ratio of 1:200 for breast cancer patients or healthy people, and reacting at 37 deg.C for 1 h;

5) after repeated washing, adding HRP-labeled goat anti-human IgG secondary antibody diluted by 1:5000 times, 50 mu l of the secondary antibody per hole, and incubating for 45min at 37 ℃;

6) repeatedly washing, adding 100 μ l of substrate color development solution TMB, reacting for 12min at room temperature in a dark place, and adding 50 μ l of 2M sulfuric acid into each hole to terminate the reaction;

7) the absorbance at OD450nm was measured using a microplate reader. The detection of each sample is a parallel multiple hole, and the result is the average value of the detection results.

(2)P53-ELISA

The method for detecting the serum 53 antibody by using the recombinant P53 protein (purchased from Abcam, Catalog number: ab82201) as the coating antigen is abbreviated as P53-ELISA. The whole experimental procedure was identical to that of the G3P20-31-ELISA, except that the antigen coated on the microplate was recombinant P53 protein and the concentration was 5. mu.g/. mu.l.

(3) Determination of cut-off value

60 cases of human healthy serum were each detected according to the previously established G3P20-31-ELISA and P53-ELISA detection systems, and cut-off values were determined for each detection method by the mean +2SD method.

As shown in FIG. 5, in 60 breast cancer patients, 13 sera from P53 positive patients were detected by the phage displaying the recombinant protein G3P20-31, the detection rate was 21.67%, and the detection efficiency was consistent with that of the recombinant P53 protein (21.67%), and the specificity of the two detection methods was 96.67% (G3P20-31-ELISA) and 95.00% (P53-ELISA), respectively. The result shows that the recombinant protein G3P20-31 has the advantages of strong specificity, high sensitivity, simple preparation and the like in the aspect of detecting the serum P53 antibody of a breast cancer patient, and can be used for detection application research of the serum P53 antibody of the breast cancer patient.

The phage display technology can link the genotype with the phenotype, so that researchers can realize in-vitro control on protein conformation at the gene level and obtain expression products with good biological activity in vitro. The invention utilizes the phage display technology to obtain a recombinant protein G3P2-31 of a minor coat Protein (PIII) display P53 protein epitope (N segment 20-31 site peptide segment), and the recombinant protein is displayed on the surface of a phage. The method has the advantages of simple preparation, low cost, high sensitivity and the like, and can be applied to the detection of the tumor patient serum p53 antibody.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> college of New county

<120> recombinant protein G3P20-31, and preparation method and application thereof

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Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn

20 25 30

Asn Val Gly Pro Gly Gly Leu Ser Leu Glu Ala Glu Thr Val Glu Ser

35 40 45

Cys Leu Ala Lys Pro His Thr Glu Asn Ser Phe Thr Asn Val Trp Lys

50 55 60

Asp Asp Lys Thr Leu Asp Arg Tyr Ala Asn Tyr Glu Gly Cys Leu Trp

65 70 75 80

Asn Ala Thr Gly Val Val Val Cys Thr Gly Asp Glu Thr Gln Cys Tyr

85 90 95

Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro Glu Asn Glu Gly Gly

100 105 110

Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

115 120 125

Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile Pro Gly Tyr Thr Tyr

130 135 140

Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly Thr Glu Gln Asn Pro

145 150 155 160

Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln Pro Leu Asn Thr Phe

165 170 175

Met Phe Gln Asn Asn Arg Phe Arg Asn Arg Gln Gly Ala Leu Thr Val

180 185 190

Tyr Thr Gly Thr Val Thr Gln Gly Thr Asp Pro Val Lys Thr Tyr Tyr

195 200 205

Gln Tyr Thr Pro Val Ser Ser Lys Ala Met Tyr Asp Ala Tyr Trp Asn

210 215 220

Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly Phe Asn Glu Asp Pro

225 230 235 240

Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln Pro Pro

245 250 255

Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu

260 265 270

Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

275 280 285

Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys

290 295 300

Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn

305 310 315 320

Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp

325 330 335

Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala

340 345 350

Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met

355 360 365

Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg

370 375 380

Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro Tyr Val

385 390 395 400

Phe Gly Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile

405 410 415

Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe

420 425 430

Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser

435 440 445

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atgaaatacc tattgcctac ggcggccgct ggattgttat tactcgcggc ccagccggcc 60

