CN103319605B - A kind of liver targeting peptides and Angiostatin fusion and preparation method thereof and application - Google Patents
A kind of liver targeting peptides and Angiostatin fusion and preparation method thereof and application Download PDFInfo
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- CN103319605B CN103319605B CN201310249186.XA CN201310249186A CN103319605B CN 103319605 B CN103319605 B CN 103319605B CN 201310249186 A CN201310249186 A CN 201310249186A CN 103319605 B CN103319605 B CN 103319605B
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Abstract
The invention discloses a kind of liver targeting peptides and Angiostatin fusion and preparation method thereof and application. Does the present invention select plasmodium circumsporozoite protein in resisting CSP? the conservative I of N end district can with the acceptor of surface of hepatocytes---heparan sulfate proteoglycan HSPG? 19 amino acid (CSP of combination specifically? I-plus), utilize gene engineering method to be merged amino or the carboxyl terminal at Angiostatin, prepare liver targeting peptides and Angiostatin fusion. This fusion energy special target suppresses new vessels in liver and forms, and improves the local concentration of medicine at lesions position, reduces whole body consumption, reduces its toxic and side effect.
Description
Technical field
The invention belongs to medical biotechnology field, more specifically, relate to a kind of liver targeting peptides and Angiogenesis suppress because ofSub-fusion and preparation method thereof and application.
Background technology
Primary hepatoma (HCC) is the incidence of disease the 5th in all cancers, the serious disease of fatal rate second, and morbidityRate increase year after year and lack effective treatment means. Carry out original new drug existing to improving liver cancer treatment with the research for the treatment of new target droneShape is significant. The growth of entity tumor and transfer depend on the proposition of new vessels generation theory, for oncotherapy is openedWard off a new approach. At present, be generated as target spot with tumor vessel, by angiogenesis inhibitor (tumorAngiogenesisinhibitors, TAI) suppress or destroy tumor vessel to generate, cut off tumors of nutrients source and " die of hunger " swellThe cancer new treatment of knurl has become study hotspot.
Tumor vessel generates regulatory mechanism and relates to the tune between Angiogenesis stimulating factor and AngiostatinSave unbalancely, vascular stimulation factor concentration rises or inhibiting factor concentration declines all can cause tumor vascular undue growth,Lead eventually oncogenic infiltration and transfer. Therefore, the methods of the many employings of target treatment to tumor blood vessel at present: one is to suppress short blood vesselGenerate the effect of the factor, comprise VEGF (VEGF), fibroblast growth factor (FGF), epidermal growth factorSon (EGF), TGF (TGF), HGF (HGF), angiogenin (Angiopoietins, AP)Deng; Another kind is to increase the self-sow amount of Angiostatin, comprise angiostatin (Angiostatin, AS),Kringles5, endostatin (Endostatin, ES), THBS1 ,-2 (TSP), TNP-470, blood plateletFactor-4 (PF-4) and NMPI (TIMP), collagen IV enzymatic fragment tumstatin,Canstatin and vastatin etc.
Liver cancer is euangiotic solid tumor, and most of liver cancer has the phenomenon of aberrant angiogenesis hyperplasia. At HCCAnd in the interstitial of periphery, often find multiple angiogenic factors overexpression, anti-angiogenesis is expressed lower, outerIt is the inhibition liver cancer Angiogenesis method of broad research that source property increases Angiostatin.
Current existing more than 40 plant angiogenesis inhibitor enters clinical testing, can be divided into two according to its mechanism of actionLarge class a: class is specific inhibitor, can specificity suppress tumor vascular endothelial cell, and to non-endothelial cell without effect.For example: angiostatin (Angiostatin), endostatin (Endostatin), kringles5, tumstatin,Canstatin, vastatin and thrombospondin-1 (TSP-1) etc., another kind of is nonspecific inhibitor, to endotheliumCell and tumour cell have inhibiting angiogenesis inhibitor, for example: interferon (interferon, IFN)And interleukin 12 (interlukin-12, IL-12) etc., and the active fragment of these Angiostatins, prominentVariant, homologue etc.
At present clinically by multiple Angiostatin or be used for the treatment of alone or in combination many with its active unitPlant tumour. But the easily diffusion degraded in vivo of these Angiostatins, causes relatively average Tissue distribution when treatment,Fail to form effective antitumour concentration at lesions position, in order to reach clinical effectiveness, only have increasing drug dose; But, addHeavy dose of can improve the cost of medicine and cause toxicity, as to physiology shapes such as women physiological period, embryo's formation, wound healingsThere is potential harmful effect in the vascularization under state. The organ targeting that improves Angiostatin is effective by medicineBe delivered to diseased region, can improve the therapeutic index of medicine, reduce its toxic and side effect.
Polypeptide guiding technique is a very fast technology of development in recent years, this technology by can with target site high degree of specificityIn conjunction with small peptide as carrier, to have the material of pharmaceutically active (as radionuclide, chemotherapeutics, toxin, enzyme, biological respinseConditioning agent, gene and virus etc.) as " bullet ", rely on the special compatibility of carrier height, by " bullet " material collection as far as possibleIn in target site, bring into play its biological function. Polypeptide guiding technique is to solve one of difficult problem in drug development for a long time: medicineThe specificity that thing distributes in vivo provides down to increasing the consumption of medicine, the cost that improves medicine and initiation toxicity notDesirable scheme. By the tumor vascular endothelial cell specific binding peptides NGR construction of fusion protein NGR-TNF that is connected with TNF, facingIn bed experiment, NGR-TNF, with respect to natural TNF, the lethality of tumour is significantly improved, and dosage is only 1/ of natural TNF30, there is good potential applicability in clinical practice. Utilize engineered method to build guidance quality interferon IFN-α 2a-NGR, bodyInterior distribution is tested and is shown with respect to common interferon, and guidance quality interferon can specificly be enriched in tumor tissues. And leadThe dosage of tropism's interferon, at 1/3 o'clock of common interferon dosage, can reach the result for the treatment of same with common interferon.
