CN106906187A - A kind of bi-functional bacteriophage and purposes - Google Patents

Abstract

The invention discloses a bifunctional phage and its use. The invention successfully obtains a WR polypeptide capable of specifically recognizing vascular endothelial growth factor VEGF through phage biopanning. Then, the WR polypeptide and the tumor targeting recognition peptide are simultaneously displayed on the filamentous phage to form a bifunctional phage. This phage can not only specifically recognize the tumor but also block the VEGF secreted by the tumor cell from entering the vascular endothelial cells around the tumor, thereby inhibiting the formation of new blood vessels around the tumor. Through this technology, tumor cells cannot proliferate due to lack of sufficient nutrients, and tumor metastasis cannot be successfully achieved through capillaries. The invention overcomes the shortcomings of traditional drugs such as lack of targeting, high cost and complicated preparation.

Description

A kind of bifunctional phage and its application

technical field

The invention belongs to the field of nanomedicine and relates to a bifunctional phage and its application in inhibiting tumor growth.

Background technique

In 1979, Dr. Folkman of Harvard University proposed the famous tumor angiogenesis hypothesis, which was accepted after nearly 30 years of research. The hypothesis holds that tumors highly express a series of endogenous angiogenic factors under conditions such as hypoxia, and quiescent endothelial cells are activated by these cytokines and undergo cell proliferation, migration and arrangement into a luminal structure. Tumor volume growth and metastasis need to rely on these new blood vessels to provide nutrients, remove metabolites and provide pathways. This hypothesis has been proved by many experiments: the growth of tumors on chick embryonic chorion (CAM) is limited in the avascular stage, but grows rapidly after vascularization; the volume of subcutaneously transplanted tumors in mice increases linearly before vascularization, and grows rapidly after vascularization. Afterwards, it increases exponentially; during the growth process of subcutaneous tumors in mice, blood vessels accounted for an increase in tumor volume, and finally reached 1.5%; in vitro spherical tumors can only reach 2mm ³ in the absence of blood vessels, and the diameter in vivo can only reach 0.4mm; breast cancer Endothelial cells proliferate 45 times faster than endothelial cells in adjacent mesenchymal vessels; there are very few circulating tumor cells in the blood before angiogenesis, and only appear in large numbers after vascularization; the neovascular basement membrane of the tumor is discontinuous and fragmented , easy to "leak" tumor cells; collagenase and plasma plasminogen activator secreted by new blood vessels also assist tumor cells to invade the surrounding knot tissue and lymphatic vessels. Therefore, angiogenesis factors are a very important target for tumor therapy: by neutralizing and inhibiting important angiogenesis factors (such as: VEGF) to inhibit tumor neovascularization, so as to achieve the purpose of "starving" tumors.

Filamentous phages are biological nanofibers of 900 nm and 6.5 nm in diameter formed by coat proteins surrounding the genome. Like other nanoparticles, it has a large specific surface area, so it can carry a large amount of molecular drugs; at the same time, its large molecular weight has strong immunogenicity, and it is easier for the body to produce an immune response and is easily eliminated by the body. Because phages are virus particles that specifically target bacteria, they are safe nanomaterials for humans. And phage can also obtain binding peptides specifically recognized by target targets (such as nanoparticles, proteins, cells and organs) through phage display technology and biopanning. Therefore, filamentous phage is a target-blocking drug carrier with great potential. However, many current studies have only used its targeting, but have not attempted to simultaneously display targeting functional peptides and therapeutic functional peptides on a phage.

Contents of the invention

The object of the present invention is to provide a bifunctional phage and its use in view of the deficiencies of the prior art.

The object of the present invention is achieved by the following technical solutions: a bifunctional phage prepared by the following method:

(1) Using a phage polypeptide library, screening out the specific binding peptide WR for human vascular endothelial growth factor through biopanning;

(2) The WR polypeptide in step (1) and the AR polypeptide capable of specifically recognizing orthotopic breast cancer tumors are respectively displayed on the p3 and p8 proteins of the double display phage fd388 to form a bifunctional phage fd388-AR-WR.

A use of the above-mentioned bifunctional phage in preparing drugs for inhibiting tumor growth.

The beneficial effect of the present invention is that the present invention uses bio-panning technology to screen and obtain a polypeptide (WR) that can specifically bind to VEGF protein, and the polypeptide and a human breast cancer-specific recognition peptide are displayed on the phage respectively by phage display technology. p8 and p3 positions and prepared a bifunctional phage that can bind tumors and VEGF at the same time; the phage can not only specifically recognize tumors but also block VEGF secreted by tumor cells from entering the vascular endothelial cells around the tumor, thereby inhibiting the vascular endothelial cells around the tumor. The formation of new blood vessels. Through this technology, tumor cells cannot proliferate due to lack of sufficient nutrients, and tumor metastasis cannot be successfully achieved through capillaries. The invention overcomes the shortcomings of traditional medicines such as lack of targeting, high cost, complicated preparation and the like.

Description of drawings

Figure 1 is a TEM image of fd388-AR-WR bifunctional phage;

Figure 2 is a statistical diagram of tumor weight after treatment with fd388-AR-WR bifunctional phage;

Figure 3 is a visualization of tumors treated with fd388-AR-WR bifunctional phage.

detailed description

The present invention will be described in further detail below through examples, and the following examples are explanations of the present invention and the present invention is not limited to the following examples.

Example 1:

1. Biological panning:

1.1. Use 0.1M NaHCO3 coating solution to dilute VEGF protein into four gradient concentrations of 20g/mL, 4g/mL, 0.8g/mL, and 0.16g/mL.

1.2. Incubate 400 μL of protein solution with a concentration of 20 g/mL in a 24-well cell culture plate overnight at 4°C.

1.3. Discard the protein solution, add 500 μL of 5 mg/mL BSA solution, block at 37°C for 2 hours; discard the blocking solution.

1.4. Add 500 μL of 5% Tween-20 in PBST solution and incubate for 6 minutes, discard and repeat 6 times.

1.5. Add 400 μL PBS containing 10 ¹⁰ pfu phage library as input, block at 37°C for 1 hour, and discard the supernatant.

1.6. Add 500 μL of 5% Tween-20 in PBST solution and incubate for 6 minutes, discard and repeat 6 times to wash away unbound and non-specifically bound phages.

1.7. Add 400 μL of 0.1M HCl with pH=2.2 and incubate for 5-10 minutes to elute the specifically bound phages; then immediately add 60 μL of 1M Tris-HCl solution with pH=9.1 for neutralization, and pass the obtained phages Infected bacteria were amplified to obtain amplified phages, and the first round of screening was completed.

