1. A targeted three-display phage is characterized in that N-terminal epitope polypeptides SV, MP and DE of a coded P53 protein are directionally cloned into PIII, PVIII and PVI genes of a filamentous phage respectively by utilizing a phage display technology to prepare targeted three-display phage-SV-MP-DE of PIII protein display peptide SV, PVIII protein display peptide MP and PVI protein display peptide DE; the amino acid sequence of the SV is shown as SEQ ID NO. 2; the amino acid sequence of the MP is shown as SEQ ID NO. 8; the amino acid sequence of the DE is shown as SEQ ID NO. 12.

2. The method for preparing a targeted three-display phage according to claim 1, comprising the following steps:

1) construction of recombinant phage vector fADL-le-SV

(1) BglI enzyme digestion vector fADL-le, obtaining enzyme digestion vector fADL-le;

(2) synthesizing P53 protein N-terminal epitope polypeptide SV;

(3) connecting the vector fADL-le subjected to enzyme digestion in the step (1) with the SV synthesized in the step (2), transforming, and screening positive clones to obtain a recombinant phage vector fADL-le-SV;

2) construction of recombinant phage vector fADL-le-SV-MP

Directionally cloning peptide MP into PVIII gene of phage by using constructed phage vector fADL-le-SV as a template and using a point mutation kit to construct and obtain recombinant phage vector fADL-le-SV-MP;

3) construction of recombinant phage vector fADL-le-SV-MP-DE

Directionally cloning peptide DE into a PVI gene of a phage by using a point mutation kit by taking the constructed phage vector fADL-le-SV-MP as a template to construct and obtain a recombinant phage vector fADL-le-SV-MP-DE;

4) preparation of phage phase-SV-MP-DE

(1) Inoculating the strain transformed with the recombinant phage vector fADL-le-SV-MP-DE to a strain containing Kar⁺In the LB liquid culture medium, shaking vigorously at 37 ℃ for 10 h;

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

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

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

(5) 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 ℃;

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

3. Use of the targeted triple-display phage of claim 1 or 2 for detecting serum P53 antibody.

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, but once the gene is mutated, the encoded mutant protein has prolonged half-life, loses cancer suppressor effect, accumulates in cells and stimulates the immune system to produce 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 the protein have the defects of complex operation, time consumption, relatively high price, low sensitivity and the like.

Therefore, the problem to be solved by the technical personnel in the field is to provide a targeting three-display phage and a preparation method and application thereof.

Disclosure of Invention

In view of the above, the invention provides a targeting three-display phage capable of being used for detecting a serum P53 antibody of a tumor patient, and the phage serving as a novel biological product has the advantages of strong specificity, high sensitivity, simplicity in preparation, low cost and the like.

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

a targeted three-display phage is characterized in that N-terminal epitope polypeptides SV, MP and DE of a coded P53 protein are directionally cloned into PIII, PVIII and PVI genes of filamentous phage respectively by utilizing a phage display technology to prepare targeted three-display phage-SV-MP-DE of PIII protein display peptide SV, PVIII protein display peptide MP and PVI protein display peptide DE; the amino acid sequence of the SV is shown as SEQ ID NO. 2; the amino acid sequence of the MP is shown as SEQ ID NO. 8; the amino acid sequence of the DE is shown as SEQ ID NO. 12.

Further, the preparation method of the targeting three-display phage comprises the following specific steps:

1) construction of recombinant phage vector fADL-le-SV

(1) BglI enzyme digestion vector fADL-le, obtaining enzyme digestion vector fADL-le;

(2) synthesizing P53 protein N-terminal epitope polypeptide SV;

(3) connecting the vector fADL-le subjected to enzyme digestion in the step (1) with the SV synthesized in the step (2), transforming, and screening positive clones to obtain a recombinant phage vector fADL-le-SV;

2) construction of recombinant phage vector fADL-le-SV-MP

Directionally cloning peptide MP into PVIII gene of phage by using constructed phage vector fADL-le-SV as a template and using a point mutation kit to construct and obtain recombinant phage vector fADL-le-SV-MP;

3) construction of recombinant phage vector fADL-le-SV-MP-DE

Directionally cloning peptide DE into a PVI gene of a phage by using a point mutation kit by taking the constructed phage vector fADL-le-SV-MP as a template to construct and obtain a recombinant phage vector fADL-le-SV-MP-DE;

4) preparation of phage phase-SV-MP-DE

(1) Inoculating the strain transformed with the recombinant phage vector fADL-le-SV-MP-DE to a strain containing Kar⁺In the LB liquid culture medium, shaking vigorously at 37 ℃ for 10 h;

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

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

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

(5) 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 ℃;

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

Further, the targeting three-display phage is applied to detection of serum P53 antibodies.

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 targeted three-display phage displaying the P53 protein epitope, which is prepared by applying the phage display technology, has the advantages of simple preparation and purification, high sensitivity, strong stability and the like, the production cost of the targeted three-display phage is far lower than that of recombinant protein prepared by the traditional genetic engineering, and the economic benefit and the social benefit are very considerable.

According to the technical scheme, compared with the prior art, the invention discloses a targeting three-display phage and a preparation method and application thereof, and the targeting three-display phage-SV-MP-DE, which is obtained by using phage display technology to display p53 protein epitope polypeptide SV of secondary coat Protein (PIII), p53 protein epitope polypeptide MP of main coat Protein (PVIII) and p53 protein epitope DE of secondary coat Protein (PVI), is obtained; the bacteriophage has the advantages of strong specificity, high sensitivity, simple preparation, low cost and the like as a novel biological product, and can be applied to the detection of the serum P53 antibody of a tumor patient.