Circumsporozoite protein (Circumsporozoiteprotein, CSP) is that plasmodium sporozoite surface is a kind of importantAntigen, thereby zygoblast utilizes the identification of itself and surface of hepatocytes acceptor interaction, sticks to surface of hepatocytes. Pass through veinInjection, recombinant C SP just can be incorporated into surface of hepatocytes within a few minutes, shows that CSP has efficient and special liver cell targeted.CSP, approximately containing 400 amino acid, is made up of the conservative I of N end district, central duplicate block and the conservative II of C end district. Research in recent years shows CSPNThe conservative I of end district can with the acceptor of surface of hepatocytes---heparan sulfate proteoglycan (heparanSulfateproteoglycans, HSPG) combination specifically, show CSPI district except comprising conserved sequence KLKQP, conservative orderHeparin sulfate binding sequence is also contained in the upstream of row, and the peptide section by this containing KLKQP and heparin sulfate binding sequence is called I-Plus. Ancsin etc. studies confirm that, CSPI-plus is with saturated mode heparin-binding and heparin sulfate gel column, and suppressThe combination of recombinant C SP and heparin gel. CSPI-plus is fixed on Macrogol Ester plastid top system by Robertson etc.Standby one-tenth is containing CSP liposome, thereby experiment in vivo and vitro all shows that it can be specifically in conjunction with HSPGs target hepatic tissue. Mouse vein is givenAfter medicine, within 15 minutes, can remove from mouse peripheral blood rapidly containing CSP liposome, quantitative analysis shows that mouse liver is containing CSP fatThe concentration of plastid is heart, kidney and lungs hundreds of times, is 12 times of spleen. Above result of study prompting CSPI-plus isA good liver targeting vector.
Summary of the invention
The object of the invention is the deficiency existing for prior art, and thin based on the efficient and special liver of CSPI-plusThe efficient anti-angiogenesis effect of born of the same parents' targeting and Angiostatin, provides a kind of liver targeting peptides and Angiogenesis to press downFactor fusion protein processed. This fusion can utilize liver targeting peptides CSPI-plus efficient special liver cell targeted by blood vesselGenerate inhibiting factor guiding liver, specificity suppresses the vascularization of liver liver cancer, can improve the therapeutic index of medicine, reduces its poisonSide effect. Provide thinking and scientific basis for developing clinically target inhibition angiogenesis treatment liver-cancer medicine, to further carryingThe treatment level of high China liver cancer is significant.
Another object of the present invention is to provide the application of liver targeting peptides and Angiostatin fusion.
Object of the present invention is achieved by the following technical programs:
A kind of liver targeting peptides and Angiostatin fusion, its amino acid sequence comprises that a kind of Angiogenesis presses downThe factor processed or its active fragment sequence and CSPI-plus sequence. Angiostatin can press down taking the multiple factor as targetThe formation of blood vessel processed. These factors comprise: MMP, various growth factor or growth factor receptors, endothelial cell etc. Be at presentOnly, existing at least 20 angiogenesis factors and exceed 300 Angiostatins and be found, wherein have at least 32 to press downThe factor processed is present inHuman bodyIn. In theory, as long as thering is inhibiting factor or its active tablet of anti-angiogenesis functionSection can realize the present invention, preferably, Angiostatin of the present invention be selected from angiostatin (Angiostatin,AS), endostatin (Endostatin, ES), human plasminogen kringles5, thrombospondin 1 (TSP-1) orA kind of in Canstatin albumen or any one the active fragment in them. Such as, can be angiostatin and CSPI-Plus sequence connects, and CSPI-plus sequence can be connected in amino or the c-terminus of angiostatin,
Liver targeting peptides of the present invention and Angiostatin antigen-4 fusion protein gene can be by any this areaThe method of knowing produces, for example: and chemical synthesis, or obtain Angiostatin gene from people's fetal hepatocytes cDNA, thenObtain antigen-4 fusion protein gene through SOE-PCR. More preferably in the latter.
A preparation method for liver targeting peptides and Angiostatin fusion, is prepared into by the following methodTo: S1. antigen-4 fusion protein gene builds: utilize amino or the c-terminus of SOE-PCR method at people's Angiostatin geneAdd CSPI-plus gene order; S2. above-mentioned antigen-4 fusion protein gene is built to recombinant expression carrier; S3. by recombinant expressed yearBody transformed host cell, the engineering bacteria of acquisition expressed fusion protein, fermented and cultured; S4. purifies and separates obtains liver targeting peptides and bloodPipe generates inhibiting factor fusion.
For by convenient the antigen-4 fusion protein gene that the obtains recombinant vector that builds, antigen-4 fusion protein gene utilizes in building described in S1SOE-PCR method also adds respectively restriction enzyme site sequence at c-terminus and the aminoterminal of Angiostatin gene.
Preferably, described fusion is liver targeting peptides and human endostatin fusion, and liver targeting peptides and people's endothelium press downThe preparation method of plain fusion protein is as follows: S1. antigen-4 fusion protein gene builds: utilize SOE-PCR method at Human endostatin geneC-terminus add the CSPI-plus gene order of nucleotides as shown in SEQIDNO:23; Utilize SOE-PCR method also to existThe aminoterminal of Human endostatin gene adds the genetic fragment of nucleotides as shown in SEQIDNO:24; S2. by above-mentioned fusion eggWhite gene constructed recombinant expression carrier; S3. by recombinant expression carrier transformed host cell, obtain the engineering of expressed fusion proteinBacterium, fermented and cultured; S4. purifies and separates obtains liver targeting peptides and recombinant human endostatin fusion; Preferably, described in S1, mergeGFP builds concrete steps: first amplify Human endostatin gene with primer sets 1, then adopt with primer sets 2 and primer sets 3Add the CSPI-of nucleotide sequence as shown in SEQIDNO:3 at the c-terminus of Human endostatin gene by SOE-PCR methodPlus gene order, adds the genetic fragment of nucleotides as shown in SEQIDNO:4, primer sets at endostatin gene aminoterminal1 is made up of primer P1 and P2, and primer sets 2 is made up of primer P3 and P4, and primer sets 3 is made up of primer P3 and P5, primer P1, P2,The nucleotide sequence of P3, P4 and P5 is as shown in SEQIDNO:5 ~ 9.
