CN1224631C - 11 segment human digestive tract tumoure blood vessel speicfic conjugated cyclopeptide GX series - Google Patents
11 segment human digestive tract tumoure blood vessel speicfic conjugated cyclopeptide GX series Download PDFInfo
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- CN1224631C CN1224631C CN 200410026137 CN200410026137A CN1224631C CN 1224631 C CN1224631 C CN 1224631C CN 200410026137 CN200410026137 CN 200410026137 CN 200410026137 A CN200410026137 A CN 200410026137A CN 1224631 C CN1224631 C CN 1224631C
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Abstract
The present invention discloses an 11 segment human digestive tract tumoure blood vessel speicfic conjugated cyclopeptide GX series. The present invention uses an annular 7 peptide library of a stochastic bacteriophage and a human metastatic tumor model under an immunosuppression mouse kidney coating to successfully sieve annular 7 peptides GX1 to GX11 which can be highly and specifically combined with the blood vessels of human squamous gullet cancer and human adenocarcinoma of stomaches with poor differentiation. The specificity of a series of the 7 peptides combined with human digestive canal tumor tissue blood vessels can be displayed through transplanted tumor tissue plaque titration, the immunohistochemical staining of transplanted tumor tissues and the tissues of human gastric cancer and oesophagus cancer, and a competition restraining experiment for showing bacteriophage in bodies through GX1 synthetic peptide and GX1. The uniqueness of the protein sequences is confirmed by sequence measurement. The polypeptide sequences have great potential value in the aspects of developing the target therapy of tumor blood vessels and the specific molecule marker reagents of the tumor blood vessels.
Description
Technical field
The invention belongs to field of biomedicine technology, relate to screening blood vessel-specific binding peptide in the body, particularly 11 sections people's digestive tract tumor blood vessel-specifics are in conjunction with cyclic peptide series.
Background technology
Though cancer of the stomach had gradually the trend that reduces in the last few years, it still occupies the 3rd of compatriots' ten big cancer cause of the death ranks, and traditional therapeutic modality is still operation, chemotherapy and radiation.1971, professor Folkman proposed tumor growth and shifts the theory that depends on blood vessel, and the blocking-up tumor-blood-vessel growth becomes a kind of method that suppresses tumor growth.Compare with traditional method, the tumor vessel suppression therapy has the following advantages: 1. be difficult for producing resistance; 2. medicine is easy to arrive target site; 3. suppress metastases; 4. hereditary-less toxicity; 5. act on strong etc.As the angiostatin (U.S.Patent No.5,801,012) that is carrying out the clinical experiment of 3 phases at present, Endostatin (U.S.Patent No.6,174,861).Yet though the tumor vessel suppression therapy has remarkable advantages, come with some shortcomings: 1. the tumor vessel specific aim is not strong yet, and tumour is difficult to be effected a radical cure fully, also has the danger of potential inhibition normal endothelial cell.Especially under situations such as wound healing, menstrual cycle, gestation, should not use.2. expression efficiency is low, and curative effect is limited.3. enter the most vasculogenesis of clinical experiment at present and suppress molecule to enter expression in vivo efficient low, can not keep treatment concentration in the tumor vessel part.Use repeatedly easily to cause immune response, and cost an arm and a leg, limited their clinical application.
Because tumor vessel and normal blood vessels all are very different on form and function, as the endotheliocyte volume-diminished, form is irregular, and the intercellular substance increases, the vessel wall that has even do not have endotheliocyte, vessel wall permeability increase or the like.The what is more important tumor vessel is expressed some specific proteinses different with normal blood vessels that are called as " vascular zipcodes ".The specific molecular that these are not only expressed in normal cell in the tumor vessel expression or trace level is expressed is the crucial target of tumor vessel treatment just.