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 is a schematic diagram of a process for preparing a targeted three-display phage phase-SV-MP-DE according to the present invention;

FIG. 2 is a map of phage vector fADL-le of the present invention;

FIG. 3 is the BglI cleavage result of fADL-le vector of the present invention;

wherein, 1, Marker; 2, fADL-le vector; 3, after enzyme digestion of the fADL-le vector BglI;

FIG. 4 is a diagram showing PCR verification of a recombinant vector fADL-le-SV positive clone according to the present invention;

wherein, 1, Marker; 2-4, positive cloning;

FIG. 5 is a partial sequencing peak diagram of the recombinant phage vector fADL-le-SV of the present invention; 884-919 is a nucleotide sequence encoding a polypeptide of interest;

FIG. 6 is a partial sequencing peak diagram of the recombinant phage vector fADL-le-SV-MP PVIII site of the invention; 491-514 is a nucleotide sequence encoding the target polypeptide;

FIG. 7 is a diagram showing partial sequencing peaks of the recombinant phage vector fADL-le-SV-MP-DE PVI site according to the present invention; 568-591 is the nucleotide sequence encoding the target polypeptide;

FIG. 8 is a drawing showing the observation of phase-SV-MP-DE by AFM according to the present invention.

FIG. 9 is a graph showing the result of detecting serum P53 antibody in breast cancer patients according to 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

The targeting triple-display phage-SV-MP-DE is characterized in that three epitope polypeptides SV, MP and DE of P53 protein are respectively displayed on PIII protein, PVIII and PVI protein of filamentous phage, and the genome sequence is as follows:

AACGCTACTACCATTAGTAGAATTGATGCCACCTTTTCAGCTCGCGCCCCAAATGAAAATATAGCTAAACAGGTTATTGACCATTTGCGAAATGTATCTAATGGTCAAACTAAATCTACTCGTTCGCAGAATTGGGAATCAACTGTTACATGGAATAAAACTTCCAGACACCGTACTTTAGTTGCATATTTAAAACATGTTGAACTACAGCACCAGATTCAGCAATTAAGCTCTAAGCCATCCGCAAAAATGACCTCTTATCAAAAGGAGCAATTAAAGGTACTGTCTAATCCTGACCTGTTGGAATTTGCTTCCGGTCTGGTTCGCTTTGAGGCTCGAATTAAAACGCGATATTTGAAGTCTTTCGGGCTTCCTCTTAATCTTTTTGATGCAATTCGCTTTGCTTCTGACTATAATAGACAGGGTAAAGACCTGATTTTTGATTTATGGTCATTCTCTTTTTCTGAACTGTTTAAAGCATTTGAGGGGGATTCAATGAATATTTATGACGATTCCGCAGTATTGGACGCTATCCAGTCTAAACATTTTACAATTACCCCCTCTGGCAAAACTTCCTTTGCAAAAGCCTCTCGCTATTTTGGTTTCTATCGTCGTCTGGTTAATGAGGGTTATGATAGTGTTGCTCTTACCATGCCTCGTAATTCCTTTTGGCGTTATGTATCTGCATTAGTTGAGTGTGGTATTCCTAAATCTCAATTGATGAATCTTTCCACCTGTAATAATGTTGTTCCGTTAGTTCGTTTTATTAACGTAGATTTTTCCTCCCAACGTCCTGACTGGTATAATGAGCCAGTTCTTAAAATCGCATAAGGTAATTCAAAATGATTAAAGTTGAAATTAAACCATCTCAAGCGCAATTTACTACCCGTTCTGGTGTTTCTCGTCAGGGCAAGCCTTATTCACTGAATGAGCAGCTTTGTTACGTTGATTTGGGTAATGAATATCCGGTGCTTGTCAAGATTACTCTCGACGAAGGTCAGCCAGCGTATGCGCCTGGTCTGTACACCGTGCATCTGTCCTCGTTCAAAGTTGGTCAGTTCGGTTCTCTTATGATTGACCGTCTGCGCCTCGTTCCGGCTAAGTAACATGGAGCAGGTCGCGGATTTCGACACAATTTATCAGGCGATGATACAAATCTCCGTTGTACTTTGTTTCGCGCTTGGTATAATCGCTGGGGGTCAAAGATGAGTGTTTTAGTGTATTCTTTCGCCTCTTTCGTTTTAGGTTGGTGCCTTCGTAGTGGCATTACGTATTTTACCCGTTTAATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCTCCGTAGCCGTTGCTACCCTCGTTCCGATGCTGTCTTTCGCTATGGAGGAGCCGCAGTCAGATCCCGCAAAAGCGGCCTTTAACTCCCTGCAAGCCTCAGCGACCGAATATATCGGTTATGCGTGGGCGATGGTTGTTGTCATTGTCGGCGCAACTATCGGTATCAAGCTGTTTAAGAAATTCACCTCGAAAGCAAGCTGATAAACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTGTCGACTAACGAGGGCAAATCATGAAATACCTATTGCCTACGGCGGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCATCAGACCTATGGAAACTACTTCCTGAAAACAACGTTGGCCCGGGAGGCCTGTCTCTAGAAGCCGAAACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATAGGTTCCGAAATAGGCAGGGTGCATTAACTGTTTATACGGGCACTGTTACTCAAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTATGACGCTTACTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATGAGGATCCATTCGTTTGTGAATATCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCGGCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGCGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGTGGCGGTTCCGGTGGCGGCTCCGGTTCCGGTGATTTTGATTATGAAAAAATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACAGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTTACCTTCTTTGCCTCAGTCGGTTGAATGTCGCCCTTATGTCTTTGGCGCTGGTAAACCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATATGTTGCCACCTTTATGTATGTATTTTCGACGTTTGCTAACATACTGCGTAATAAGGAGTCTTAATCATGCCAGTTCTTTTGGGTATTCCGTTATTATTGCGTTTCCTCGGTTTCCTTCTGGTAACTTTGTTCGGCTATCTGCTTACTTTCCTTAAAAAGGGCTTCGGTAAGATAGCTATTGCTATTTCATTGTTTCTTGCTCTTATTATTGGGCTTAACTCAATTCTTGTGGGTTATCTCTCTGATATTAGCGCACAATTACCCTCTGATTTTGTTCAGGGCGTTCAGTTAATTCTCCCGTCTAATGCGCTTCCCTGTTTTTATGTTATTCTCTCTGTAAAGGCTGCTATTTTCATTTTTGACGTTAAACAAAAAATCGTTTCTTATTTGGATTGGGATAAAGATATTGAACAATGGTTCACTGAATAAATATGGCTGTTTATTTTGTAACTGGCAAATTAGGCTCTGGAAAGACGCTCGTTAGCGTTGGTAAGATTCAGGATAAAATTGTAGCTGGGTGCAAAATAGCAACTAATCTTGATTTAAGGCTTCAAAACCTCCCGCAAGTCGGGAGGTTCGCTAAAACGCCTCGCGTTCTTAGAATACCGGATAAGCCTTCTATTTCTGATTTGCTTGCTATTGGTCGTGGTAATGATTCCTACGACGAAAATAAAAACGGTTTGCTTGTTCTTGATGAATGCGGTACTTGGTTTAATACCCGTTCATGGAATGACAAGGAAAGACAGCCGATTATTGATTGGTTTCTTCATGCTCGTAAATTGGGATGGGATATTATTTTTCTTGTTCAGGATTTATCTATTGTTGATAAACAGGCGCGTTCTGCATTAGCTGAACACGTTGTTTATTGTCGCCGTCTGGACAGAATTACTTTACCCTTTGTCGGCACTTTATATTCTCTTGTTACTGGCTCAAAAATGCCTCTGCCTAAATTACATGTTGGTGTTGTTAAATATGGTGATTCTCAATTAAGCCCTACTGTTGAGCGTTGGCTTTATACTGGTAAGAATTTATATAACGCATATGACACTAAACAGGCTTTTTCCAGTAATTATGATTCAGGTGTTTATTCATATTTAACCCCTTATTTATCACACGGTCGGTATTTCAAACCATTAAATTTAGGTCAGAAGATGAAATTAACTAAAATATATTTGAAAAAGTTTTCTCGCGTTCTTTGTCTTGCGATAGGATTTGCATCAGCATTTACATATAGTTATATAACCCAACCTAAGCCGGAGGTTAAAAAGGTAGTCTCTCAGACCTATGATTTTGATAAATTCACTATTGACTCTTCTCAGCGTCTTAATCTAAGCTATCGCTATGTTTTCAAGGATTCTAAGGGAAAATTAATTAATAGCGACGATTTACAGAAGCAAGGTTATTCCATCACATATATTGATTTATGTACTGTTTCAATTAAAAAAGGTAATTCAAATGAAATTGTTAAATGTAATTAATTTTGTTTTCTTGATGTTTGTTTCATCATCTTCTTTTGCTCAAGTAATTGAAATGAATAATTCGCCTCTGCGCGATTTCGTGACTTGGTATTCAAAGCAAACAGGTGAATCTGTTATTGTCTCACCTGATGTTAAAGGTACAGTGACTGTATATTCCTCTGACGTTAAGCCTGAAAATTTACGCAATTTCTTTATCTCTGTTTTACGTGCTAATAATTTTGATATGGTTGGCTCTAATCCTTCCATAATTCAGAAATATAACCCAAATAGTCAGGATTATATTGATGAATTGCCATCATCTGATATTCAGGAATATGATGATAATTCCGCTCCTTCTGGTGGTTTCTTTGTTCCGCAAAATGATAATGTTACTCAAACATTTAAAATTAATAACGTTCGCGCAAAGGATTTAATAAGGGTTGTAGAATTGTTTGTTAAATCTAATACATCTAAATCCTCAAATGTATTATCTGTTGATGGTTCTAACTTATTAGTAGTTAGCGCCCCTAAAGATATTTTAGATAACCTTCCGCAATTTCTTTCTACTGTTGATTTGCCAACTGACCAGATATTGATTGAAGGATTAATTTTCGAGGTTCAGCAAGGTGATGCTTTAGATTTTTCCTTTGCTGCTGGCTCTCAGCGCGGCACTGTTGCTGGTGGTGTTAATACTGACCGTCTAACCTCTGTTTTATCTTCTGCGGGTGGTTCGTTCGGTATTTTTAACGGCGATGTTTTAGGGCTATCAGTTCGCGCATTAAAGACTAATAGCCATTCAAAAATATTGTCTGTGCCTCGTATTCTTACGCTTTCAGGTCAGAAGGGTTCTATTTCTGTTGGCCAGAATGTCCCTTTTATTACTGGTCGTGTAACTGGTGAATCTGCCAATGTAAATAATCCATTTCAGACAATTGAGCGTCAAAATGTTGGTATTTCTATGAGTGTTTTTCCCGTTGCAATGGCTGGCGGTAATATTGTTTTAGATATAACCAGTAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATTGCGACAACGGTTAATTTGCGTGATGGTCAGACTCTTTTGCTCGGTGGCCTCACTGATTACAAAAACACTTCTCAAGATTCTGGTGTGCCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGTTCTGATTCTAACGAGGAAAGCACGTTGTACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCTCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGATCTCGGGAAAAGCGTTGGTGACCAAAGGTGCCTTTTATCATCACTTTAAAAATAAAAAACAATTACTCAGTGCCTGTTATAAGCAGCAATTAATTATGATTGATGCCTACATCACAACAAAAACTGATTTAACAAATGGTTGGTCTGCCTTAGAAAGTATATTTGAACATTATCTTGATTATATTATTGATAATAATAAAAACCTTATCCCTATCCAAGAAGTGATGCCTATCATTGGTTGGAATGAACTTGAAAAAATTAGCCTTGAATACATTACTGGTAAGGTAAACGCCATTGTCAGCAAATTGATCCAAGAGAACCAACTTAAAGCTTATGATGATGATGTGCTTAAAAACTTACTCAATGGCTGGTTTATGCATATCGCAATACATGCGAAAAACCTAAAAGAGCTTGCCGATAAAAAAGGCCAATTTATTGCTATTTACCGCGGCTTTTTATTGAGCTTGAAAGATAAATAAAATAGATAGGTTTTATTTGAAGCTAAATCTTCTTTATCGTAAAAAATGCCCTCTTGGGTTATCAAGAGGGTCATTATATTTCGCGGAATAAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTTGTTGACAAAGGGAATCATAGATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCTCCAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCACAACTAACCCGGCCTATTCTTTTGATTTATAAGGATTTTTGTCATTTTCTGCTTACTGGTTAAAAAATAAGCTGATTTAACAAATATTTAACGCGAAATTTAACAAAACATTAACGTTTACAATTTAAATATTTGCTTATACAATCATCCTGTTTTTGGGGCTTTTCTGATTATCAATCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACTTTCAGGTAATGACCTGATAGCCTTTGTAGACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATTTATCAGCTAGAACGGTTGAATATCATATTGACGGTGATTTGACTGTCTCCGGCCTTTCTCACCCGTTTGAATCTTTGCCTACTCATTACTCCGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTATCCCTGCGTTGAAATTAAGGCTTCACCAGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAACTCTCTGCCTTGCTTGTACGATTTATTGGATGTT；SEQ ID NO.1。