Use ProtParam that CDD program in NCBI server and http://expasy.org website provide,The associated biomolecule such as ProtScale, NPS informatics analysis tool is melted the liver targeting peptides and the recombinant human endostatin that prepareThe physicochemical properties of hop protein, functional domain, hydrophobicity, secondary protein structure carry out forecast analysis. Result shows ES-CSPFor cationic protein, containing 211 amino acid, wherein acidic amino acid residue (Asp+Glu) and alkaline amino acid residue (Arg+ Lys) content is respectively 18 and 27, and molecular weight is 23326.4Da, and theoretical isoelectric point is 9.69, and instability index is39.19, protein is more stable, is 30h at mammal Half-life in vivo, and in yeast and Escherichia coli, the half-life respectively canBe greater than 20h and 10h, GRAVY index (amphipathic index) is-0.465, carries out hydrophilic/hydrophobic analysis aobvious with ProtScaleThe hydrophilic amino acid quantity of showing this albumen is greater than hydrophobic amino acid, is hydrophilic protein, and NPS analyzes and shows this albumen secondary knotStructure, taking alpha-helix, extended chain and random coil as primary structure element, shows, this fusion is applicable to in-vitro recombination expression.
A kind of recombinant expression carrier, inserts liver targeting peptides described above and Angiogenesis suppresses by the MCS of carrierFactor protein gene. The present invention relates to the fusion that a kind of exogenous protein expression system is expressed this invention, for example protokaryonExpression system, yeast expression system, mammal cell line. Preferably, described prokaryotic expression system is e. coli bl21. InstituteStating expression vector is pET21b. Introduce in detail the construction step of recombinant expression carrier as an example of human endostatin example: in successThe prokaryotic expression plasmid of further gene fusion construct on the basis of ground clone's fusion, to the recombinant plasmid successfully constructingPMD20-T-ES-CSP and prokaryotic expression carrier pET21b carry out respectively XhoI and Nde III double digestion and connect with T4DNAConnect after enzyme connects and transform. Through bacterium colony PCR Preliminary Identification, positive clone shakes bacterium, extracts plasmid and carries out XhoI and Nde III pairEnzyme is cut qualification, and result shows that recombinant plasmid contains and expection band of the same size (Fig. 5), through DNA sequencing analysis, without base mistakeJoin, finally determine that recombinant expression plasmid pET21b-ES-CSP successfully constructs.
A kind of recombinant strain, contains recombinant expression carrier as above. Introduce in detail as an example of human endostatin example heavilyThe concrete construction step of group strain is: the recombined pronucleus expression plasmid successfully constructing is transformed to Host Strains E.coliBL21 (DE3),Utilize isopropyl-β-D-thiogalactoside (Isopropyl-β-D-thiogalactopyranoside, IPTG) induction tableReach, adopt lauryl sodium sulfate-poly amic acid gel electrophoresis (SodiumDodecylSulfate-Polyacrylaminegelelectrophoresis, SDS-PAGE), Western blotting (WesternBlot) method his-and-hers watchesReach product and carry out Analysis and Identification, the albumen of result Explicit Expression is destination protein ES-CSP, shows expressing fusion protein success. PointOther on affecting conventional expression condition (inducing temperature, induction thalline initial concentration, derivant pH, the derivant of exogenous gene expressionConcentration, induction time) be optimized, find ES-CSP/pET21b recombinant plasmid in e. coli bl21 (DE3) Host StrainsGood expression condition is: inducing temperature is that 37 DEG C, starter bacteria concentration are that the final concentration of OD600 approximately 0.6, derivant IPTG is0.06mM, induction time are 4 hours. The destination protein of abduction delivering exists with inclusion body form, carrying out ultrasonic bacteria breaking, and bag is collected in washingContain body, with 6M guanidine hydrochloride dissolution inclusion body, then gradient renaturation after dilution. Because destination protein contains 6 × His amino acid, useHisTrapHP affinity column carries out separation and purification, more respectively with washing containing the Tris salt of 50mM, 100mM, 300mM imidazolesDe-liquid wash-out destination protein, Fractional Collections eluent, SDS-PAGE analysis result shows, in the eluent of 100mM imidazoles, containsA large amount of destination proteins, contain destination protein hardly in the eluent of 50mM, 300mM imidazoles. Destination protein after purifying is through desaltingRear concentrated for subsequent use.
Fusion of the present invention comprises and comes from plasmodium falciparum circumsporozoite protein CSPN end I-plus district and have liverCell targeted 19 amino acid sequences and at least one Angiostatin or its activity unit, described blood vesselGeneration inhibiting factor is selected from: angiostatin (Angiostatin, AS) and endostatin (Endostatin, ES), people's fibreThe former kringles5 of lyase, thrombospondin (TSP-1) Arresten, Canstatin and TNF and active fragment thereof, prominentVariant, homologue etc. More preferably, fusion comprises CSPN end I-plus district and has 19 liver cell targeted ammoniaThe novel recombinant human endothelium that base acid sequence (SEQIDNO:1) and amino terminal are added with 9 amino acid sequences (MGGSHHHHH) presses downElement (SEQIDNO:2).
Fusion of the present invention can be prepared by any methods known in the art, for example: chemical synthesis or fromExpression of nucleic acid produces. More preferably, this fusion is expressed and is produced by the fusion (SEQIDNO:3) of this albumen.
Compared with prior art, the present invention has following beneficial effect:
The present invention is creatively by raw to the liver targeting peptides CSPI-plus and the blood vessel that derive from plasmodium circumsporozoite protein in resistingBecome inhibiting factor to merge, utilize efficient special liver cell targeted Angiostatin is led of CSPI-plusLiver, makes it in liver enrichment, thereby improves the specificity of blood vessel inhibiting factor anti-angiogenesis, reaches the effect of Hepatoma therapyReally, reduce whole body consumption, reduce toxicity, improve amount effect ratio.
Brief description of the drawings
Fig. 1 .ES gene magnification and fusion restructuring schematic diagram.
Fig. 2. sleeve type PCR is cloned the nucleotide fragments that contains ES gene; M:DL2000DNAMarker;
1,2,3,4 be respectively taking 2.0,1.5,1.0,0.0 μ lcDNA as template the DNA that contains ES gene of pcr amplificationFragment.
Fig. 3 .SOEPCR restructuring ES-CSP fusion; M::DL2000DNAMarker; 1,2,3,4,5 be respectively with0.0,2.0,1.5,1.0,0.5 μ lES genetic fragment is template, the ES-CSP fusion of SOEPCR amplification.
Fig. 4 .ES-CSP fusion TA clone PCR and double digestion qualification; M:DL10000DNAMarker; 1,2: restructuringPlasmid pMD20-T-ES-CSP double digestion; The ES-CSP fusion of 3:SOEPCR amplification.