1.8. Use protein solutions with concentrations of 4g/mL, 0.8g/mL, and 0.16g/mL in turn to complete the 2nd-4th round of screening according to steps 1.2-1.7, and the input for each screening is the amplification obtained in the previous round. of bacteriophages.

1.9. After four rounds of screening, some phages specifically combined with VEGF were obtained, and then the genomes of these phages were subjected to DNA sequencing.

2. Verify affinity: perform phage ELISA and dot hybridization experiments on the phages and VEGF proteins that appear frequently in DNA sequencing in step 1. The experimental results showed that the phage carrying the WR polypeptide had the highest affinity to VEGF. The amino acid sequence of the WR-carrying polypeptide is shown in SEQ ID NO.1.

3. Phage display: Add PstI/HindIII restriction sites to both ends of the double-stranded complementary DNA sequence encoding the WR polypeptide, and encode the DNA of breast cancer tumor-specific recognition polypeptide (AR, whose amino acid sequence is shown in SEQ ID NO.9) Add SfiI restriction sites at both ends of the sequence. The double-stranded complementary DNA sequence corresponding to the WR polypeptide is shown in SEQ ID NO.2 and SEQ ID NO.11, and the double-stranded complementary DNA sequence corresponding to the AR polypeptide is shown in SEQ ID NO.3 and SEQ ID NO.12; the fd388 The phage display vector was digested with PstI and HindIII, and ligated with the designed DNA fragment encoding the WR polypeptide with PstI/HindIII restriction sites through T4 ligase; the fd388-WR corresponding to the DNA of the WR polypeptide was successfully inserted, and then Use SfiI to digest, and connect with the designed DNA fragment encoding AR polypeptide with SfiI restriction site to form the genome of fd388-AR-WR bifunctional phage; then transform the genome into Escherichia coli for transcription, Translated and assembled into a bifunctional phage, the fd388-AR-WR dual display phage recombinant p3 protein and recombinant p8 protein amino acid sequences are shown in SEQ ID NO.4 and SEQ ID NO.5; finally the constructed phage Purify by adding into a solution containing PEG8000 and NaCl, the volume ratio of the phage to the solution is 6:1, the mass concentration of PEG8000 in the solution is 16.7%, and the molar concentration of NaCl is 3.3M.

The fd388 phage display vector is prepared by the following steps:

(a) Obtain recombinant gene3: Add fUSE55 vector (sequence shown in SEQ ID NO.6) according to New England Biolab's BsrGI/BamHI double enzyme digestion system for 3 hours at 37°C; use Qiagen's plasmid recovery kit to recover A small fragment was obtained to obtain a 1222bp DNA fragment 1 containing the recombinant gene3. Wherein, the fUSE55 is obtained by inserting a DNA fragment containing two SfiI restriction sites (SEQ ID NO.8: GCCGACGTGGCCCGGCCTCTGGGGCC) into the 5' end of gene3 of wild fd phage through an overlap PCR experiment.

(b) Obtain other parts of the phage genome: add the f88-4 vector (sequence shown in SEQ ID NO.7) according to the BsrGI/BamHI double enzyme digestion system of New England Biolab Company and perform double enzyme digestion at 37°C for 3 hours; use the plasmid from Qiagen Company The recovery kit recovers large fragments to obtain DNA fragment 2 with a length of 8034bp remaining after removing DNA fragment 1 in the phage genome. The f88-4 vector is obtained by inserting the PstI and HindIII restriction sites into the 5' end of the wild gene8 by overlapping PCR reaction to obtain the engineered gene8, and inserting the engineered gene8 into the wild fd phage.

(c) Construction of double display vector fd388: DNA fragment 1 and DNA fragment 2 were ligated at a ratio of 1:3 by T4 DNA ligase overnight at 16°C.

(d) Transform the ligation product into JM109 competent cells, and smear it on a tetracycline-resistant plate at 37°C overnight to obtain positive single clones, and obtain a phage double display vector capable of ectopically displaying two polypeptides, and the phage double display vector The sequence is shown in SEQ ID NO.10.

4. Establishment of an orthotopic xenogeneic breast cancer mouse model: First, a 0.36mg/pellet 17βestradiol sustained-release tablet (Innovative Research of America) was implanted on the back of a 4-week-old immunodeficient mouse; the next day Inject 100 μL of 10 ⁷ MCF-7 cells in the fat pad of the second pair of mammary glands.

5. Tumor growth inhibition monitoring: 10 days after the successful establishment of the orthotopic tumor model in step 4, inject 100 μL of the bifunctional phage purified in step 3 into the tail vein of the mouse every other day, and measure the length and width of the tumor and mouse body weight; use PBS solution as a control; after 10 times of injection treatment, the tumor was stripped out and weighed, and HE staining and immunohistochemistry were performed to verify the blood vessel density; the blood of the mice was collected to detect the hemolytic effect of phage in vitro; the main organs ( Such as kidney, liver and spleen) for HE staining and immunohistochemistry. Fig. 2 and Fig. 3 are tumor weight statistics and visual diagrams of tumor-bearing mice treated with fd388-AR-WR bifunctional phage, WT wild-type phage (negative control) and PBS (blank control group). The results showed that fd388-AR-WR bifunctional phage could inhibit tumor growth more significantly than WT wild-type phage and PBS.

SEQUENCE LISTING

<110> Zhejiang University

<120> A bifunctional phage and its use

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gcgtgatggt cagactcttt tgctcggtgg cctcactgat tacaaaaaca cttctcaaga 5400

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gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 5580

ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttctccg 5640

gctttccccg tcaagctcta aatcggggga tctcgggaaa agcgttggtg accaaaggtg 5700

ccttttatca tcactttaaa aataaaaaac aattactcag tgcctgttat aagcagcaat 5760

taattatgat tgatgcctac atcacaacaa aaactgattt aacaaatggt tggtctgcct 5820

tagaaagtat atttgaacat tatcttgatt atattattga taataataaa aaccttatcc 5880

ctatccaaga agtgatgcct atcattggtt ggaatgaact tgaaaaaatt agccttgaat 5940

aactactgg taaggtaaac gccattgtca gcaaattgat ccaagagaac caacttaaag 6000

cttatgatga tgatgtgctt aaaaacttac tcaatggctg gtttatgcat atcgcaatac 6060

atgcgaaaaa cctaaaagag cttgccgata aaaaaggcca atttattgct atttaccgcg 6120

gctttttatt gagcttgaaa gataaataaa atagataggt tttatttgaa gctaaatctt 6180

ctttatcgta aaaaatgccc tcttggggtta tcaagagggt catttatattt cgcggaataa 6240

catcatttgg tgacgaaata actaagcact tgtctcctgt ttactcccct gagcttgagg 6300

ggttaacatg aaggtcatcg atagcaggat aataatacag taaaacgcta aaccaataat 6360

ccaaatccag ccatcccaaa ttggtagtga atgattataa ataacagcaa acagtaatgg 6420

gccaataaca ccggttgcat tggtaaggct caccaataat ccctgtaaag caccttgctg 6480

atgactcttt gtttggatag acatcactcc ctgtaatgca ggtaaagcga tcccaccacc 6540

agccaataaa attaaaacag ggaaaactaa ccaaccttca gatataaacg ctaaaaaggc 6600

aaatgcacta ctatctgcaa taaatccgag cagtactgcc gttttttcgc cccattagt 6660

ggctattctt cctgccacaa aggcttggaa tactgagtgt aaaagaccaa gacccgctaa 6720

tgaaaagcca accatcatgc tattccatcc aaaacgattt tcggtaaata gcacccacac 6780

cgttgcggga atttggccta tcaattgcgc tgaaaaataa ataatcaaca aaatgggcat 6840

cgttttaaat aaagtgatgt ataccgaatt cgattgcgtc tcaacccccta cttcggtatc 6900

tgtattatca cgtgtatttt tggtttcacg gaaccaaaac ataaccacaa ggaaagtgac 6960

aatatttagc aacgcagcga taaaaaaggg actatgcggt gaaatctctc ctgcaaaacc 7020

accaataata ggccccgcta ttaaaccaag cccaaaactt gcccctaacc aaccgaacca 7080

cttcacgcgt tgagaagctg aggtggtatc ggcaatgacc gatgccgcga cagccccagt 7140

agctcctgtg atccctgaaa gcaaacggcc taaatacagc atccaaagcg cacttgaaaa 7200

agccagcaat aagtaatcca gcgatgcgcc tattaatgac aacaacagca ctgggcgccg 7260

accaaatcgg tcagacattt ttccaagcca aggagcaaag ataacctgca ttaacgcata 7320

aagtgcaagc aatacgccaa agtggttagc gatatcttcc gaagcaataa attcacgtaa 7380

taacgttggc aagactggca tgataaggcc aatccccatg gcatcgagta acgtaattac 7440

caatgcgatc tttgtcgaac tattcatttc acttttctct atcactgata gggagtggta 7500

aaataactct atcaatgata gagtgtcaac aaaaattagg aattaatgat gtctagatta 7560

gataaaagta aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt 7620

ttaacaaccc gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat 7680

gtaaaaaata agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat 7740

actcactttt gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa 7800

agttttagat gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg 7860

cctacagaaa aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt 7920

ttttcactag agaatgcatt atatgcactc agcgctgtgg ggcattttac tttaggttgc 7980

gtattggaag atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat 8040

agtatgccgc cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca 8100

gccttcttat tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa 8160

agtgggtctt aaaagcagca taaccttttt ccgtgatggt aacttcacgg taaccaagat 8220

gtcgagttaa ccacccttta gattcataaa gcgaaaataa tgcggctcca acgtacccac 8280

ctaaatggaa acggcgttca ctccaatcta aacacgcaca acagaatttta cgtgaatgtt 8340

tggaaggaac gtcaattccc atttcatgaa aatattgaat accacttaat gtgatcattg 8400

aaccattttc agtgatccat tgctgttgac aaagggaatc atagatccct ttagggttcc 8460

gatttagtgc tttacggcac ctcgacctcc aaaaacttga tttgggtgat ggttcacgta 8520

gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 8580

atagtggact cttgttccaa actggaacaa cactcacaac taactcggcc tattcttttg 8640

atttataagg atttttgtca ttttctgctt actggttaaa aaataagctg atttaacaaa 8700

tattaacgc gaaatttaac aaaacattaa cgtttacaat ttaaatattt gcttatacaa 8760

tcatcctgtt tttggggctt ttctgattat caatcggggt acatatgatt gacatgctag 8820

ttttacgatt accgttcatc gattctcttg tttgctccag actttcaggt aatgacctga 8880

tagcctttgt agacctctca aaaatagcta ccctctccgg catgaattta tcagctagaa 8940

cggttgaata tcatattgac ggtgatttga ctgtctccgg cctttctcac ccgtttgaat 9000

ctttgcctac tcattactcc ggcattgcat ttaaaatata tgagggttct aaaaattttt 9060

atccctgcgt tgaaattaag gcttcaccag caaaagtatt acagggtcat aatgtttttg 9120

gtacaaccga tttagcttta tgctctgagg ctttattgct taattttgct aactctctgc 9180

cttgcttgta cgattattg gatgtt 9206

<210> 7

<211> 9234

<212>DNA

<213> artificial design

<400> 7

aacgctacta ccttagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60

atagctaaac aggttatga ccatttgcga aatgtatcta atggtcaaac taaatctact 120

cgttcgcaga attgggaatc aactgttaca tggaatgaaa cttccagaca ccgtacttta 180

gttgcatatt taaaacatgt tgaactacag caccagattc agcaattaag ctctaagcca 240

tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactgtctaa tcctgacctg 300

ttggaatttg cttccggtct ggttcgcttt gaggctcgaa ttgaaacgcg atatttgaag 360

tctttcgggc ttcctcttaa tctttttgat gcaattcgct ttgcttctga ctataataga 420

cagggtaaag acctgatttt tgattatgg tcattctcgt tttctgaact gtttaaagca 480

tttgagggggg attcaatgaa tatttatgac gattccgcag tatggacgc tatccagtct 540

aaacatttta caattaccccc 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 aaaccgtctc aagcgcaatt tactacccgt tctggtgttt 900

ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960

aatatccggt gcttgtcaag attackctcg 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 tctttcgctg ctgagggtga 1380