wherein, in SEQ ID NO.1, the nucleotide sequence shown in position 1301-1369 is a signal peptide encoding the PVIII protein; the nucleotide sequence shown in the 1370-1393 th site is the nucleotide sequence coding the epitope polypeptide MP of the P53 protein; the nucleotide sequence shown in the 1394-1525 th site is a nucleotide sequence for coding PVIII protein; the nucleotide sequence shown in position 1592-1657 is a signal peptide encoding a PIII protein; the nucleotide sequence shown in the position 1658-1693 is a nucleotide sequence coding the P53 protein epitope polypeptide SV; the nucleotide sequence shown in position 1694-1717 is Linker; the nucleotide sequence shown in position 1718-2935 is a nucleotide sequence coding the PIII protein; the nucleotide sequence shown in position 2941-3276 is a nucleotide sequence coding the PVI protein; the nucleotide sequence shown in the 3277-3300 position is the nucleotide sequence coding the epitope polypeptide DE of the P53 protein. The steps for preparing the targeting three-display phage phase-SV-MP-DE (schematic diagram shown in figure 1) are as follows: (I) construction of recombinant phage vector fADL-le-SV

1) Extraction of phage vector fADL-le (vector map shown in FIG. 2)

A commercial phage vector fADL-le (Antibody Design laboratories, Catalog number: PD020) is extracted by using an Axygen plasmid miniprep kit, and the specific steps are as follows:

(1) 6ml of bacterial liquid JM109 transformed with a carrier fADL-le cultured in an LB medium overnight is taken, centrifuged at 12000 Xg for 1min, and the supernatant is discarded;

(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 of Buffer S2, gently and fully turning over for 4-6 times, and mixing to fully crack the thallus until a transparent 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 mul of Buffer W1 is added, 12000 Xg is added for centrifugation for 1min, and the filtrate is discarded;

(7) the prepared tube is put back into a centrifuge tube, 700 mul of Buffer W2 is added, 12000 Xg is added for centrifugation for 1min, and the filtrate is discarded; the mixture was washed once more with 700. mu.l Buffer W2 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:

the enzyme digestion reaction condition is water bath for 2h at 37 ℃. The PIII gene of phage vector fADL-le was digested with Bgl I, and the result is shown in FIG. 3. The fADL-le vector mainly exists in a circular form and a linear form, and is changed into a linear form after enzyme digestion, and the success of enzyme digestion is preliminarily proved.

3) Recovery of enzyme digestion vectors

The agarose gel electrophoresis proves that the carrier which is completely digested is cut and recovered, and the operation is carried out according to the instruction of a DNA gel recovery kit (AXYGEN, Cat.No. AP-GX-50), and the specific steps are 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 Buffer W1, centrifuging at 12000 Xg for 1min, and removing the filtrate;

(6) the prepared tube is put back into a centrifuge tube, 700 mul of Buffer W2 is added, 12000 Xg is added for centrifugation for 1min, and the filtrate is discarded; the mixture was washed once more with 700. mu.l Buffer W2 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

Two complementary DNA fragments were synthesized which code for amino acids SDLWKLLPENNV (SEQ ID NO. 2; SV for short) at positions 20-31 of the N-terminus of the P53 protein:

5’-CGGCCATGGCATCAGACCTATGGAAACTACTTCCTGAAAACAACGTTGGCCCGGG-3’；SEQ ID NO.3；

5’-GGGCCAACGTTGTTTTCAGGAAGTAGTTTCCATAGGTCTGATGCCATGGCCGGCT-3’；SEQ ID NO.4；

the two fragments are dissolved and mixed in an equimolar way, and are denatured at 94 ℃ for 5min and then renatured at 58 ℃ for 4min, so that the two fragments are complementarily combined into a double chain.

5) The target fragment is connected with a fADL-le enzyme digestion vector to form fADL-le-SV

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

6) recombinant vector fADL-le-SV 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 (3) selecting a recombinant vector fADL-le-SV positive clone, 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 1): 5'-ccgtgcatctgtcctcgttcaa-3', respectively; SEQ ID No. 5;

downstream primer (PR 1): 5'-GTTTTCAGGAAGTAGTTTCCATAGGTC-3', respectively; SEQ ID No. 6;

an upstream primer PF1 is designed at about 700bp upstream of the pIII gene insertion site of the vector fADL-le, an antisense strand of an insertion fragment is used as a downstream primer for PCR verification, a specific target band appears at 700bp after PCR of positive clone, and no specific band appears after PCR of negative clone.