Fig. 5. recombined pronucleus expression plasmid ES-CSP/pET21b single endonuclease digestion, double digestion qualification; M:DL10000DNAMarker; 1: expression plasmid pET21b-ES-CSP double digestion; 2: expression plasmid pET21b-ES-CSP single endonuclease digestion.
Fig. 6. the SDS-PAGE of fusion ES-CSP and Western-blotting qualification; M: protein standard Marker;1,3,5, B1: the not recombinant bacterial strain whole bacterial protein of induction; 2,4,6, B2: the recombinant bacterial strain whole bacterial protein of induction.
Fig. 7. fusion ES-CSP is cell targeted to human liver cancer cell HepG2; A:DAPI dyes core; B:PE markAnti-Anti-ES.
Fig. 8. the inhibitory action of fusion ES-CSP to HUVEC.
Fig. 9. the inhibitory action of fusion ES-CSP to HUVEC on cell migration.
Figure 10. fusion ES-CSP suppresses the formation of chick chorioallantoic membrane new vessels.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is made further and being elaborated, but embodiment is not rightThe present invention limits in any form.
The preparation of embodiment 1 liver targeting peptides and endostatin fusion
Liver targeting peptides and ES Fusion gene construction process are shown in Fig. 1
The clone of S1.ES gene
Cell is cultivated and total RNA extracts: fetal hepatocytes L-02 cultivates and containing dual anti-(100U/ml penicillin, 100ug/Ml streptomysin) and 1640 cell culture fluids of 10% hyclone in, be paved with to cell, carry by the total RNA of TIANGENTRNzolGet kit description operation, every 10cm2Area adds 1mlTRNzol, extracts cell total rna.
The detection of RNA: get RNA sample 2 μ L, add sample loading buffer, 80V1.0% agarose gel electrophoresis 20min.EB dyeing, gel imaging instrument detects RNA integrality; Use without the ultra-pure water of RNA enzyme with 1: 50 total RNA product of dilution, nucleic acid eggWhite analyzer reading, concentration and the purity of analysis RNA sample. 1.8≤OD260/OD280≤ 2.0 is pure rna.
Design of primers: the collagen X VIII of announcing according to NCBI and gene order and the plasmodium falciparum of carboxyl terminal ES thereofCSPI-plus gene order, with PrimerPremier5.0 biosoftware design primer P1 ~ P5, primer P1, P2, P3, P4With the nucleotide sequence of P5 as shown in SEQIDNO:25 ~ 29.
P1(20bp):CCGCACCACAGCTCCTACGT（SEQIDNO：25）；P2(20bp):TACTGCACCCTGCCTGACCC（SEQIDNO：26）；P3(55bp):GGAATTCCATATGGGGGGTTCTCATCACCATCACCATCACAGCCACCGCGACTTC（SEQIDNO：27）；P4(55bp):TTTTATGTTTTGGTTTCCTTAATTTCTCGTTGTCCTTGGAGGCAGTCATGAAGCT（SEQIDNO：28）；P5(55bp):GGCCGCTCGAGTTAACCATCCGCTGGTTGCTTTAATTTTTTATGTTTTGGTTTCC（SEQIDNO：29); Sleeve type PCR clone ES gene: extract total RNA from fetal hepatocytes L-02, by the reverse transcription kit of InvitrogenIllustrate and do reverse transcription reaction (taking oligodT as primer), the cDNA obtaining does pcr template, with P1 and P2 for drawingThing, amplification contains ES genetic fragment, and size is the sequence (see figure 2) of 877bp.
To adding item by item following composition in 0.2mlEppendorf pipe:
The preparation of PCR reaction system is all carried out in ice bath, and pcr amplification parameter is: 94 DEG C of denaturation 5min, 94 DEG C of sex changeReaction 60sec, 60 DEG C of annealing reaction 40sec, 72 DEG C of amplified reaction 90sec, carry out 30 circulations, extend 10min in 72 DEG C. WithDNAPurificationKit purified pcr product, DNA sequencing analysis, the gene order of the upper ES of ES gene order and GenebankRow consistent (as Fig. 2).
S2.SOEPCR restructuring ES-CSP fusion (as Fig. 1)
The 877bp sequence increasing taking P1, P2 is as template, with primer P3, P4, P5 amplification ES-CSP fusion. Primer P3For ESN terminal sequence, and ' end adds gene order and the NdeI restriction endonuclease sites of the MGGSHHHHH that encodes, primer 5P4 contains ESC end complementary series and CSPI-plus19 amino acid whose partial sequence of coding, and primer P5 contains identical with P4Sequence, CSPI-plus19 amino acid whose remainder sequence of coding and XhoI restriction endonuclease sites, detailed processAs follows: Mg2+plus
To adding item by item following table ingredients listed in 0.2mlEppendorf pipe:
The preparation of PCR reaction system is all carried out in ice bath, and pcr amplification parameter is: 94 DEG C of reaction of degeneration (RD) 60sec, 67 DEG CAnnealing reaction 40sec, 72 DEG C of amplified reaction 90sec, carry out 10 circulations, take out Eppendorf pipe and add:
94 DEG C of reaction of degeneration (RD) 60sec, 68 DEG C of annealing reaction 40sec, 72 DEG C of amplified reaction 90sec, carry out 25 circulations, in72 DEG C are extended 10min. Use DNAPurificationKit purified pcr product, object fragment be cloned into pMD20-T carrier,Obtain recombinant plasmid pMD20-T-ES-CSP, transform bacillus coli DH 5 alpha, cut mirror through blue hickie screening, bacterium liquid PCR qualification, enzymeAfter fixed and DNA sequencing is analyzed, show to obtain the fusion that contains nrhES and CSPI-plus sequence, the order splicing and combiningAnd direction is entirely true, illustrate that fusion recombinates successfully (as Fig. 3 and Fig. 4), liver targeting peptides and endostatin fusion(ES-CSP) amino acid sequence is as shown in SEQIDNO:1, and its nucleotide sequence is as SEQIDNO:12.
The structure of S3.ES-CSP fusion gene expression plasmid
Recombinant plasmid pMD20-T-ES-CSP and expression vector pET21b are used respectively to restriction enzyme NdeI and XhoICarry out double digestion, separate through 1.2% agarose gel electrophoresis, reclaim kit with Ago-Gel and reclaim ES-CSP fusionFragment and carrier pET21b, then connect and spend the night with T4 ligase, connects product Transformed E .coliDH5 α competent cell, turnsChange bacterial classification and be applied to the flat board that contains ampicillin, cultivate after 16 ~ 18h, picking list bacterium colony carries out bacterium liquid PCR after cultivating, enzyme is cutQualification and DNA sequencing checking (as Fig. 5), result shows that fusion gene expression plasmid pET21b-ES-CSP successfully constructs.