cgatcccgca aaagcggcct ttgactccct gcaagcctca gcgaccgaat atatcggtta 1440

tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1500

attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560

tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc 1620

tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aacctcatac agaaaattca 1680

tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggctgt 1740

ctgtggaatg ctacaggcgt tgtggtttgt actggtgacg aaactcagtg ttacggtaca 1800

tgggttccta ttgggcttgc tatccctgaa aatgagggtg gtggctctga gggtggcggt 1860

tctgagggtg gcggttctga gggtggcggt actaaacctc ctgagtacgg tgatacacct 1920

attccgggct atacttatat caaccctctc gacggcactt atccgcctgg tactgagcaa 1980

aaccccgcta atcctaatcc ttctcttgag gagtctcagc ctcttaatac tttcatgttt 2040

cagaataata ggttccgaaa taggcagggt gcattaactg tttatacggg cactgttact 2100

caaggcactg accccgttaa aacttattac cagtacactc ctgtatcatc aaaagccatg 2160

tatgacgctt actggaacgg taaattcaga gactgcgctt tccattctgg ctttaatgag 2220

gatccattcg tttgtgaata tcaaggccaa tcgtctgacc tgcctcaacc tcctgtcaat 2280

gctggcggcg gctctggtgg tggttctggt ggcggctctg agggtggcgg ctctgagggt 2340

ggcggttctg agggtggcgg ctctgagggt ggcggttccg gtggcggctc cggttccggt 2400

gattttgatt atgaaaaaat ggcaaacgct aataaggggg ctatgaccga aaatgccgat 2460

gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt 2520

gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact 2580

ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct 2640

ttaatgaata atttccgtca atatttacct tctttgcctc agtcggttga atgtcgccct 2700

tatgtctttg gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta 2760

ttccgtggtg tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttcgacg 2820

tttgctaaca tactgcgtaa taaggagtct taatcatgcc agttcttttg ggtattccgt 2880

tattattgcg tttcctcggt ttccttctgg taactttgtt cggctatctg cttactttcc 2940

ttaaaaaggg cttcggtaag atagctattg ctatttcatt gtttcttgct cttattattg 3000

ggcttaactc aattcttgtg ggttatctct ctgatattag cgcacaatta ccctctgatt 3060

ttgttcaggg cgttcagtta attctcccgt ctaatgcgct tccctgtttt tatgttattc 3120

tctctgtaaa ggctgctatt ttcatttttg acgttaaaca aaaaatcgtt tcttatttgg 3180

attgggataa ataaatatgg ctgtttattt tgtaactggc aaattaggct ctggaaagac 3240

gctcgttagc gttggtaaga ttcaggataa aattgtagct gggtgcaaaa tagcaactaa 3300

tcttgatta aggcttcaaa acctcccgca agtcgggagg ttcgctaaaa cgcctcgcgt 3360

tcttagaata ccggataagc cttctatttc tgatttgctt gctattggtc gtggtaatga 3420

ttcctacgac gaaaataaaa acggtttgct tgttcttgat gaatgcggta cttggtttaa 3480

tacccgttca tggaatgaca aggaaagaca gccgattatt gattggtttc ttcatgctcg 3540

taaattggga tgggatatta tttttcttgt tcaggattta tctattgttg ataaacaggc 3600

gcgttctgca ttagctgaac acgttgttta ttgtcgccgt ctggacagaa ttactttacc 3660

ctttgtcggc actttatatt ctcttgttac tggctcaaaa atgcctctgc ctaaattaca 3720

tgttggtgtt gttaaatatg gtgattctca attaagccct actgttgagc gttggcttta 3780

tactggtaag aatttatata acgcatatga cactaaacag gctttttcca gtaattatga 3840

ttcaggtgtt tattcatatt taacccctta tttatcacac ggtcggtatt tcaaaccatt 3900

aaatttaggt cagaagatga aattaactaa aatatatttg aaaaagtttt ctcgcgttct 3960

ttgtcttgcg ataggatttg catcagcatt tacatatagt tatataaccc aacctaagcc 4020

ggaggttaaa aaggtagtct ctcagaccta tgattttgat aaattcacta ttgactcttc 4080

tcagcgtctt aatctaagct atcgctatgt tttcaaggat tctaagggaa aattaattaa 4140

tagcgacgat ttacagaagc aaggttattc catcacatat attgattat gtactgtttc 4200

aattaaaaaa ggtaattcaa atgaaattgt taaatgtaat taattttgtt ttcttgatgt 4260

ttgtttcatc atcttctttt gctcaagtaa ttgaaatgaa taattcgcct ctgcgcgatt 4320

tcgtgacttg gtattcaaag caaacaggtg aatctgttat tgtctcacct gatgttaaag 4380

gtacagtgac tgtatattcc tctgacgtta agcctgaaaa tttacgcaat ttctttatct 4440

ctgttttacg tgctaataat tttgatatgg ttggctcaat tccttccata attcagaaat 4500

ataacccaaa tagtcaggat tatattgatg aattgccatc atctgatatt caggaatatg 4560

atgataattc cgctccttct ggtggtttct ttgttccgca aaatgataat gttactcaaa 4620

catttaaaat taataacgtt cgcgcaaagg atttaataag ggttgtagaa ttgtttgtta 4680

aatctaatac atctaaatcc tcaaatgtat tatctgttga tggttctaac ttattagtag 4740

ttagcgcccc taaagatatt ttagataacc ttccgcaatt tctttctact gttgatttgc 4800

caactgacca gatattgatt gaaggattaa ttttcgaggt tcagcaaggt gatgctttag 4860

attttcctt tgctgctggc tctcagcgcg gcactgttgc tggtggtgtt aatactgacc 4920

gtctaacctc tgttttatct tctgcgggtg gttcgttcgg tatttttaac ggcgatgttt 4980

tagggctatc agttcgcgca ttaaagacta atagccattc aaaaatattg tctgtgcctc 5040

gtattcttac gctttcaggt cagaagggtt ctatttctgt tggccagaat gtccctttta 5100

ttactggtcg tgtaactggt gaatctgcca atgtaaataa tccattcag acggttgagc 5160

gtcaaaatgt tggtatttct atgagtgttt ttcccgttgc aatggctggc ggtaatattg 5220

ttttagatat aaccagtaag gccgatagtt tgagttcttc tactcaggca agtgatgtta 5280

ttactaatca aagaagtatt gcgacaacgg ttaatttgcg tgatggtcag actcttttgc 5340

tcggtggcct cactgattac aaaaacactt ctcaagattc tggtgtgccg ttcctgtcta 5400

aaatcccttt aatcggcctc ctgtttagct cccgttctga ttctaacgag gaaagcacgt 5460

tgtacgtgct cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg 5520

ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 5580

ttcgctttct tcccttcctt tctcgccacg ttctccggct ttccccgtca agctctaaat 5640

cggggggagct cgagcttact ccccatcccc ctgttgacaa ttaatcatcg gctcgtataa 5700

tgtgtggaat tgtgagcgga taacaatttc ttaatggaaa cttcctcatg aaaaagtctt 5760