The PCR reaction system is as follows:

the PCR reaction program is: 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.

After the reaction was completed, the results were shown in FIG. 4 by agarose gel electrophoresis; PCR results show that a specific target band appears between 500bp and 750bp, and the successful insertion of the coding fragment of the exogenous peptide SV into the pIII gene of the phage is preliminarily proved.

The screened positive clones are sent to Shanghai Biotechnology service company Limited for sequencing, and the sequencing result of the recombinant vector fADL-le-SV is as follows:

sequencing results show that the target fragment (the black bold underlined part and FIG. 5) is successfully cloned into the PIII gene of the phage vector fADL-le, and is completely consistent with the original sequence of the connection, which indicates that the recombinant vector fADL-le-SV is successfully constructed.

(II) construction of recombinant phage vector fADL-le-SV-MP

The constructed phage vector fADL-le-SV is used as a template, and a point mutation kit (Vazyme, cat # C215) is utilized to directionally clone peptide MP (encoding amino acids MEEPQSDP of 1 st to 8 th positions at the N end of P53 protein, SEQ ID NO. 8; MP for short) into the PVIII gene of the phage to construct recombinant phagemid fADL-le-SV-MP, and the specific steps are as follows:

1) amplification of vector fADL-le-SV

The kit is used for carrying out PCR amplification by using a reagent, and the reaction system is as follows:

the sequences of primers PF2 and PR2 used in the amplification reaction are as follows:

upstream primer (PF 2): 5'-atggaggagccgcagtcagatcccGCAAAAGCGGCC-3', respectively; SEQ ID No. 9;

downstream primer (PR 2): 5'-tgactgcggctcctccatAGCGAAAGACAGCATCGGAA-3', respectively; SEQ ID No. 10;

wherein, the lower case letters are partial sequences of the coding exogenous peptide MP and are used for the later homologous recombination to form the circular recombinant phagemid fADL-le-SV-MP, and the upper case letters are used for amplifying the sequence of the vector fADL-le-SV.

2) Digestion of amplification product Dpn I

Because the amplification product of the last step contains the original template plasmid, 1 mu l of Dpn I is added after the PCR amplification reaction is finished, and the mixture is digested for 2h at 37 ℃ to prevent the original template plasmid from forming a false positive transformant after the transformation, so that the methylated template plasmid is removed.

3) Recombination to form fADL-le-SV-MP

The recombination reaction system is as follows (reaction at 37 ℃ for 30 min):

4) fADL-le-SV-MP transformed Escherichia coli JM109 cells

(1) Adding 10 μ l of recombinant 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 (3) selecting a recombinant vector fADL-le-SV-DP positive clone, and sending the screened positive clone to Shanghai Biotechnology service company Limited for sequencing.

The sequencing results were as follows:

sequencing results show that the target fragment (black bold underlined part and FIG. 6) encoding the peptide MP is successfully cloned into the PVIII gene of the phage vector fADL-le-SV and completely consistent with the original sequence of the ligation, indicating that the recombinant phage vector fADL-le-SV-MP is successfully constructed.

(III) construction of recombinant phage vector fADL-le-SV-MP-DE

The constructed phage vector fADL-le-SV-MP is used as a template, peptide DE (encoding amino acids 49-56 of the N end of P53 protein DIEQWFTE, SEQ ID NO. 12; DE for short) is directionally cloned into a PVI gene of a phage by using a point mutation kit (Vazyme, cat # C215) to construct and obtain a recombinant phage vector fADL-le-SV-MP-DE, the whole operation process is consistent with the construction of the recombinant phage vector fADL-le-SV-MP, and the used amplification primers are as follows:

upstream primer (PF 3): 5'-attgaacaatggttcactgaaTAAATATGGCTGTTTATTTTGTAACTGG-3', respectively; SEQ ID No. 13;

downstream primer (PR 3): 5'-cagtgaaccattgttcaatatcTTTATCCCAATCCAAATAAGAAACG-3', respectively; SEQ ID No. 14;

wherein, the lower case letters are partial sequences of coding exogenous peptide DE and are used for forming circular recombinant phagemid fADL-le-SV-MP-DE by later homologous recombination, and the upper case letters are used for amplifying vector fADL-le-SV sequences.

The sequencing results were as follows:

sequencing results show that a target fragment (a black bold underlined part and FIG. 7) of the coded peptide DE is successfully cloned into a PVI gene of the phage vector fADL-le-SV-MP and is completely consistent with a connected original sequence, and the recombinant phage vector fADL-le-SV-MP-DE is successfully constructed and can be used for preparing a phage in the next step.

(IV) preparation of phage phase-SV-MP-DE and Atomic Force Microscope (AFM) observation analysis

1) Preparation of phage phase-SV-MP-DE

(1) Mu.l of JM109 correctly sequenced transformed fADL-le-SV-MP-DE was inoculated into a medium containing 100ml of LB liquid medium (100. mu.g/ml Kar)⁺) In the test tube, shaking vigorously for 10h at 37 ℃;

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

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

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

(5) 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 ℃;

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

2) Atomic Force Microscope (AFM) observation phase-SV-MP-DE

phage-SV-MP-DE was diluted to 10 with PBS buffer⁷Mu.l/ml was added dropwise to a mica plate by pipetting, spin-coated at a low speed for 1min, and then observed by AFM.

AFM results show (figure 8), the targeting three-display phage phase-SV-MP-DE is about 900nm long and 7nm wide, has a relatively flexible structure, and the display of exogenous peptides has no influence on the morphological structure.