S4. the expression of fusion ES-CSP and qualification
S41. pET21b-ES-CSP plasmid is transformed to the expressive host bacterium E.coliBL21(DE3 having prepared) competenceCell, through ammonia benzyl resistance screening, the positive single bacterium colony of picking pET21b-ES-CSP in 5mlLB fluid nutrient medium, 37 DEG C,220rpm jolting is spent the night, and then this bacterium liquid is inoculated in fresh LB fluid nutrient medium to 37 DEG C, 220rpm in 1:100 ratioJolting is to OD600Approximately 0.6 o'clock, adding final concentration was the IPTG induction 4h of 1.0mmol/L. Centrifugal collection bacterial cell adds 1 × SDS-PAGEBuffer, boils 5min, centrifugal, gets supernatant and carries out SDS-PAGE. SDS-PAGE result shows: after induction, thalline existsNear 23KD, there is obvious band of expression to conform to expection. Because fusion contains 6 × His label, adopt mouse source His monoclonalAntibody is that primary antibodie is carried out WesternBlotting, and result shows having at 23kDa place containing expression of recombinant plasmid bacterial strain after inductionA specific band and not having of induction further confirmed that the albumen of expressing is the destination protein (knot with His labelFruit sees Fig. 6).
S42. the optimization of fusion ES-CSP expression condition and the preparation of activated protein
On affecting conventional expression condition (induction thalline initial concentration, inducing temperature, the inducing culture of exogenous gene expressionPH, derivant concentration, induction time) be optimized. Transformed bacteria is inoculated in to the LB culture medium containing 100 μ g/ml ampicillinsIn, 37 DEG C of shaken cultivation are spent the night. Next day, according to the ratio of volume ratio 1:100, incubated overnight bacterium liquid is joined and 50ml is housed containsShaken cultivation in the 250ml triangular flask of the LB culture medium of 100 μ g/ml ampicillins, grows at cell concentration respectivelyOD600 approximately 0.2,0.4,0.6,0.8,1.0,2.0 o'clock, add respectively derivant IPTG to final concentration be 0.00,0.06,0.12,0.24,0.48,0.96,1.92mmol is placed under 42 DEG C, 37 DEG C, 32 DEG C and 28 DEG C of cultivation temperature and carries out abduction delivering, andReceive bacterium respectively at 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h after induction. Expression product, through 15%SDS-PAGE electrophoresis detection, is tiedFruit shows that pET21b-ES-CSP recombinant plasmid optimum condition of the expression in e. coli bl21 (DE3) Host Strains is: inducing temperatureBe that 37 DEG C, starter bacteria concentration are OD600Approximately 0.6, the final concentration of derivant IPTG is that 0.06mM, induction time are 4 hours.
By the condition induced gene engineering recombinant bacterium after optimizing, expression product, after renaturation, utilizes HisTrapHP parentCarry out affinity chromatography purifying with chromatographic column, RP-HPLC detects the purity of the ES-CSP expressing, and result shows through abduction delivering multipleThe purity of the ES-CSP fusion after property purifying reaches 95%, can carry out next step activity experiment.
The biological information analysis of S43.ES-CSP fusion encoding fusion protein
Use ProtParam that CDD program in NCBI server and http://expasy.org website provide,The physicochemical properties of the associated biomolecule such as ProtScale, NPS informatics analysis tool to fusion, functional domain, hydrophobicity,Secondary protein structure carries out forecast analysis. Result shows that ES-CSP is cationic protein, containing 211 amino acid, wherein acidAmino acid residue (Asp+Glu) and alkaline amino acid residue (Arg+Lys) content are respectively 18 and 27, and molecular weight is23326.4Da, theoretical isoelectric point is 9.69, and instability index is 39.19, and protein is more stable, in mammalian body halfThe phase of declining is 30h, and in yeast and Escherichia coli, the half-life can be greater than respectively 20h and 10h, GRAVY index (amphipathic index) is-0.465, carry out hydrophilic/hydrophobic analysis with ProtScale and show that the hydrophilic amino acid quantity of this albumen is greater than hydrophobic aminoAcid, is hydrophilic protein, and NPS analyzes and shows that this albumen secondary structure is taking alpha-helix, extended chain and random coil as main knotConstitutive element part, shows, this fusion is applicable to in-vitro recombination expression. Use the CDD program in NCBI server to guard knot to itStructure territory is searched for and is shown that this fusion contains ES domain and ligand binding site; And use SWISS-MODEL full-automaticThe space conformation of pattern model prediction ES-CSP, result shows: the template code of mating most finding is: 1bn1D (2.90);The matching degree of sequence is: 99.44%; Evaluating: 5.64e-100, shows that the fusion that the method is expressed has endothelium inhibitionElement ES active structure domain.
S5. fusion ES-CSP is to liver cell targeting effect
The Cell binding experiment of S51.ES-CSP: with the culture medium training containing 5.0,2.5,1.25 μ g/mlES, ES-CSPSupport normal hepatocytes, the heart, spleen, lung and kidney derived cell and human liver cancer cell, select different time point collecting cells, add successivelyTwo of Anti-ES and PE mark resists, and carries out cellular immunofluorescence test, adopts fluorescence microscope and flow cytometer to detect cellThe fluorescence intensity (the results are shown in Figure 7) on surface, the HepG2 cell that contains as seen ES-CSP has shiny red fluorescence, prompting ES-CSPHuman liver cancer cell HepG2 cell is had to higher combination activity.
S52. HepG2 cell lines is injected under Balb/c nude mice armpit, builds hepatocellular carcinoma in nude mice transplantable tumor model,Be divided at random blank (physiological saline) group, ES group, ES-CSP group. By mouse tail vein administration, respectively at after administration 15,30,60, the blood sampling of 120min eye socket, centrifugation serum, ELISA method detects human endostatin concentration in serum. After mouse blood samplingPut to death immediately, get respectively liver, kidney, heart, spleen, lung and tumor tissues and prepare homogenate, ELISA measures human endostatin in each sampleConcentration, and calculate liver target and hepatoma-targeting index. Make Mouse Liver, kidney, heart, spleen, lung and tumor tissue section, add successivelyEnter two of Anti-ES and PE mark and resist, in fluorescence microscopy images analytical system, carry out graphical analysis.