Detect the specific molecular marker of tumor vessel and exist a lot of difficulties,, do not exist in the normal blood vessels because these molecules only are present in tumor vessel.And the amount of expressing is less, be difficult to detect, more can't purifying.Just because of this, the successful Application of phage random peptide library has solved this difficult problem (US.Patent.No.5,866,363).Adopt the phage random peptide library technology, can be under the indefinite situation of target molecules structure of desire screening, need not to know in advance that screen in interactional zone and interactional character between them, thus filter out some can with target molecule bonded specific polypeptide.Arap.W. wait adopt the phage random peptide library scientific discovery can with the little peptide of tumor of prostate blood vessel-specific bonded " SMSIARL " (US.Patent.No.6,610,651)
At present, most of screening methods all adopt in-vitro screening, but for tumor vascular endothelial cell, directly separate and vitro culture people tumor vascular endothelial cell, the one, the difficulty of isolation technique operation, the 2nd, long-term vitro culture makes endotheliocyte lose intravital living environment, thereby makes endotheliocyte cell forfeiture tissue specificity.And the nude mice model of human tumor cell line or transplanted tissue's knurl piece is adopted in some intravital experiments of carrying out mostly, also there are some to adopt transgenic mice to carry out, the growth of xenografted cycle is longer, the form and the viability of tumour cell are changed, perhaps lose the humanized, thereby it is perhaps also imperfect to have influence on such experimental result.
Method was set up CTX immunosuppression Balb/c mouse people's cancer of the stomach or esophageal carcinoma transplanted tumor model in 6 days, because the growth of xenografted cycle is shorter, the growth and cause logical again with people's stomach organization blood vessel though the mouse vascular endothelial cell begins to sprout, guaranteed partial blood supply, but, the vascular endothelial cell of knurl piece inside was not replaced by mouse vascular endothelial cell institute in 6 days fully, had also kept the humanized mostly.This institute once utilized 6 days methods to set up CTX immunosuppression Balb/c mouse people cancer of the stomach transplanted tumor model cancer of the stomach was screened, but it is not really perfect to be used for next step method for screening because of the picking clone, so the specificity of little peptide is not really desirable, and can not carry out little peptide carried out that competition suppresses experiment in the body that activity in vivo detects.The applicant adopts the successful ring-type 7 peptide GX series that can combine with cancer of the stomach and esophageal carcinoma blood vessel-specific that filter out in vivo of triage techniques in the improved phage random peptide library body, present result of study shows that this series peptide section can be with the gastric cancer vascular specific combination, and with the debond of normal spleen blood vessel, with liver cancer, angiomatous blood vessel is not seen the specificity combination yet.
Although its sequence of the little peptide of the simulation that the applicant filters out and natural known molecular there is no similarity, can not illustrate that like this it does not have can not deny that with the similar function of natural molecule it does not have the ability that can combine with natural molecule.As filtering out the little peptide of simulation ring-type (U.S.Patent No.5 the small peptide storehouse at random from phage surface, 835,382), though the sequence of its sequence and natural EPO does not also have similarity, but it can combine with EPO acceptor high affinity really, and the function of simulation erythropoietin (EPO).Similarly, filter out and to be significant at aspects such as neoplasm targeted therapies with the digestive tract tumor blood vessel-specific bonding polypeptide.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing phage random peptide library to carry out screening and the active detection of The selection result in the body, and obtain 11 can with the aminoacid sequence of tumor vessel specificity bonded ring-type 7 peptides.
The technical scheme that realizes the foregoing invention purpose is: at first prepare transplanted tumor model under the immunosuppressed mice people tumor tissues kidney peplos, therefrom filter out have can with the polypeptide of the vascular endothelial cell high combination activity of tumour; And utilize plaque to form the specificity homing ability of experiment, immunohistochemical methods detection GX series, the interior competition of tumor-bearing mice body suppressed the combination activity that experiment etc. has detected GX series; Confirm to obtain GX series amino acid sequence of polypeptide, and the uniqueness of proof corresponding sequence.
The present invention selects triage techniques in the phage random peptide library body for use, the specificity of successful acquisition people poorly differentiated adenocarcinoma cancer and people's esophagus squama cancer blood vessel is in conjunction with ring-type 7 peptide GX series, and obtain its unique aminoacid sequence, will be at target treatment to tumor blood vessel, lay a good foundation in the development aspect of tumor vessel specificity molecular marker reagent.
Description of drawings
Fig. 1 is 11 sections aminoacid sequences that can specificity be incorporated into the cyclic peptide on people's digestive tract tumor vascular endothelial cell of the present invention.
Fig. 2 is GX of the present invention
1Sequence compares with the highest protein sequence of its homology.
Fig. 3 is GX of the present invention
2Sequence compares with the highest protein sequence of its homology.
Fig. 4 is GX of the present invention
3Sequence compares with the highest protein sequence of its homology.
Fig. 5 is GX of the present invention
4Sequence compares with the highest protein sequence of its homology.