(IV) results of detection of serum P53 antibody from cancer patient by phage phase-SV-MP-DE

(1)phage-SV-MP-DE-ELISA

The detection of serum P53 antibody was carried out on 60 breast cancer patients and 60 healthy persons (negative control) by ELISA method using phase-SV-MP-DE as detection antigen, and the specific steps were as follows:

1) taking the phase-SV-MP-DE as a coating antigen (short for phase-SV-MP-DE-ELISA) to coat a 96-hole enzyme label plate, wherein the concentration is 60 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 is identical to the phase-SV-MP-DE-ELISA except that the antigen coated on the ELISA plate is recombinant P53 protein and the concentration is 5 mug/ml.

(3) Determination of cut-off value

60 cases of healthy human serum were detected according to the established phase-SV-MP-DE-ELISA and P53-ELISA detection systems, and cut-off values of each detection method were determined by the mean +2SD method.

As shown in FIG. 9, in 60 breast cancer patients, 15 sera from P53 antibody-positive patients were detected by the phase-SV-MP-DE at a detection rate of 25% which was higher than the detection efficiency of the recombinant P53 protein (21.67%), and the specificity of the two detection methods was 95% (phase-SV-MP-DE-ELISA) and 95.00% (P53-ELISA), respectively. The result shows that the phase-SV-MP-DE 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 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> targeting three-display phage and preparation method and application thereof