S6. fusion ES-CSP suppresses angiogenesis function
S61.MTT method is measured the inhibitory action of ES-CSP to cell
Taking Human umbilical vein endothelial cells (HUVEC) as research object. The cell of taking the logarithm growth period, adds after cell countIn 96 porocyte culture plates, after cell attachment, add 10.0,5.0,2.5,1.25,0.625,0.312,0.156,0.0 μ g/mlES, ES-CSP, 6 multiple holes, cultivate 48h, and mtt assay is measured the OD of 490nm place value, calculates the growth inhibition ratio of cell. Inhibiting rate (%)=(the average OD of the average OD/ control group of 1-experimental group) × 100%(the results are shown in Figure 8). Result demonstration, ES-CSP can suppressThe propagation of HUVEC, and there is certain concentration dependent.
S62. the impact of Flow cytometry ES-CSP cell growth cycle and apoptosis
Taking Human umbilical vein endothelial cells (HUVEC) as research object, the cell in the growth period of taking the logarithm, adjusts after cell countTo cell concentration be 5 × 104Individual/ml, adds in (2ml/ hole) in 6 porocyte culture plates, after cell attachment, adds respectively necessarily denseES, the ES-CSP of degree, establish blank group, collecting cell, adopts PI mono-Flow Cytometry Assay cycle situation of change of dying, and adoptsThe two Apoptosis by Flow Cytometry situations of dying of AnnexinV and PI.
S63. scratch experiment, Transwell detect the impact of ES-CSP on cell migration
Taking Human umbilical vein endothelial cells (HUVEC) as research object.
Scratch experiment: first use marker pen at 6 orifice plates behind, comparing with ruler, evenly must draw horizontal line, approximately every 0.5 ~1cm together, crosses via hole. Every hole is at least through 5 lines. In hole, add approximately 5 × 105Individual cell. Within second day, comparing with rifle headRuler hangs down as for horizontal line cut behind as far as possible, and it is vertical that rifle head is wanted, and can not tilt. Wash cell 3 times with PBS, remove draw lower carefullyBorn of the same parents, add respectively the free serum culture of the ES serum free medium that contains 1.25 μ g/ml and the ES-CSP that contains 1.25 μ g/mlBase arranges blank simultaneously, does not contain the serum free medium of any material. Put into 37 DEG C, 5%CO2Incubator is cultivated. PressSampling in 0,6,12,24 hours, take pictures (result is as Fig. 9), result shows, ES-CSP has the biology that ES suppresses HUVEC migration and livesProperty.
Transwell: the cell in the growth period of taking the logarithm, being adjusted to cell concentration after cell count is 1 × 106Individual/ml,The indoor cell suspension 100 μ l that add respectively serum-free medium on Transwell cell, lower chamber adds containing 10%FBS and medicineConditioned medium 600 μ l, incubator cultivate 18h. Take out chamber liquid on cell reject, go up to the greatest extent chamber with cotton swab wiping and do not wear the thin of filmBorn of the same parents, the fixing 30min of 10% formaldehyde under room temperature, conventional brazilwood extract dyeing, counts the migrating cell number in 5 visuals field under 200 times of light borders,Get its mean value, computation migration inhibiting rate. Every group is repeated 3 times. Inhibiting rate (%)=(1-experimental group is worn theca cell number/control group and is wornTheca cell number) × 100%.
S64. the effect that chick chorioallantoic membrane (CAM) modelling verification ES-CSP forms new vessels
Get the chicken embryo of the 7th day, find embryo head by illumination, peel off gently the eggshell that diameter is about 1cm with hand drill, carefulRemove shell membrane, expose chick chorioallantoic membrane. Add respectively 30 μ l physiological saline (NS), 50 and 250 μ g/mlES, ES-CSP in chickenOn embryo chorioallantoic membrane, seal breach with sealed membrane, put into insulating box and cultivate (37 DEG C, humidity 70%). After 72h, take out chicken embryo, officePortion adopts acetone and the fixing 10min of absolute ethyl alcohol. Cut chick chorioallantoic membrane photograph and observe (result is as Figure 10), visible, ES-CSPCan suppress the formation of chick chorioallantoic membrane new vessels.
The preparation of embodiment 2 liver targeting peptides and angiostatin fusion (AS-CSP)
Design primer, utilizes SOE-PCR method to add CSPI-plus base at amino or the c-terminus of angiostatin geneBecause of sequence; While adding aminoterminal, the amino acid sequence of fusion is as shown in SEQIDNO:2, its corresponding nucleotide sequence asShown in SEQIDNO:13; While adding c-terminus, the amino acid sequence of fusion is as shown in SEQIDNO:3, its corresponding coreNucleotide sequence is as shown in SEQIDNO:14. Antigen-4 fusion protein gene after connecting is carried out to eukaryotic expression, and purifying obtains merging eggIn vain, concrete grammar is with embodiment 1.
The preparation of embodiment 3 liver targeting peptides and human plasminogen kringles5 fusion
Design primer, utilizes SOE-PCR method to add at amino or the c-terminus of human plasminogen kringles5 geneCSPI-plus gene order; While adding aminoterminal, the amino acid sequence of fusion is as shown in SEQIDNO:4, and it is correspondingNucleotide sequence is as shown in SEQIDNO:15; While adding c-terminus, the amino acid sequence of fusion is as SEQIDNO:5 instituteShow, its corresponding nucleotide sequence is as shown in SEQIDNO:16. Antigen-4 fusion protein gene after connecting is carried out to eukaryotic expression, pureChange and obtain fusion, concrete grammar is with embodiment 1.
The preparation of embodiment 4 liver targeting peptides and tumor chalone fusion
Design primer, utilizes SOE-PCR method to add CSPI-plus base at amino or the c-terminus of tumor chalone geneBecause of sequence; While adding aminoterminal, the amino acid sequence of fusion is as shown in SEQIDNO:6, its corresponding nucleotide sequence asShown in SEQIDNO:17; While adding c-terminus, the amino acid sequence of fusion is as shown in SEQIDNO:7, its corresponding coreNucleotide sequence is as shown in SEQIDNO:18. Antigen-4 fusion protein gene after connecting is carried out to eukaryotic expression, and purifying obtains merging eggIn vain, concrete grammar is with embodiment 1.