Fig. 6 is GX of the present invention
5Sequence compares with the highest protein sequence of its homology.
Fig. 7 is GX of the present invention
6Sequence compares with the highest protein sequence of its homology.
Fig. 8 is GX of the present invention
7Sequence compares with the highest protein sequence of its homology.
Fig. 9 is GX of the present invention
8Sequence compares with the highest protein sequence of its homology.
Figure 10 is GX of the present invention
9Sequence compares with the highest protein sequence of its homology.
Figure 11 is GX of the present invention
10Sequence compares with the highest protein sequence of its homology.
Figure 12 is GX of the present invention
11Sequence compares with the highest protein sequence of its homology.
Aminoacid sequence
GX
1：Gly Asn Ser Asn Pro Lys Ser
GX
2：Pro His Asn Leu Thr Lys Leu
GX
3：Tyr Ser Phe Asn Ser Trp Met
GX
4：Pro Asn Pro Asn Asn Ser Thr
GX
5：Tyr Ser Ile Asn Asp Trp His
GX
6：Leu Phe Ala Met Pro Asn Ser
GX
7：Ser Thr Val Ala Thr Ser Gln
GX
8：Tyr Val Thr Pro Tyr Asp Ile
GX
9：Ser Pro Ala Asn Leu Phe Thr
GX
10：Pro Met Asn Ala Asp Asn Leu
GX
11：Ser Arg His Asp Leu Asn Ser
Embodiment
The present invention is described in further detail below in conjunction with accompanying drawing.
Tumor vascular endothelial cell molecular surface marker is the basis of tumor vessel treatment, for this reason, the applicant selects triage techniques in the phage random peptide library body for use, the specificity of successful acquisition lineup poorly differentiated adenocarcinoma cancer and people's esophagus squama cancer blood vessel is in conjunction with ring-type 7 peptide GX series, and utilize plaque to form the specificity homing ability of experiment, immunohistochemical methods detection GX series, the interior competition of tumor-bearing mice body suppresses experiment and waits the combination that has detected GX series active, and this series GX peptide has the higher ability in conjunction with the digestive tract tumor vascular endothelial cell.
1. technical scheme and route thereof
1.1 the foundation of transplanted tumor model under people's tumor tissues kidney peplos:
1.1.1 prepare before the art:
Balb/c mouse inbred lines male and female half and half (available from The Fourth Military Medical University's Experimental Animal Center), 6 ages in week, body weight 20g-24g, 24h mouse peritoneal injection endoxan (CTX before the experiment, available from the permanent auspicious limited-liability company in Jiangsu) 150mg/kg once, make the CTX immunosuppressed mice.
1.1.2 the processing of fresh cancerous tissue:
In surgical procedure, get the tumor tissues of cancer of the stomach or esophageal carcinoma patient with operation respectively, patient with operation all before art pathological diagnosis be poorly differentiated adenocarcinoma cancer or esophagus squama cancer, after exsomatizing, immediately it is dropped among the serum-free RPMI1640 (available from Gibco BRL company) tumor tissue, removing knot in Bechtop forms and necrotic tissue, soak 15min among two anti-RPMI-1640 (two anti-: the concentration of penicillin is 100U/ml with containing, the concentration of Streptomycin sulphate is 100 μ g/ml), be cut into 2mm then
3About fritter, wash repeatedly with serum-free RPMI1640 again, place RPMI-1640 standby.
1.1.3 kidney peplos is transplanted down:
With reference to Wang Yu method row SRCA method: vetanarcol 150mg/kg (Shanghai chemical reagent packing factory) intraperitoneal injection of anesthesia mouse, sterilization, aseptic condition exposes down; free kidney; the careful separation kidney peplos is implanted the tissue block of well cutting under the mouse kidney peplos, sews up the incision.
1.2 wash in a pan sieve in the phage mouse body
1.2.1 wash in a pan the sieve process in the body:
Postoperative the 6th day is got a lotus knurl BALB/c mouse, vetanarcol 150mg/kg intraperitoneal injection of anesthesia, 75% alcohol immersion 5min.To contain 10
11200 μ l RPMI1640 of pfu C7C phage random peptide library (available from NEWENGLAND BIOLABS company), inject mouse tail vein, get tumor tissue and other control tissue behind the 5min, weigh, adding ice 1ml RPMI1640-PI (containing 1Mm PMSF, 20ug/ml aprotinin, 1ug/ml leupeptin) grinds with tissue homogenizer, homogenate is moved into the 50ml centrifuge tube, add 5ml ice RPMI1640-PI (containing 1%BSA) washing 4 times after.With the static infection 30min of 1ml intestinal bacteria ER2738 (OD=0.5), add 9ml LB nutrient solution incubated at room 30min, mixed solution shop is made (contain 4%X-gal, 5%IPTG), 37 ℃ are spent the night on the LB agarose plate.