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8054

<212> DNA

<213> Artificial Sequence

<400> 1

aacgctacta ccattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60

atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 120

cgttcgcaga attgggaatc aactgttaca tggaataaaa cttccagaca ccgtacttta 180

gttgcatatt taaaacatgt tgaactacag caccagattc agcaattaag ctctaagcca 240

tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactgtctaa tcctgacctg 300

ttggaatttg cttccggtct ggttcgcttt gaggctcgaa ttaaaacgcg atatttgaag 360

tctttcgggc ttcctcttaa tctttttgat gcaattcgct ttgcttctga ctataataga 420

cagggtaaag acctgatttt tgatttatgg tcattctctt tttctgaact gtttaaagca 480

tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct 540

aaacatttta caattacccc ctctggcaaa acttcctttg caaaagcctc tcgctatttt 600

ggtttctatc gtcgtctggt taatgagggt tatgatagtg ttgctcttac catgcctcgt 660

aattcctttt ggcgttatgt atctgcatta gttgagtgtg gtattcctaa atctcaattg 720

atgaatcttt ccacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 780

tcctcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 840

aaatgattaa agttgaaatt aaaccatctc aagcgcaatt tactacccgt tctggtgttt 900

ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960

aatatccggt gcttgtcaag attactctcg acgaaggtca gccagcgtat gcgcctggtc 1020

tgtacaccgt gcatctgtcc tcgttcaaag ttggtcagtt cggttctctt atgattgacc 1080

gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1140

caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1200

caaagatgag tgttttagtg tattctttcg cctctttcgt tttaggttgg tgccttcgta 1260

gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320

caaagcctcc gtagccgttg ctaccctcgt tccgatgctg tctttcgcta tggaggagcc 1380

gcagtcagat cccgcaaaag cggcctttaa ctccctgcaa gcctcagcga ccgaatatat 1440

cggttatgcg tgggcgatgg ttgttgtcat tgtcggcgca actatcggta tcaagctgtt 1500

taagaaattc acctcgaaag caagctgata aaccgataca attaaaggct ccttttggag 1560

cctttttttt gtcgactaac gagggcaaat catgaaatac ctattgccta cggcggccgc 1620

tggattgtta ttactcgcgg cccagccggc catggcatca gacctatgga aactacttcc 1680

tgaaaacaac gttggcccgg gaggcctgtc tctagaagcc gaaactgttg aaagttgttt 1740

agcaaaacct catacagaaa attcatttac taacgtctgg aaagacgaca aaactttaga 1800

tcgttacgct aactatgagg gctgtctgtg gaatgctaca ggcgttgtgg tttgtactgg 1860

tgacgaaact cagtgttacg gtacatgggt tcctattggg cttgctatcc ctgaaaatga 1920

gggtggtggc tctgagggtg gcggttctga gggtggcggt tctgagggtg gcggtactaa 1980

acctcctgag tacggtgata cacctattcc gggctatact tatatcaacc ctctcgacgg 2040

cacttatccg cctggtactg agcaaaaccc cgctaatcct aatccttctc ttgaggagtc 2100

tcagcctctt aatactttca tgtttcagaa taataggttc cgaaataggc agggtgcatt 2160

aactgtttat acgggcactg ttactcaagg cactgacccc gttaaaactt attaccagta 2220

cactcctgta tcatcaaaag ccatgtatga cgcttactgg aacggtaaat tcagagactg 2280

cgctttccat tctggcttta atgaggatcc attcgtttgt gaatatcaag gccaatcgtc 2340

tgacctgcct caacctcctg tcaatgctgg cggcggctct ggtggtggtt ctggtggcgg 2400

ctctgagggt ggcggctctg agggtggcgg ttctgagggt ggcggctctg agggtggcgg 2460

ttccggtggc ggctccggtt ccggtgattt tgattatgaa aaaatggcaa acgctaataa 2520

gggggctatg accgaaaatg ccgatgaaaa cgcgctacag tctgacgcta aaggcaaact 2580

tgattctgtc gctactgatt acggtgctgc tatcgatggt ttcattggtg acgtttccgg 2640

ccttgctaat ggtaatggtg ctactggtga ttttgctggc tctaattccc aaatggctca 2700

agtcggtgac ggtgataatt cacctttaat gaataatttc cgtcaatatt taccttcttt 2760

gcctcagtcg gttgaatgtc gcccttatgt ctttggcgct ggtaaaccat atgaattttc 2820

tattgattgt gacaaaataa acttattccg tggtgtcttt gcgtttcttt tatatgttgc 2880

cacctttatg tatgtatttt cgacgtttgc taacatactg cgtaataagg agtcttaatc 2940

atgccagttc ttttgggtat tccgttatta ttgcgtttcc tcggtttcct tctggtaact 3000

ttgttcggct atctgcttac tttccttaaa aagggcttcg gtaagatagc tattgctatt 3060

tcattgtttc ttgctcttat tattgggctt aactcaattc ttgtgggtta tctctctgat 3120

attagcgcac aattaccctc tgattttgtt cagggcgttc agttaattct cccgtctaat 3180

gcgcttccct gtttttatgt tattctctct gtaaaggctg ctattttcat ttttgacgtt 3240

aaacaaaaaa tcgtttctta tttggattgg gataaagata ttgaacaatg gttcactgaa 3300

taaatatggc tgtttatttt gtaactggca aattaggctc tggaaagacg ctcgttagcg 3360

ttggtaagat tcaggataaa attgtagctg ggtgcaaaat agcaactaat cttgatttaa 3420

ggcttcaaaa cctcccgcaa gtcgggaggt tcgctaaaac gcctcgcgtt cttagaatac 3480

cggataagcc ttctatttct gatttgcttg ctattggtcg tggtaatgat tcctacgacg 3540

aaaataaaaa cggtttgctt gttcttgatg aatgcggtac ttggtttaat acccgttcat 3600

ggaatgacaa ggaaagacag ccgattattg attggtttct tcatgctcgt aaattgggat 3660

gggatattat ttttcttgtt caggatttat ctattgttga taaacaggcg cgttctgcat 3720

tagctgaaca cgttgtttat tgtcgccgtc tggacagaat tactttaccc tttgtcggca 3780

ctttatattc tcttgttact ggctcaaaaa tgcctctgcc taaattacat gttggtgttg 3840

ttaaatatgg tgattctcaa ttaagcccta ctgttgagcg ttggctttat actggtaaga 3900

atttatataa cgcatatgac actaaacagg ctttttccag taattatgat tcaggtgttt 3960

attcatattt aaccccttat ttatcacacg gtcggtattt caaaccatta aatttaggtc 4020

agaagatgaa attaactaaa atatatttga aaaagttttc tcgcgttctt tgtcttgcga 4080

taggatttgc atcagcattt acatatagtt atataaccca acctaagccg gaggttaaaa 4140

aggtagtctc tcagacctat gattttgata aattcactat tgactcttct cagcgtctta 4200

atctaagcta tcgctatgtt ttcaaggatt ctaagggaaa attaattaat agcgacgatt 4260

tacagaagca aggttattcc atcacatata ttgatttatg tactgtttca attaaaaaag 4320

gtaattcaaa tgaaattgtt aaatgtaatt aattttgttt tcttgatgtt tgtttcatca 4380

tcttcttttg ctcaagtaat tgaaatgaat aattcgcctc tgcgcgattt cgtgacttgg 4440

tattcaaagc aaacaggtga atctgttatt gtctcacctg atgttaaagg tacagtgact 4500

gtatattcct ctgacgttaa gcctgaaaat ttacgcaatt tctttatctc tgttttacgt 4560

gctaataatt ttgatatggt tggctctaat ccttccataa ttcagaaata taacccaaat 4620

agtcaggatt atattgatga attgccatca tctgatattc aggaatatga tgataattcc 4680

gctccttctg gtggtttctt tgttccgcaa aatgataatg ttactcaaac atttaaaatt 4740

aataacgttc gcgcaaagga tttaataagg gttgtagaat tgtttgttaa atctaataca 4800

tctaaatcct caaatgtatt atctgttgat ggttctaact tattagtagt tagcgcccct 4860

aaagatattt tagataacct tccgcaattt ctttctactg ttgatttgcc aactgaccag 4920

atattgattg aaggattaat tttcgaggtt cagcaaggtg atgctttaga tttttccttt 4980

gctgctggct ctcagcgcgg cactgttgct ggtggtgtta atactgaccg tctaacctct 5040

gttttatctt ctgcgggtgg ttcgttcggt atttttaacg gcgatgtttt agggctatca 5100

gttcgcgcat taaagactaa tagccattca aaaatattgt ctgtgcctcg tattcttacg 5160

ctttcaggtc agaagggttc tatttctgtt ggccagaatg tcccttttat tactggtcgt 5220

gtaactggtg aatctgccaa tgtaaataat ccatttcaga caattgagcg tcaaaatgtt 5280