atggcatcag acctatggaa actacttcct gaaaacaacg ttggcccggg aggcctgtct 120

ctagaagccg aaactgttga aagttgttta gcaaaacctc atacagaaaa ttcatttact 180

aacgtctgga aagacgacaa aactttagat cgttacgcta actatgaggg ctgtctgtgg 240

aatgctacag gcgttgtggt ttgtactggt gacgaaactc agtgttacgg tacatgggtt 300

cctattgggc ttgctatccc tgaaaatgag ggtggtggct ctgagggtgg cggttctgag 360

ggtggcggtt ctgagggtgg cggtactaaa cctcctgagt acggtgatac acctattccg 420

ggctatactt atatcaaccc tctcgacggc acttatccgc ctggtactga gcaaaacccc 480

gctaatccta atccttctct tgaggagtct cagcctctta atactttcat gtttcagaat 540

aataggttcc gaaataggca gggtgcatta actgtttata cgggcactgt tactcaaggc 600

actgaccccg ttaaaactta ttaccagtac actcctgtat catcaaaagc catgtatgac 660

gcttactgga acggtaaatt cagagactgc gctttccatt ctggctttaa tgaggatcca 720

ttcgtttgtg aatatcaagg ccaatcgtct gacctgcctc aacctcctgt caatgctggc 780

ggcggctctg gtggtggttc tggtggcggc tctgagggtg gcggctctga gggtggcggt 840

tctgagggtg gcggctctga gggtggcggt tccggtggcg gctccggttc cggtgatttt 900

gattatgaaa aaatggcaaa cgctaataag ggggctatga ccgaaaatgc cgatgaaaac 960

gcgctacagt ctgacgctaa aggcaaactt gattctgtcg ctactgatta cggtgctgct 1020

atcgatggtt tcattggtga cgtttccggc cttgctaatg gtaatggtgc tactggtgat 1080

tttgctggct ctaattccca aatggctcaa gtcggtgacg gtgataattc acctttaatg 1140

aataatttcc gtcaatattt accttctttg cctcagtcgg ttgaatgtcg cccttatgtc 1200

tttggcgctg gtaaaccata tgaattttct attgattgtg acaaaataaa cttattccgt 1260

ggtgtctttg cgtttctttt atatgttgcc acctttatgt atgtattttc gacgtttgct 1320

aacatactgc gtaataagga gtct 1344

<210>3

<211>55

<212>DNA

<213>Artificial Sequence

<400>3

cggccatggc atcagaccta tggaaactac ttcctgaaaa caacgttggc ccggg 55

<210>4

<211>55

<212>DNA

<213>Artificial Sequence

<400>4

gggccaacgt tgttttcagg aagtagtttc cataggtctg atgccatggc cggct 55

<210>5

<211>22

<212>DNA

<213>Artificial Sequence

<400>5

ccgtgcatct gtcctcgttc aa 22

<210>6

<211>27

<212>DNA

<213>Artificial Sequence

<400>6

gttttcagga agtagtttcc ataggtc 27

<210>7

<211>966

<212>DNA

<213>Artificial Sequence

<400>7

tcctgactgg tataatgagc cagttcttaa aatcgcataa ggtaattcaa aatgattaaa 60

gttgaaatta aaccatctca agcgcaattt actacccgtt ctggtgtttc tcgtcagggc 120

aagccttatt cactgaatga gcagctttgt tacgttgatt tgggtaatga atatccggtg 180

cttgtcaaga ttactctcga cgaaggtcag ccagcgtatg cgcctggtct gtacaccgtg 240

catctgtcct cgttcaaagt tggtcagttc ggttctctta tgattgaccg tctgcgcctc 300

gttccggcta agtaacatgg agcaggtcgc ggatttcgac acaatttatc aggcgatgat 360

acaaatctcc gttgtacttt gtttcgcgct tggtataatc gctgggggtc aaagatgagt 420

gttttagtgt attctttcgc ctctttcgtt ttaggttggt gccttcgtag tggcattacg 480

tattttaccc gtttaatgga aacttcctca tgaaaaagtc tttagtcctc aaagcctccg 540

tagccgttgc taccctcgtt ccgatgctgt ctttcgctgc tgagggtgac gatcccgcaa 600

aagcggcctt taactccctg caagcctcag cgaccgaata tatcggttat gcgtgggcga 660

tggttgttgt cattgtcggc gcaactatcg gtatcaagct gtttaagaaa ttcacctcga 720

aagcaagctg ataaaccgat acaattaaag gctccttttg gagccttttt tttgtcgact 780

aacgagggca aatcatgaaa tacctattgc ctacggcggc cgctggattg ttattactcg 840

cggcccagcc ggccatggca tcagacctat ggaaactact tcctgaaaac aacgttggcc 900

cgggaggcct gtctctagaa gccgaaactg ttgaaagttg tttagcaaaa cctcatacag 960

aaaatc 966

Claims

1. A recombinant protein G3P20-31, wherein the N segment of the P53 protein, amino acids SDLWKLLPENNV at positions 20-31, is displayed on the filamentous phage minor coat protein PIII; the amino acid sequence of the recombinant protein G3P20-31 is shown in SEQ ID NO. 1.

2. The recombinant protein G3P20-31 of claim 1, wherein the nucleotide sequence encoding the recombinant protein G3P20-31 is shown in SEQ ID No. 2.

3. The method for preparing the recombinant protein G3P20-31 according to claim 1 or 2, wherein the method comprises the following steps:

(2) synthetic phage display epitope SDLWKLLPENNV:

synthesizing two complementary DNA fragments which code the 20 th to 31 th amino acids SDLWKLLPENNV at the N end of the P53 protein;

5’-CGGCCATGGCATCAGACCTATGGAAACTACTTCCTGAAAACAACGTTGGCCCGGG-3’；SEQ IDNO.3；

5’-GGGCCAACGTTGTTTTCAGGAAGTAGTTTCCATAGGTCTGATGCCATGGCCGGCT-3’；SEQ IDNO.4；

4. Use of the recombinant protein G3P20-31 according to claims 1-3 in the preparation of a biological product for detecting serum P53 antibodies in tumor patients.