The preparation of the fusion of embodiment 5 liver targeting peptides and Canstatin albumen
Design primer, utilizes SOE-PCR method to add CSPI-at amino or the c-terminus of Canstatin GFPPlus gene order; While adding aminoterminal, the amino acid sequence of fusion is as shown in SEQIDNO:8, its corresponding nucleotidesSequence is as shown in SEQIDNO:19; While adding c-terminus, the amino acid sequence of fusion is as shown in SEQIDNO:9, its phaseThe nucleotide sequence of answering is as shown in SEQIDNO:20. Antigen-4 fusion protein gene after connecting is carried out to eukaryotic expression, and purifying obtainsFusion, concrete grammar is with embodiment 1.
The preparation of the fusion of embodiment 6 liver targeting peptides and thrombospondin-1
Design primer, utilizes SOE-PCR method to add CSPI-at amino or the c-terminus of Canstatin GFPPlus gene order; While adding aminoterminal, the amino acid sequence of fusion is as shown in SEQIDNO:10, its corresponding nucleosidesAcid sequence is as shown in SEQIDNO:21; While adding c-terminus, the amino acid sequence of fusion is as shown in SEQIDNO:11,Its corresponding nucleotide sequence is as shown in SEQIDNO:22. Antigen-4 fusion protein gene after connecting is carried out to eukaryotic expression, purifyingObtain fusion, concrete grammar is with embodiment 1.
The fusion that mensuration embodiment 2 ~ 6 prepares is to hepatocellular targeting, and mensuration embodiment 2 ~ 6 systemsThe standby fusion obtaining is to suppressing angiogenesis function, and concrete operation step is with step S5 and the step S6 of embodiment 1. ResultShow, after the amino of Angiostatin or c-terminus add liver targeting peptides CSPI-plus gene order, can obviously increaseAdd the liver targeting of Angiostatin, and the CSPI-plus gene order adding can not affect AngiogenesisThe inhibitory action of inhibiting factor to Angiogenesis.
SEQUENCELISTING
<110>Guangdong Pharmaceutical University
<120>a kind of liver targeting peptides and Angiostatin fusion and preparation method thereof and application
<130>
<160>29
<170>PatentInversion3.3
<210>1
<211>211
<212>PRT
<213>liver targeting peptides and recombinant human endostatin fusion amino acid sequence
<400>1
MetGlyGlySerHisHisHisHisHisHisSerHisArgAspPheGln
151015
ProValLeuHisLeuValAlaLeuAsnSerProLeuSerGlyGlyMet
202530
ArgGlyIleArgGlyAlaAspPheGlnCysPheGlnGlnAlaArgAla
354045
ValGlyLeuAlaGlyThrPheArgAlaPheLeuSerSerArgLeuGln
505560
AspLeuTyrSerIleValArgArgAlaAspArgAlaAlaValProIle
65707580
ValAsnLeuLysAspGluLeuLeuPheProSerTrpGluAlaLeuPhe
859095
SerGlySerGluGlyProLeuLysProGlyAlaArgIlePheSerPhe
100105110
AspGlyLysAspValLeuArgHisProThrTrpProGlnLysSerVal
115120125
TrpHisGlySerAspProAsnGlyArgArgLeuThrGluSerTyrCys
130135140
GluThrTrpArgThrGluAlaProSerAlaThrGlyGlnAlaSerSer
145150155160
LeuLeuGlyGlyArgLeuLeuGlyGlnSerAlaAlaSerCysHisHis
165170175
AlaTyrIleValLeuCysIleGluAsnSerPheMetThrAlaSerLys
180185190
AspAsnGluLysLeuArgLysProLysHisLysLysLeuLysGlnPro
195200205
AlaAspGly
210
<210>2
<211>413
<212>PRT
<213>liver targeting peptides and angiostatin fusion amino acid sequence 1
<400>2
AspAsnGluLysLeuArgLysProLysHisLysLysLeuLysGlnPro
151015
AlaAspGlyMetAlaGluAsnArgLysSerSerIleIleIleArgMet
202530
ArgAspValValLeuPheGluLysLysValTyrLeuSerGluCysLys
354045
ThrGlyAsnGlyLysAsnTyrArgGlyThrMetSerLysThrLysAsn
505560
GlyIleThrCysGlnLysTrpSerSerThrSerProHisArgProArg
65707580
PheSerProAlaThrHisProSerGluGlyLeuGluGluAsnTyrCys
859095
ArgAsnProAspAsnAspProGlnGlyProTrpCysTyrThrThrAsp
100105110
ProGluLysArgTyrAspTyrCysAspIleLeuGluCysGluGluGlu
115120125
CysMetHisCysSerGlyGluAsnTyrAspGlyLysIleSerLysThr
130135140
MetSerGlyLeuGluCysGlnAlaTrpAspSerGlnSerProHisAla
145150155160
HisGlyTyrIleProSerLysPheProAsnLysAsnLeuLysLysAsn
165170175
TyrCysArgAsnProAspArgGluLeuArgProTrpCysPheThrThr
180185190
AspProAsnLysArgTrpGluLeuCysAspIleProArgCysThrThr
195200205
ProProProSerSerGlyProThrTyrGlnCysLeuLysGlyThrGly
210215220
GluAsnTyrArgGlyAsnValAlaValThrValSerGlyHisThrCys
225230235240
GlnHisTrpSerAlaGlnThrProHisThrHisAsnArgThrProGlu
245250255
AsnPheProCysLysAsnLeuAspGluAsnTyrCysArgAsnProAsp
260265270
GlyLysArgAlaProTrpCysHisThrThrAsnSerGlnValArgTrp
275280285
GluTyrCysLysIleProSerCysAspSerSerProValSerThrGlu
290295300
GlnLeuAlaProThrAlaProProGluLeuThrProValValGlnAsp