tagttcttaa agcatctgtt gctgttgcga ctcttgttcc tatgctaagc tttgccaacg 5820

tccctgcaga aggtgatgac ccggctaaag ctgcttttga ctctcttcag gcttctgcta 5880

ctgaatacat cggctacgct tgggctatgg tggttgttat cgttggtgct actattggca 5940

tcaaactttt caaaaaattc acttctaaag cgtcttaatg aactcagata cccagcccgc 6000

ctaatgagcg ggcttttttt taagctagct tatgatgatg atgtgcttaa aaacttactc 6060

aatggctggt ttatgcatat cgcaatacat gcgaaaaacc taaaagagct tgccgataaa 6120

aaaggccaat ttattgctat ttaccgcggc tttttatga gcttgaaaga taaataaaat 6180

agataggttt tatttgaagc taaatcttct ttatcgtaaa aaatgccctc ttgggttatc 6240

aagagggtca ttatatttcg cggaataaca tcatttggtg acgaaataac taagcacttg 6300

tctcctgttt actcccctga gcttgagggg ttaacatgaa ggtcatcgat agcaggataa 6360

taatacagta aaacgctaaa ccaataatcc aaatccagcc atcccaaatt ggtagtgaat 6420

gattataaat aacagcaaac agtaatgggc caataacacc ggttgcattg gtaaggctca 6480

ccaataatcc ctgtaaagca ccttgctgat gactctttgt ttggatagac atcactccct 6540

gtaatgcagg taaagcgatc ccaccaccag ccaataaaat taaaacaggg aaaactaacc 6600

aaccttcaga tataaacgct aaaaaggcaa atgcactact atctgcaata aatccgagca 6660

gtactgccgt tttttcgccc catttagtgg ctattcttcc tgccacaaag gcttggaata 6720

ctgagtgtaa aagaccaaga cccgctaatg aaaagccaac catcatgcta ttccatccaa 6780

aacgattttc ggtaaatagc accccacaccg ttgcgggaat ttggcctatc aattgcgctg 6840

aaaaataaat aatcaacaaa atgggcatcg ttttaaataa agtgatgtat accgaattcg 6900

attgcgtctc aacccctact tcggtatctg tattatcacg tgtatttttg gtttcacgga 6960

accaaaacat aaccacaagg aaagtgacaa tatttagcaa cgcagcgata aaaaagggac 7020

tatgcggtga aatctctcct gcaaaaccac caataatagg ccccgctatt aaaccaagcc 7080

caaaacttgc ccctaaccaa ccgaaccact tcacgcgttg agaagctgag gtggtatcgg 7140

caatgaccga tgccgcgaca gccccagtag ctcctgtgat ccctgaaagc aaacggccta 7200

aatacagcat ccaaagcgca cttgaaaaag ccagcaataa gtaatccagc gatgcgccta 7260

ttaatgacaa caacagcact gggcgccgac caaatcggtc agacattttt ccaagccaag 7320

gagcaaagat aacctgcatt aacgcataaa gtgcaagcaa tacgccaaag tggttagcga 7380

tatcttccga agcaataaat tcacgtaata acgttggcaa gactggcatg ataaggccaa 7440

tccccatggc atcgagtaac gtaattacca atgcgatctt tgtcgaacta ttcatttcac 7500

ttttctctat cactgatagg gagtggtaaa ataactctat caatgataga gtgtcaacaa 7560

aaattaggaa ttaatgatgt ctagattaga taaaagtaaa gtgattaaca gcgcattaga 7620

gctgcttaat gaggtcggaa tcgaaggttt aacaacccgt aaactcgccc agaagctagg 7680

tgtagagcag cctacattgt attggcatgt aaaaaataag cgggctttgc tcgacgcctt 7740

agccattgag atgttagata ggcaccatac tcacttttgc cctttagaag gggaaagctg 7800

gcaagatttt ttacgtaata acgctaaaag ttttagatgt gctttactaa gtcatcgcga 7860

tggagcaaaa gtacatttag gtacacggcc tacagaaaaa cagtatgaaa ctctcgaaaa 7920

tcaattagcc tttttatgcc aacaaggttt ttcactagag aatgcattat atgcactcag 7980

cgctgtgggg cattttactt taggttgcgt attggaagat caagagcatc aagtcgctaa 8040

agaagaaagg gaaacaccta ctactgatag tatgccgcca ttattacgac aagctatcga 8100

attatttgat caccaaggtg cagagccagc cttcttattc ggccttgaat tgatcatatg 8160

cggattagaa aaacaactta aatgtgaaag tgggtcttaa aagcagcata acctttttcc 8220

gtgatggtaa cttcacggta accaagatgt cgagttaacc accctttaga ttcataaagc 8280

gaaaataatg cggctccaac gtacccacct aaatggaaac ggcgttcact ccaatctaaa 8340

cacgcacaac agattttacg tgaatgtttg gaaggaacgt caattcccat ttcatgaaaa 8400

tattgaatac cacttaatgt gatcattgaa ccattttcag tgatccatg ctgttgacaa 8460

agggaatcat agatcccttt agggttccga tttagtgctt tacggcacct cgacctccaa 8520

aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 8580

cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 8640

ctcacaacta actcggccta ttcttttgat ttataaggat ttttgtcatt ttctgcttac 8700

tggttaaaaa ataagctgat ttaacaaata tttaacgcga aatttaacaa aacattaacg 8760

tttacaattt aaatatttgc ttatacaatc atcctgtttt tggggctttt ctgattatca 8820

atcggggtac atatgattga catgctagtt ttacgattac cgttcatcga ttctcttgtt 8880

tgctccagac tttcaggtaa tgacctgata gcctttgtag acctctcaaa aatagctacc 8940

ctctccggca tgaatttatc agctagaacg gttgaatatc atattgacgg tgatttgact 9000

gtctccggcc tttctcaccc gtttgaatct ttgcctactc attackccgg cattgcattt 9060

aaaatatatg agggttctaa aaatttttat ccctgcgttg aaattaaggc ttcaccagca 9120

aaagtattac agggtcataa tgtttttggt acaaccgatt tagctttatg ctctgaggct 9180

ttattgctta attttgctaa ctctctgcct tgcttgtacg atttattgga tgtt 9234

<210> 8

<211> 26

<212>DNA

<213> artificial design

<400> 8

gccgacgtgg cccggcctct ggggcc 26

<210> 9

<211> 15

<212> PRT

<213> artificial design

<400> 9

Ala Arg Glu Tyr Gly Thr Arg Phe Ser Leu Ile Gly Gly Tyr Arg

1 5 10 15

<210> 10

<211> 9257

<212>DNA

<213> artificial design

<400> 10

aacgctacta ccttagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60

atagctaaac aggttatga ccatttgcga aatgtatcta atggtcaaac taaatctact 120

cgttcgcaga attgggaatc aactgttaca tggaatgaaa cttccagaca ccgtacttta 180

gttgcatatt taaaacatgt tgaactacag caccagattc agcaattaag ctctaagcca 240

tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactgtctaa tcctgacctg 300

ttggaatttg cttccggtct ggttcgcttt gaggctcgaa ttgaaacgcg atatttgaag 360

tctttcgggc ttcctcttaa tctttttgat gcaattcgct ttgcttctga ctataataga 420

cagggtaaag acctgatttt tgattatgg tcattctcgt tttctgaact gtttaaagca 480

tttgagggggg attcaatgaa tatttatgac gattccgcag tatggacgc tatccagtct 540

aaacatttta caattaccccc 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 aaaccgtctc aagcgcaatt tactacccgt tctggtgttt 900

ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960

aatatccggt gcttgtcaag attackctcg 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 tctttcgctg ctgagggtga 1380

cgatcccgca aaagcggcct ttgactccct gcaagcctca gcgaccgaat atatcggtta 1440

tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1500

attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560

tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc 1620

tattctcact cggccgacgt ggcccggcct ctggggccga aactgttgaa agttgtttag 1680

caaaacctca tacagaaaat tcatttacta acgtctggaa agacgacaaa actttagatc 1740

gttacgctaa ctatgagggc tgtctgtgga atgctacagg cgttgtggtt tgtactggtg 1800

acgaaactca gtgttacggt acatgggttc ctattgggct tgctatccct gaaaatgagg 1860

gtggtggctc tgagggtggc ggttctgagg gtggcggttc tgagggtggc ggtactaaac 1920

ctcctgagta cggtgataca cctattccgg gctatactta tatcaaccct ctcgacggca 1980

cttatccgcc tggtactgag caaaaccccg ctaatcctaa tccttctctt gaggagtctc 2040

agcctcttaa tactttcatg tttcagaata ataggttccg aaataggcag ggtgcattaa 2100

ctgtttatac gggcactgtt actcaaggca ctgaccccgt taaaacttat taccagtaca 2160

ctcctgtatc atcaaaagcc atgtatgacg cttactggaa cggtaaattc agagactgcg 2220

ctttccattc tggctttaat gaggatccat tcgtttgtga atatcaaggc caatcgtctg 2280

acctgcctca acctcctgtc aatgctggcg gcggctctgg tggtggttct ggtggcggct 2340

ctgagggtgg cggctctgag ggtggcggtt ctgagggtgg cggctctgag ggtggcggtt 2400

ccggtggcgg ctccggttcc ggtgattttg attatgaaaa aatggcaaac gctaataagg 2460

gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc tgacgctaaa ggcaaacttg 2520