1.2.2 the amplification in vitro of phage:
With toothpick about 250 phage clones that present locus coeruleus of picking at random, each clone adds 5mlLB nutrient solution in shaking tube, and 37 ℃ of following 250rpm cultivate 12～14h.Then culture is mixed, 4 ℃ of centrifugal 15min of 10000rpm move into supernatant in the one aseptic triangular pyramidal bottle, add PEG/NaCl (available from Beijing ancient cooking vessel state biotech development center) solution of 1/6 volume, 4 ℃ of standing over night.With 4 ℃ of sedimentation liquids that spend the night in 4 ℃ of centrifugal 15min of following 10000rpm, supernatant discarded.With the resuspended throw out of 1ml RPMI1640, add 160 μ l PEG/NaCl solution once more, ice bath 1h, 4 ℃ of centrifugal 10min of following 10000rpm, supernatant discarded contains 0.02%NaN with 200 μ l
3The resuspended throw out of RPMI1640 solution at (available from Beijing ancient cooking vessel state biotech development center), this is the phage of first round amplification.It is stored in 4 ℃, is used for the next round screening.Wash in a pan sieve 4 in the adenocarcinoma of stomach organizer and take turns, esophagus squama cancerous tissue is washed in a pan sieve 5 and is taken turns.
1.2.3 the selecting and checking order of positive phage clones:
The 4th takes turns after adenocarcinoma of stomach tissue or the 5th takes turns esophagus squama cancerous tissue screening and finish, under the aseptic condition with toothpick at random picking 14 (cancer of the stomach) or 13 (esophageal carcinoma) separate good blue plaques and add to respectively in the 2ml LB nutrient solution, 37 ℃ of following 250rpm cultivate 2h.To get culture supernatant behind 4 ℃ of centrifugal 10min of 10000rpm of phage nutrient solution.The single stranded DNA extraction test kit of producing with Hua Shun company carries out the extraction of phage single-chain DNA, operate by process specifications: in containing the LB nutrient solution of single phage clone, add precipitated liquid, obtain the phage particle precipitation behind the high speed centrifugation, add lysate lytic phage coat protein, through resin purification, wash-out obtains phage single-chain DNA.With the single stranded DNA is template, and the sequencing primer that is provided in the phage random peptide library test kit is provided ,-96gIII, 5 '-
HOCCC TCATAG TTA GCG TAA CG-3 ' delivers to the order-checking of Shanghai biotechnology company limited.The result shows below table and Fig. 1: with its difference called after GX1-GX11, have 2 routine samples not measure in the esophagus cancer.