ggtatttcta tgagtgtttt tcccgttgca atggctggcg gtaatattgt tttagatata 5340

accagtaagg ccgatagttt gagttcttct actcaggcaa gtgatgttat tactaatcaa 5400

agaagtattg cgacaacggt taatttgcgt gatggtcaga ctcttttgct cggtggcctc 5460

actgattaca aaaacacttc tcaagattct ggtgtgccgt tcctgtctaa aatcccttta 5520

atcggcctcc tgtttagctc ccgttctgat tctaacgagg aaagcacgtt gtacgtgctc 5580

gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 5640

tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 5700

cccttccttt ctcgccacgt tctccggctt tccccgtcaa gctctaaatc gggggatctc 5760

gggaaaagcg ttggtgacca aaggtgcctt ttatcatcac tttaaaaata aaaaacaatt 5820

actcagtgcc tgttataagc agcaattaat tatgattgat gcctacatca caacaaaaac 5880

tgatttaaca aatggttggt ctgccttaga aagtatattt gaacattatc ttgattatat 5940

tattgataat aataaaaacc ttatccctat ccaagaagtg atgcctatca ttggttggaa 6000

tgaacttgaa aaaattagcc ttgaatacat tactggtaag gtaaacgcca ttgtcagcaa 6060

attgatccaa gagaaccaac ttaaagctta tgatgatgat gtgcttaaaa acttactcaa 6120

tggctggttt atgcatatcg caatacatgc gaaaaaccta aaagagcttg ccgataaaaa 6180

aggccaattt attgctattt accgcggctt tttattgagc ttgaaagata aataaaatag 6240

ataggtttta tttgaagcta aatcttcttt atcgtaaaaa atgccctctt gggttatcaa 6300

gagggtcatt atatttcgcg gaataaacca attaaccaat tctgattaga aaaactcatc 6360

gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa 6420

aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc 6480

ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc 6540

gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa 6600

tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc 6660

atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg 6720

aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag 6780

gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg 6840

gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat 6900

aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc 6960

atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc 7020

gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca 7080

tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg agcaagacgt 7140

ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt 7200

tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac 7260

aacgtggctt ttgttgacaa agggaatcat agatcccttt agggttccga tttagtgctt 7320

tacggcacct cgacctccaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc 7380

cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 7440

tgttccaaac tggaacaaca ctcacaacta acccggccta ttcttttgat ttataaggat 7500

ttttgtcatt ttctgcttac tggttaaaaa ataagctgat ttaacaaata tttaacgcga 7560

aatttaacaa aacattaacg tttacaattt aaatatttgc ttatacaatc atcctgtttt 7620

tggggctttt ctgattatca atcggggtac atatgattga catgctagtt ttacgattac 7680

cgttcatcga ttctcttgtt tgctccagac tttcaggtaa tgacctgata gcctttgtag 7740

acctctcaaa aatagctacc ctctccggca tgaatttatc agctagaacg gttgaatatc 7800

atattgacgg tgatttgact gtctccggcc tttctcaccc gtttgaatct ttgcctactc 7860

attactccgg cattgcattt aaaatatatg agggttctaa aaatttttat ccctgcgttg 7920

aaattaaggc ttcaccagca aaagtattac agggtcataa tgtttttggt acaaccgatt 7980

tagctttatg ctctgaggct ttattgctta attttgctaa ctctctgcct tgcttgtacg 8040

atttattgga tgtt 8054

<210> 2

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 2

Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val

1 5 10

<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> 626

<212> DNA

<213> Artificial Sequence

<400> 7

acaatttatc aggcgatgat acaaatctcc gttgtacttt gtttcgcgct tggtataatc 60

gctgggggtc aaagatgagt gttttagtgt attctttcgc ctctttcgtt ttaggttggt 120

gccttcgtag tggcattacg tattttaccc gtttaatgga aacttcctca tgaaaaagtc 180

tttagtcctc aaagcctccg tagccgttgc taccctcgtt ccgatgctgt ctttcgctgc 240

tgagggtgac gatcccgcaa aagcggcctt taactccctg caagcctcag cgaccgaata 300

tatcggttat gcgtgggcga tggttgttgt cattgtcggc gcaactatcg gtatcaagct 360

gtttaagaaa ttcacctcga aagcaagctg ataaaccgat acaattaaag gctccttttg 420

gagccttttt tttgtcgact aacgagggca aatcatgaaa tacctattgc ctacggcggc 480

cgctggattg ttattactcg cggcccagcc ggccatggca tcagacctat ggaaactact 540

tcctgaaaac aacgttggcc cgggaggcct gtctctagaa gccgaaactg ttgaaagttg 600

tttagcaaaa cctcatacag aaaatc 626

<210> 8

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 8

Met Glu Glu Pro Gln Ser Asp Pro

1 5

<210> 9

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 9

atggaggagc cgcagtcaga tcccgcaaaa gcggcc 36

<210> 10

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 10

tgactgcggc tcctccatag cgaaagacag catcggaa 38

<210> 11

<211> 688

<212> DNA

<213> Artificial Sequence

<400> 11

tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt caaagatgag 60

tgtttagtgt attctttcgc ctctttcgtt ttaggttggt gccttcgtag tggcattacg 120

tattttaccc gtttaatgga aacttcctca tgaaaaagtc tttagtcctc aaagcctccg 180

tagccgttgc taccctcgtt ccgatgctgt ctttcgctat ggaggagccg cagtcagatc 240

ccgcaaaagc ggcctttaac tccctgcaag cctcagcgac cgaatatatc ggttatgcgt 300

gggcgatggt tgttgtcatt gtcggcgcaa ctatcggtat caagctgttt aagaaattca 360

cctcgaaagc aagctgataa accgatacaa ttaaaggctc cttttggagc cttttttttg 420

tcgactaacg agggcaaatc atgaaatacc tattgcctac ggcggccgct ggattgttat 480

tactcgcggc ccagccggcc atggcatcag acctatggaa actacttcct gaaaacaacg 540

ttggcccggg aggcctgtct ctagaagccg aaactgttga aagttgttta gcaaaacctc 600

atacagaaaa ttcatttact aacgtctgga aagacgacaa aactttagat cgttacgcta 660

actatgaggg ctgtctgtgg aatgctac 688

<210> 12

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 12

Asp Ile Glu Gln Trp Phe Thr Glu

1 5

<210> 13

<211> 49

<212> DNA

<213> Artificial Sequence

<400> 13

attgaacaat ggttcactga ataaatatgg ctgtttattt tgtaactgg 49

<210> 14

<211> 47

<212> DNA

<213> Artificial Sequence

<400> 14

cagtgaacca ttgttcaata tctttatccc aatccaaata agaaacg 47

<210> 15

<211> 460

<212> DNA

<213> Artificial Sequence

<400> 15

tttcattgtt tcttgctctt attattgggc ttaactcaat tcttgtgggt tatctctctg 60

atattagcgc acaattaccc tctgattttg ttcagggcgt tcagttaatt ctcccgtcta 120

atgcgcttcc ctgtttttat gttattctct ctgtaaaggc tgctattttc atttttgacg 180

ttaaacaaaa aatcgtttct tatttggatt gggataaaga tattgaacaa tggttcactg 240

aataaatatg gctgtttatt ttgtaactgg caaattaggc tctggaaaga cgctcgttag 300

cgttggtaag attcaggata aaattgtagc tgggtgcaaa atagcaacta atcttgattt 360

aaggcttcaa aacctcccgc aagtcgggag gttcgctaaa acgcctcgcg ttcttagaat 420

accggataag ccttctattt ctgatttgct tgctattggt 460