attctgtcgc tactgattac ggtgctgcta tcgatggttt cattggtgac gtttccggcc 2580

ttgctaatgg taatggtgct actggtgatt ttgctggctc taattcccaa atggctcaag 2640

tcggtgacgg tgataattca cctttaatga ataatttccg tcaatatta ccttctttgc 2700

ctcagtcggt tgaatgtcgc ccttatgtct ttggcgctgg taaaccatat gaattttcta 2760

ttgattgtga caaaataaac ttattccgtg gtgtctttgc gtttctttta tatgttgcca 2820

cctttatgta tgtattttcg acgtttgcta acatactgcg taataaggag tcttaatcat 2880

gccagttctt ttgggtattc cgttattatt gcgtttcctc ggtttccttc tggtaacttt 2940

gttcggctat ctgcttactt tccttaaaaa gggcttcggt aagatagcta ttgctatttc 3000

attgtttctt gctcttatta ttgggcttaa ctcaattctt gtgggttatc tctctgatat 3060

tagcgcacaa ttaccctctg attttgttca gggcgttcag ttaattctcc cgtctaatgc 3120

gcttccctgt tttatgtta ttctctctgt aaaggctgct attttcattt ttgacgttaa 3180

acaaaaaatc gtttcttatt tggattggga taaataaata tggctgttta ttttgtaact 3240

ggcaaattag gctctggaaa gacgctcgtt agcgttggta agattcagga taaaattgta 3300

gctgggtgca aaatagcaac taatcttgat ttaaggcttc aaaacctccc gcaagtcggg 3360

aggttcgcta aaacgcctcg cgttcttaga ataccggata agccttctat ttctgatttg 3420

cttgctattg gtcgtggtaa tgattcctac gacgaaaata aaaacggttt gcttgttctt 3480

gatgaatgcg gtacttggtt taatacccgt tcatggaatg acaaggaaag acagccgatt 3540

attgattggt ttcttcatgc tcgtaaattg ggatgggata ttaatttttct tgttcaggat 3600

ttatctattg ttgataaaca ggcgcgttct gcattagctg aacacgttgt ttaattgtcgc 3660

cgtctggaca gaattacttt accctttgtc ggcactttat attctcttgt tactggctca 3720

aaaatgcctc tgcctaaatt acatgttggt gttgttaaat atggtgattc tcaattaagc 3780

cctactgttg agcgttggct ttatactggt aagaatttat ataacgcata tgacactaaa 3840

caggcttttt ccagtaatta tgattcaggt gtttaattcat atttaaccccc ttattatca 3900

cacggtcggt atttcaaacc attaaattta ggtcagaaga tgaaattaac taaaatatat 3960

ttgaaaaagt tttctcgcgt tctttgtctt gcgataggat ttgcatcagc atttacatat 4020

agttatataa cccaacctaa gccggaggtt aaaaaggtag tctctcagac ctatgatttt 4080

gataaattca ctattgactc ttctcagcgt cttaatctaa gctatcgcta tgttttcaag 4140

gattctaagg gaaaattaat taatagcgac gatttacaga agcaaggtta ttccatcaca 4200

tatattgatt tatgtactgt ttcaattaaa aaaggtaatt caaatgaaat tgttaaatgt 4260

aattaatttt gttttcttga tgtttgtttc atcatcttct tttgctcaag taattgaaat 4320

gaataattcg cctctgcgcg atttcgtgac ttggtattca aagcaaacag gtgaatctgt 4380

tattgtctca cctgatgtta aaggtacagt gactgtatat tcctctgacg ttaagcctga 4440

aaatttacgc aatttcttta tctctgtttt acgtgctaat aattttgata tggttggctc 4500

aattccttcc ataattcaga aatataaccc aaatagtcag gattatattg atgaattgcc 4560

atcatctgat attcaggaat atgatgataa ttccgctcct tctggtggtt tctttgttcc 4620

gcaaaatgat aatgttactc aaacatttaa aattaataac gttcgcgcaa aggatttaat 4680

aagggttgta gaattgtttg ttaaatctaa tacatctaaa tcctcaaatg tattatctgt 4740

tgatggttct aacttattag tagttagcgc ccctaaagat attttagata accttccgca 4800

atttctttct actgttgatt tgccaactga ccagatattg attgaaggat taattttcga 4860

ggttcagcaa ggtgatgctt tagatttttc ctttgctgct ggctctcagc gcggcactgt 4920

tgctggtggt gttaatactg accgtctaac ctctgtttta tcttctgcgg gtggttcgtt 4980

cggtattttt aacggcgatg ttttagggct atcagttcgc gcattaaaga ctaatagcca 5040

ttcaaaaata ttgtctgtgc ctcgtattct tacgctttca ggtcagaagg gttctatttc 5100

tgttggccag aatgtccctt ttaattactgg tcgtgtaact ggtgaatctg ccaatgtaaa 5160

taatccattt cagacggttg agcgtcaaaa tgttggtatt tctatgagtg tttttcccgt 5220

tgcaatggct ggcggtaata ttgttttaga tataaccagt aaggccgata gtttgagttc 5280

ttctactcag gcaagtgatg ttattactaa tcaaagaagt attgcgacaa cggttaattt 5340

gcgtgatggt cagactcttt tgctcggtgg cctcactgat tacaaaaaca cttctcaaga 5400

ttctggtgtg ccgttcctgt ctaaaatccc tttaatcggc ctcctgttta gctcccgttc 5460

tgattctaac gaggaaagca cgttgtacgt gctcgtcaaa gcaaccatag tacgcgccct 5520

gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 5580

ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttctccg 5640

gctttccccg tcaagctcta aatcggggga gctcgagctt actccccatc cccctgttga 5700

caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat ttcttaatgg 5760

aaacttcctc atgaaaaagt ctttagttct taaagcatct gttgctgttg cgactcttgt 5820

tcctatgcta agctttgcca acgtccctgc agaaggtgat gacccggcta aagctgcttt 5880

tgactctctt caggcttctg ctactgaata catcggctac gcttgggcta tggtggttgt 5940

tatcgttggt gctactattg gcatcaaact tttcaaaaaa ttcacttcta aagcgtctta 6000

atgaactcag atacccagcc cgcctaatga gcgggctttt ttttaagcta gcttatgatg 6060

atgatgtgct taaaaactta ctcaatggct ggtttatgca tatcgcaata catgcgaaaa 6120

acctaaaaga gcttgccgat aaaaaaggcc aatttattgc tattaccgc ggctttttat 6180

tgagcttgaa agataaataa aatagatagg ttttatttga agctaaatct tctttatcgt 6240

aaaaaatgcc ctcttgggtt atcaagaggg tcatttatatt tcgcggaata acatcatttg 6300

gtgacgaaat aactaagcac ttgtctcctg tttactcccc tgagcttgag gggttaacat 6360

gaaggtcatc gatagcagga taataataca gtaaaacgct aaaccaataa tccaaatcca 6420

gccatcccaa attggtagtg aatgattata aataacagca aacagtaatg ggccaataac 6480

accggttgca ttggtaaggc tcaccaataa tccctgtaaa gcaccttgct gatgactctt 6540

tgtttggata gacatcactc cctgtaatgc aggtaaagcg atcccaccac cagccaataa 6600

aattaaaaca gggaaaacta accaaccttc agatataaac gctaaaaagg caaatgcact 6660

actatctgca ataaatccga gcagtactgc cgttttttcg ccccatttag tggctattct 6720

tcctgccaca aaggcttgga atactgagtg taaaagacca agacccgcta atgaaaagcc 6780

aaccatcatg ctattccatc caaaacgatt ttcggtaaat agcacccaca ccgttgcggg 6840

aatttggcct atcaattgcg ctgaaaaata aataatcaac aaaatgggca tcgttttaaa 6900

taaagtgatg tataccgaat tcgattgcgt ctcaacccct acttcggtat ctgtattatc 6960

acgtgtatttttggtttcac ggaaccaaaa cataaccaca aggaaagtga caatatttag 7020

caacgcagcg ataaaaaagg gactatgcgg tgaaatctct cctgcaaaac caccaataat 7080

aggccccgct attaaaccaa gcccaaaact tgcccctaac caaccgaacc acttcacgcg 7140

ttgagaagct gaggtggtat cggcaatgac cgatgccgcg acagccccag tagctcctgt 7200

gatccctgaa agcaaacggc ctaaatacag catccaaagc gcacttgaaa aagccagcaa 7260

taagtaatcc agcgatgcgc ctattaatga caacaacagc actgggcgcc gaccaaatcg 7320

gtcagacatt tttccaagcc aaggagcaaa gataacctgc attaacgcat aaagtgcaag 7380

caatacgcca aagtggttag cgatatcttc cgaagcaata aattcacgta ataacgttgg 7440

caagactggc atgataaggc caatccccat ggcatcgagt aacgtaatta ccaatgcgat 7500

ctttgtcgaa ctattcattt cacttttctc tatcactgat agggagtggt aaaataactc 7560

tatcaatgat agagtgtcaa caaaaattag gaattaatga tgtctagatt agataaaagt 7620

aaagtgatta acagcgcatt agagctgctt aatgaggtcg gaatcgaagg tttaacaacc 7680

cgtaaactcg cccagaagct aggtgtagag cagcctacat tgtattggca tgtaaaaaat 7740

aagcgggctt tgctcgacgc cttagccatt gagatgttag ataggcacca tactcacttt 7800

tgccctttag aaggggaaag ctggcaagat tttttacgta ataacgctaa aagttttaga 7860

tgtgctttac taagtcatcg cgatggagca aaagtacatt taggtacacg gcctacagaa 7920

aaacagtatg aaactctcga aaatcaatta gcctttttat gccaacaagg tttttcacta 7980

gagaatgcat tatatgcact cagcgctgtg gggcatttta ctttaggttg cgtattggaa 8040

gatcaagagc atcaagtcgc taaagaagaa agggaaacac ctactactga tagtatgccg 8100

ccattattac gacaagctat cgaattattt gatcaccaag gtgcagagcc agccttctta 8160

ttcggccttg aattgatcat atgcggatta gaaaaacaac ttaaatgtga aagtgggtct 8220

taaaagcagc ataacctttt tccgtgatgg taacttcacg gtaaccaaga tgtcgagtta 8280

accaccccttt agattcataa agcgaaaata atgcggctcc aacgtaccca cctaaatgga 8340