| Sample number | The sample source | Present sequence | Name |
| A 3，A 4，A 5，A 6， B 3，C 1，D 3，D 4， E 2，E 1，G 1，G 3 | Cancer of the stomach | GNSNPKS | GX 1 |
| A 10，B 1 | Cancer of the stomach | PHNLTKL | GX 2 |
| EB 1 | The esophageal carcinoma | YSFNSWM | GX 3 |
| ED 2/Ee | The esophageal carcinoma | PNPNNST | GX 4 |
| EE 3 | The esophageal carcinoma | YSINDWH | GX 5 |
| EF 1 | The esophageal carcinoma | LPAMPNS | GX 6 |
| EG 1/Ed | The esophageal carcinoma | STVATSQ | GX 7 |
| Eb | The esophageal carcinoma | YPTPYDI | GX 8 |
| Eg | The esophageal carcinoma | SPANLFT | GX 9 |
| Eh | The esophageal carcinoma | PMNADNL | GX 10 |
| Ei | The esophageal carcinoma | SRHDLNS | GX 11 |
1.3 present the evaluation of phage homing ability
1.3.1 locus coeruleus counting
Get and transplant the lotus knurl BALB/c mouse that people's adenocarcinoma of stomach or esophagus squama cancer are arranged, with the GX series mono-clonal that obtains respectively with 10
11Pfu 200 μ l RPMI1640 inject mouse tail vein, and other working method are the same, bluer plaque quantity after spending the night in 37 ℃ of incubators.The result shows: GX
1The blue plaque that obtains from tumor tissue is its 4.6～137.26 times in control tissue; GX
2The blue plaque that obtains from tumor tissue is its 3.98～180.74 times in control tissue; GX
3The blue plaque that obtains from tumor tissue is its 3.01～44.57 times in control tissue; GX
4The blue plaque that obtains from tumor tissue is its 1.43～3.26 times in control tissue; GX
5The blue plaque that obtains from tumor tissue is its 5.16～70.68 times in control tissue; GX
6The blue plaque that obtains from tumor tissue is its 1.69～9.42 times in control tissue; GX
7The blue plaque that obtains from tumor tissue is its 3.47～12.39 times in control tissue; GX
8The blue plaque that obtains from tumor tissue is its 1.18～4.45 times in control tissue; GX
9The blue plaque that obtains from tumor tissue is its 1.15～3.16 times in control tissue; GX
10The blue plaque that obtains from tumor tissue is its 1.28～5.71 times in control tissue; GX
11The blue plaque that obtains from tumor tissue is its 1.44～3.13 times in control tissue.From result GX series good going back to the nest in tumor vascular characteristic arranged all.
1.3.2 immunohistochemical methods
1.3.2.1 the expression of phage M13 albumen in transplanted tumor
Get lotus cancer of the stomach or esophageal carcinoma mouse, the tail vein injects GX series phage 10 respectively
11Pfu, lavation mouse whole body, method is the same.Get tumor tissues and control tissue, 4% paraformaldehyde is fixed, paraffin embedding, section.Adopt conventional SP method dyeing (SP test kit available from Beijing Zhong Shan Bioisystech Co., Ltd), one anti-for mouse anti M13 monoclonal antibody (available from Pharmacia company, working concentration is 1: 80), negative control adopts normal goats serum to replace M13 one anti-, surplus step is the same, observations after the mounting.The result shows: the presenting in the blood vessel that phage all rests on transplanted tumor of GX series, M13 expresses higher, and does not almost have expression on other control tissue.
1.3.2.2VIII the expression of factor related antigen in transplanted tumor
As above method is got the transplanted tumor tissue, and 4% paraformaldehyde is fixed, paraffin embedding, section.Adopt conventional SP method dyeing (SP test kit available from Beijing Zhong Shan Bioisystech Co., Ltd), one anti-ly is the anti-people VIII of rabbit factor polyclonal antibody (available from DAKO company, working concentration is 1: 300), and surplus step is the same, observations after the mounting.The result shows: the colour developing of the VIII factor is stronger in the transplanted tumor, rich blood vessel in the transplanted tumor is described, M13 dyeing confirms also that with the VIII factor related antigen comparative result M13GX series presents phage expression and VIII factor expression scope is similar, illustrate GX series present peptide can specific combination on blood vessel.
1.3.2.3 present the expression of phage GX series on people's cancer of the stomach and esophagus cancer
Get people's poorly differentiated adenocarcinoma cancer or people's esophagus squama cancer sample, paraffin embedding, section.Employing presents phage GX for people's poorly differentiated adenocarcinoma cancer
1With GX
2As one anti-, adopt GX for people's esophagus squama cancer
3～GX
11As one anti-, mouse anti M13 monoclonal antibody is anti-as two, and other programs are identical with the SP method, control tissue employing people liver cancer tissue, people's vascular tumor, people's spleen.Observations shows: GX
1With GX
2Be incorporated on people's poorly differentiated adenocarcinoma cancer blood vessel endothelium, and GX
3～GX
11Be incorporated on people's adenocarcinoma of esophagus vascular endothelial cell, and no specificity combination on other control tissue.GX is described
1, GX
2Present the phage specific combination on people's gastric cancer vascular endotheliocyte, and GX
3～GX
11Present the phage specific combination on people's esophagus squama cancer vascular endothelial cell.