aacggcgttc actccaatct aaacacgcac aacagatttt acgtgaatgt ttggaaggaa 8400

cgtcaattcc catttcatga aaatattgaa taccacttaa tgtgatcatt gaaccatttt 8460

cagtgatcca ttgctgttga caaagggaat catagatccc tttagggttc cgattagtg 8520

ctttacggca cctcgacctc caaaaacttg atttgggtga tggttcacgt agtgggccat 8580

cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 8640

tcttgttcca aactggaaca acactcacaa ctaactcggc ctattctttt gatttataag 8700

gatttttgtc attttctgct tactggttaa aaaataagct gatttaacaa atatttaacg 8760

cgaaatttaa caaaacatta acgtttacaa tttaaatatt tgcttataca atcatcctgt 8820

ttttggggct tttctgatta tcaatcgggg tacatatgat tgacatgcta gttttacgat 8880

taccgttcat cgattctctt gtttgctcca gactttcagg taatgacctg atagcctttg 8940

tagacctctc aaaaatagct accctctccg gcatgaattt atcagctaga acggttgaat 9000

atcatattga cggtgatttg actgtctccg gcctttctca cccgtttgaa tctttgccta 9060

ctcattactc cggcattgca tttaaaatat atgagggttc taaaaatttt tatccctgcg 9120

ttgaaattaa ggcttcacca gcaaaagtat tacagggtca taatgttttt ggtacaaccg 9180

atttagcttt atgctctgag gctttattgc ttaattttgc taactctctg ccttgcttgt 9240

acgattatt ggatgtt 9257

<210> 11

<211> 43

<212>DNA

<213> artificial design

<400> 11

ccacgctgcg caagctcaaa attataagga agatgccagg caa 43

<210> 12

<211> 51

<212>DNA

<213> artificial design

<400> 12

tgcgatagcc tccgatgagg ctaaacctgg tgccatactc cctcgcttcg t 51

Claims (5)

1. A bifunctional phage, characterized in that it is prepared by the following method: (1) Using a phage polypeptide library, screening out the specific binding peptide WR for human vascular endothelial growth factor through biopanning; (2) The WR polypeptide in step (1) and the AR polypeptide capable of specifically recognizing orthotopic breast cancer tumors are respectively displayed on the p3 and p8 proteins of the double display phage fd388 to form a bifunctional phage fd388-AR-WR. 2. The bifunctional phage according to claim 1, wherein said step 1 comprises the following substeps: (1.1) Dilute the VEGF protein into four gradient concentrations of 20g/mL, 4g/mL, 0.8g/mL, and 0.16g/mL using 0.1M NaHCO ₃ coating solution. (1.2) Incubate 400 μL of protein solution with a concentration of 20 g/mL in a 24-well cell culture plate at 4° C. overnight. (1.3) Discard the protein solution, add 500 μL of 5 mg/mL BSA solution, and block at 37°C for 2 hours; discard the blocking solution. (1.4) Add 500 μL of Tween-20 in 5% PBST solution and incubate for 6 minutes, discard and repeat 6 times. (1.5) Add 400 μL of PBS containing 10 ¹⁰ pfu of phage library as input, block at 37°C for 1 hour, and discard the supernatant. (1.6) Add 500 μL Tween-20 with a volume concentration of 5% PBST solution and incubate for 6 minutes, discard and repeat 6 times to wash away unbound and non-specifically bound phages. (1.7) Add 400 μL of hydrochloric acid with a pH=2.2 and a concentration of 0.1M and incubate for 5-10 minutes to elute the specifically bound phages; then immediately add 60 μL of a Tris-HCl solution with a pH=9.1 and a concentration of 1M to neutralize, The obtained phages are amplified by infecting bacteria to obtain the amplified phages, and the first round of screening is completed. (1.8) Complete the 2nd to 4th rounds of screening according to steps 1.2-1.7 with the protein solution whose concentration is 4g/mL, 0.8g/mL, and 0.16g/mL in step 1.1, and the input of each round of screening is obtained in the previous round amplified phage. (1.9) After four rounds of screening, some phages specifically binding to VEGF were obtained, and then the genomes of these phages were subjected to DNA sequencing. (1.10) Perform phage ELISA and dot hybridization experiments on the phage and VEGF proteins with high DNA sequencing frequency in step 1.9, and the phage with the highest affinity to VEGF is the phage carrying the WR polypeptide; the amino acid sequence of the WR polypeptide As shown in SEQ ID NO.1. 3. The bifunctional phage according to claim 1, wherein the step 2 is specifically: adding PstI/HindIII enzyme cutting sites to both ends of the double-stranded complementary DNA sequence encoding the WR polypeptide, encoding breast cancer tumor SfiI restriction sites are added to both ends of the DNA sequence of the specific recognition polypeptide (AR, whose amino acid sequence is shown in SEQ ID NO.9). The double-stranded complementary DNA sequence corresponding to the WR polypeptide is shown in SEQ ID NO.2 and SEQ ID NO.11, and the double-stranded complementary DNA sequence corresponding to the AR polypeptide is shown in SEQ ID NO.3 and SEQ ID NO.12; the fd388 The phage display vector was digested with PstI and HindIII, and ligated with the DNA fragment encoding the WR polypeptide with the PstI/HindIII restriction site by T4 ligase; successfully inserted into the fd388-WR of the DNA corresponding to the WR polypeptide, and then carried out using SfiI Enzyme digestion, and ligation with the DNA fragment encoding the AR polypeptide with the SfiI restriction site to form the genome of the fd388-AR-WR bifunctional phage; then transform the genome into Escherichia coli for transcription, translation, and assembly into bifunctional Finally, the constructed phage was added to a solution containing PEG8000 and NaCl for purification, and the volume ratio of the phage to the solution was 6:1; in the solution, the mass concentration of PEG8000 was 16.7%, and the molar concentration of NaCl was 3.3M. The sequence of the phage double display vector is shown in SEQ ID NO.10. 4. The bifunctional phage according to claim 3, wherein the bifunctional phage is prepared by the following method: (a) Based on the BsrGI single restriction site at position 1022 and the BamHI single restriction site at position 2244 of the genome of fd filamentous phage fUSE55, a 1223bp gene fragment containing recombinant gene3 was obtained by double digestion with BsrGI/BamHI, said The sequence of fUSE55 is shown in SEQ ID NO.6. (b) Based on the BsrG I single restriction site at position 1022 and the BamHI single restriction site at position 2221 in the genome of fd filamentous bacteriophage f88-4. The wild gene3 of f88-4 was knocked out by BsrGI/BamHI double enzyme digestion, and the remaining 8034bp gene fragment was obtained. The sequence of f88-4 is shown in SEQ ID NO.7. (c) Construction of dual display vector fd388: The 1223bp gene fragment obtained in step 1 and the 8034bp gene fragment obtained in step 2 were ligated at a ratio of 1:3 by T4 DNA ligase overnight at 16°C. (d) Transform the ligation product into JM109 competent cells, and smear it on a tetracycline-resistant plate overnight at 37°C to obtain positive single clones, thereby obtaining a phage double display vector fd388 and fd388 with a length of 9257 bp that can display two polypeptides ectopically The sequence of is shown in SEQ ID NO.10. 5. A use of the bifunctional phage according to claim 1 in the preparation of a drug for inhibiting tumor growth.