1.3.3 competition suppresses experiment in the body
Below be that the applicant chooses the best GX of experimental result
1Be example, other GX
2～GX
11Carry out according to this method equally;
Synthetic GX
1Polypeptide (biochemical company limited is synthetic by the Shanghai gill), preparation lotus people poorly differentiated adenocarcinoma cancer mouse model, method is the same.Inject GX by mouse tail vein
1Synthetic peptide, dosage is respectively 12.5 μ g, 25 μ g, 50 μ g, 100 μ g, 200 μ g and 400 μ g, behind 5～15min again from tail vein injection GX1 phage 10
11Pfu gets tumor tissue behind the 5min, concrete operation method is the same, bed board, and 37 ℃ are spent the night, and calculate the locus coeruleus number.The result shows: along with GX
1The rising of synthetic peptide dosage, GX
1The plaque number of phage descends, and GX is described
1Present peptide and can block GX
1Present phage and combine, GX is described with tumor vascular
1The peptide specific combination is on tumor vessel.
1.3.4 the amino acid identity analysis of peptide sequence
After determining that order-checking is errorless,, identify amino acid whose homology by following technical scheme.
In non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF Protein Data Bank, carry out amino acid sequence homology analysis (Search for short, nearlyexact matches): the result shows there be not to find the known protein sequence identical with the little peptide of ring-type of GX series in existing protein polypeptide storehouse.GX
1The highest with the homology that is numbered a segment structure in the gi/44554133/gb/EAK06478.1/ sequence, be 100%, but this sequence be agnoprotein in the environment and this sequence for linear, be not the cyclic peptide (Fig. 2) that obtains in our experiment yet.GX
2With the people's liver cancer rise factor that is numbered gi/19923540/ref/NP_060177.2/ of linearity homology being arranged, is linear but people's liver cancer raises the factor, and GX
2Be ring type polypeptide (Fig. 3).GX
3Illusion albumen gi/7497287/pir/T19889 homology with nematode is the highest, and this imagination albumen also belongs to the albumen (Fig. 4) of not clear and definite function and character.GX
4With a segment structure among the illusion albumen gi/19113611/ref/NP_596819 among the Schizosaccharomyces pombe (fission yeast) homology (Fig. 5) is arranged, still, this albumen still belongs to linear structure, but not the ring-type of applicant's test-results.GX
5The partial sequence of agnoprotein gi/44477743/gb/EAJ54860.1/ in 6 peptides in 7 peptides and the environment becomes 100% identical (Fig. 6), but GX
5Ring texture but do not have homologous sequence.GX
67 peptide sequences and a kind of possible metalloprotease gi/32473780/ref/NP_866774.1/ identical (Fig. 7), but this albumen is linear structure, is possible metalloprotease, its concrete function is still not really clear.GX
77 peptide sequences with the partial amino-acid series that is numbered in the imaginary albumen of gi/39593478/emb/CAE61770.1/ 100% homology (Fig. 8) is arranged, but should still belong to linear agnoprotein by imagination albumen.GX
8In 6 peptides and the Turbatrix imaginary albumen that is numbered gi/39597108/emb/CAE59335.1/ 100% homology (Fig. 9) is arranged, still, this albumen still belongs to linear structure, and belongs to the albumen of not clear and definite function and character.GX
9With the agnoprotein that is numbered gi/44043120/gb/EAG81516.1/ in the environment 100% homology (Figure 10) is arranged, but this sequence be agnoprotein in the environment and this sequence for linear, be not the ring texture that obtains in our experiment yet.GX
10There are 6 peptides and imaginary membranin gi/29349446/ref/NP_812949.1/ that 100% homology (Figure 11) is arranged on aminoacid sequence in 7 peptides, but still are linear imaginary albumen.GX
11In 6 peptides be numbered gi/32488243/emb/CAE03063.1/ 100% homology (Figure 12) arranged, but and the situation of the several peptides in front similar, this albumen still is linearity, and GX
11Be a ring-type 7 peptides, so even higher homology is arranged between the two sequence, but actual space structure and function and inequality.Therefore, say accurately: do not find the consistent known protein of homologous, function and structure for these 11 sections ring-type 7 peptides, these 11 sections ring-type 7 peptides belong to new albumen, and they are with tangible relation is arranged between the digestive tract tumor.(seeing accompanying drawing 2-12)
2. technique effect
The present invention adopts phage random ring 7 peptide storehouses screening people's cancer of the stomach or esophageal carcinoma blood vessel-specific cyclic peptide, filters out and can combine ring-type 7 peptide GX with vascular endothelial cell bonded high specific
1～GX
11, organize plaque titration through transplanted tumor, transplanted tumor tissue and people's cancer of the stomach, human esophageal carcinoma immunohistochemical staining, GX
1Synthetic peptide and GX
1Present phage competition inhibition experiment in vivo and all show these series 7 peptides and people's digestive tract tumor tissue blood vessel bonded specificity.The discovery of these cyclic peptide will be at target treatment to tumor blood vessel, and the development aspect of tumor vessel specificity molecular marker reagent will have huge potential value.
In addition, the aminoacid sequence of coding GX series cyclic peptide do not find the gene order identical with the present invention, so the present invention has uniqueness and autonomous intellecture property behind homology analysis.
3. preferred forms
3.1 the structure of targeted drug:
3.1.1 the structure of blood-vessels target sexual gland virus mediation siRNA
Is the gene therapy vector of selectively targeted gastric cancer vascular endothelium with the specificity cyclic peptide of finding among the present invention with the adenovirus pAdeasy system reform, and by siRNA specificity importing cancer of the stomach or the esophageal carcinoma vascular endothelial cell of this carrier with VEGFR, Raf-1, disturb the growth signals of cancer of the stomach or esophageal carcinoma vascular endothelial cell by the RNAi mode, suppress cancer of the stomach or esophageal carcinoma vascular endothelial cell proliferation and migration, finally reach the purpose that suppresses the cancer of the stomach or the esophageal carcinoma.
3.1.2 the structure of the compound peptide of blood-vessels target
With the specificity cyclic peptide found among the present invention with a kind of biologically active peptides
D(KLAKLAK)
2Connect, utilize the ability and the biologically active peptides of cyclic peptide specificity combining with vascular endothelial cell in this discovery
D(KLAKLAK)
2Selective action makes compound peptide target destroy the vascular endothelial cell of the cancer of the stomach or the esophageal carcinoma in the effect of mitochondrial membrane, thereby reaches the purpose that suppresses the cancer of the stomach or the esophageal carcinoma.
3.2 the structure of tumor vessel specificity molecular marker test kit
The specificity cyclic peptide of finding among synthetic the present invention, immune Balb/c mouse or new zealand rabbit prepare monoclonal antibody according to a conventional method, thereby make up the monoclonal antibody reagent kit of tumor vessel specificity molecular marker.
Claims (1)
1. people's digestive tract tumor blood vessel-specific is characterized in that in conjunction with cyclic peptide series, comprises 11 sections cyclic peptide that can specificity be incorporated on people's digestive tract tumor vascular endothelial cell, and concrete aminoacid sequence is as follows:
Gly Asn Ser Asn Pro Lys Ser
Pro His Asn Leu Thr Lys Leu
Tyr Ser Phe Ash Ser Trp Met
Pro Asn Pro Asn Asn Ser Thr
Tyr Ser Ile Asn Asp Trp His
Leu Phe Ala Met Pro Asn Ser
Ser Thr Val Ala Thr Ser Gln
Tyr Val Thr Pro Tyr Asp Ile
Ser Pro Ala Asn Leu Phe Thr
Pro Met Asn Ala Asp Asn Leu
Ser Arg His Asp Leu Asn Ser
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| CN100349914C (en) * | 2005-06-14 | 2007-11-21 | 中国人民解放军第四军医大学 | Human stomache cancer endothelial-cell specific combination short peptide series |
| CN100475836C (en) * | 2005-12-09 | 2009-04-08 | 中国人民解放军军事医学科学院野战输血研究所 | Mesenchymal stem cell affinity peptide screening and use |
| CN101113164B (en) * | 2007-07-12 | 2010-11-24 | 南开大学 | 12 peptide specifically combined with tumor metastasis related protein PRL-3 and uses thereof |
| CN101161674B (en) * | 2007-07-13 | 2010-08-11 | 中国科学院昆明植物研究所 | Cyclopeptide, preparation method and uses thereof |
| US8247378B2 (en) * | 2009-07-29 | 2012-08-21 | The Chinese University Of Hong Kong | Homing peptide for tumor vasculature |
| CN107361024A (en) * | 2017-07-18 | 2017-11-21 | 郑州大学 | A kind of method for building up of esophageal squamous cell carcinoma early stage metastasis model |
| CN112717136B (en) * | 2021-02-19 | 2021-09-14 | 黑龙江中医药大学 | Medicine for treating gastric cancer and preparation method thereof |
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