305310315320
CysTyrHisGlyAspGlyGlnSerTyrArgGlyThrSerSerThrThr
325330335
ThrThrGlyLysLysCysGlnSerTrpSerSerMetThrProHisArg
340345350
HisGlnLysThrProGluAsnTyrProAsnAlaGlyLeuThrMetAsn
355360365
TyrCysArgAsnProAspAlaAspLysGlyProTrpCysPheThrThr
370375380
AspProSerValArgTrpGluTyrCysAsnLeuLysLysCysSerGly
385390395400
ThrGluAlaSerValValAlaProProProValValLeu
405410
<210>3
<211>413
<212>PRT
<213>liver targeting peptides and angiostatin fusion amino acid sequence 2
<400>3
MetAlaGluAsnArgLysSerSerIleIleIleArgMetArgAspVal
151015
ValLeuPheGluLysLysValTyrLeuSerGluCysLysThrGlyAsn
202530
GlyLysAsnTyrArgGlyThrMetSerLysThrLysAsnGlyIleThr
354045
CysGlnLysTrpSerSerThrSerProHisArgProArgPheSerPro
505560
AlaThrHisProSerGluGlyLeuGluGluAsnTyrCysArgAsnPro
65707580
AspAsnAspProGlnGlyProTrpCysTyrThrThrAspProGluLys
859095
ArgTyrAspTyrCysAspIleLeuGluCysGluGluGluCysMetHis
100105110
CysSerGlyGluAsnTyrAspGlyLysIleSerLysThrMetSerGly
115120125
LeuGluCysGlnAlaTrpAspSerGlnSerProHisAlaHisGlyTyr
130135140
IleProSerLysPheProAsnLysAsnLeuLysLysAsnTyrCysArg
145150155160
AsnProAspArgGluLeuArgProTrpCysPheThrThrAspProAsn
165170175
LysArgTrpGluLeuCysAspIleProArgCysThrThrProProPro
180185190
SerSerGlyProThrTyrGlnCysLeuLysGlyThrGlyGluAsnTyr
195200205
ArgGlyAsnValAlaValThrValSerGlyHisThrCysGlnHisTrp
210215220
SerAlaGlnThrProHisThrHisAsnArgThrProGluAsnPhePro
225230235240
CysLysAsnLeuAspGluAsnTyrCysArgAsnProAspGlyLysArg
245250255
AlaProTrpCysHisThrThrAsnSerGlnValArgTrpGluTyrCys
260265270
LysIleProSerCysAspSerSerProValSerThrGluGlnLeuAla
275280285
ProThrAlaProProGluLeuThrProValValGlnAspCysTyrHis
290295300
GlyAspGlyGlnSerTyrArgGlyThrSerSerThrThrThrThrGly
305310315320
LysLysCysGlnSerTrpSerSerMetThrProHisArgHisGlnLys
325330335
ThrProGluAsnTyrProAsnAlaGlyLeuThrMetAsnTyrCysArg
340345350
AsnProAspAlaAspLysGlyProTrpCysPheThrThrAspProSer
355360365
ValArgTrpGluTyrCysAsnLeuLysLysCysSerGlyThrGluAla
370375380
SerValValAlaProProProValValLeuAspAsnGluLysLeuArg
385390395400
LysProLysHisLysLysLeuLysGlnProAlaAspGly
405410
<210>4
<211>101
<212>PRT
<213>liver targeting peptides and human plasminogen kringles5 fusion amino acid sequence 1
<400>4
AspAsnGluLysLeuArgLysProLysHisLysLysLeuLysGlnPro
151015
AlaAspGlyMetPheGlyAsnGlyLysGlyTyrArgGlyLysArgAla
202530
ThrThrValThrGlyThrProCysGlnAspTrpAlaAlaGlnGluPro
354045
HisArgHisSerIlePheThrProGluThrAsnProArgAlaGlyLeu
505560
GluLysAsnTyrCysArgAsnProAspGlyAspValGlyGlyProTrp
65707580
CysTyrThrThrAsnProArgLysLeuTyrAspTyrCysAspValPro
859095
GlnCysAlaAlaPro
100
<210>5
<211>101
<212>PRT
<213>liver targeting peptides and human plasminogen kringles5 fusion amino acid sequence 2
<400>5
MetPheGlyAsnGlyLysGlyTyrArgGlyLysArgAlaThrThrVal
151015
ThrGlyThrProCysGlnAspTrpAlaAlaGlnGluProHisArgHis
202530
SerIlePheThrProGluThrAsnProArgAlaGlyLeuGluLysAsn
354045
TyrCysArgAsnProAspGlyAspValGlyGlyProTrpCysTyrThr
505560
ThrAsnProArgLysLeuTyrAspTyrCysAspValProGlnCysAla
65707580
AlaProAspAsnGluLysLeuArgLysProLysHisLysLysLeuLys
859095
GlnProAlaAspGly
100
<210>6
<211>264
<212>PRT
<213>liver targeting peptides and tumor chalone fusion amino acid sequence 1
<400>6
AspAsnGluLysLeuArgLysProLysHisLysLysLeuLysGlnPro
151015
AlaAspGlyProGlyLeuLysGlyLysArgGlyAspSerGlySerPro
202530
AlaThrTrpThrThrArgGlyPheValPheThrArgHisSerGlnThr
354045
ThrAlaIleProSerCysProGluGlyThrValProLeuTyrSerGly
505560
PheSerPheLeuPheValGlnGlyAsnGlnArgAlaHisGlyGlnAsp
65707580
LeuGlyThrLeuGlySerCysLeuGlnArgPheThrThrMetProPhe
859095
LeuPheCysAsnValAsnAspValCysAsnPheAlaSerArgAsnAsp
100105110
TyrSerTyrTrpLeuSerThrProAlaLeuMetProMetAsnMetAla
115120125
ProIleThrGlyArgAlaLeuGluProTyrIleSerArgCysThrVal
130135140
CysGluGlyProAlaIleAlaIleAlaValHisSerGlnThrThrAsp
145150155160
IleProProCysProHisGlyTrpIleSerLeuTrpLysGlyPheSer
165170175
PheIleMetPheThrSerAlaGlySerGluGlyThrGlyGlnAlaLeu
180185190
AlaSerProGlySerCysLeuGluGluPheArgAlaSerProPheLeu
195200205
GluCysHisGlyArgGlyThrCysAsnTyrTyrSerAsnSerTyrSer
210215220
PheTrpLeuAlaSerLeuAsnProGluArgMetPheArgLysProIle
225230235240
ProSerThrValLysAlaGlyGluLeuGluLysIleIleSerArgCys
245250255
GlnValCysMetLysLysArgHis
260
<210>7
<211>264
<212>PRT
<213>liver targeting peptides and tumor chalone fusion amino acid sequence 2
<400>7
ProGlyLeuLysGlyLysArgGlyAspSerGlySerProAlaThrTrp
151015
ThrThrArgGlyPheValPheThrArgHisSerGlnThrThrAlaIle
202530
ProSerCysProGluGlyThrValProLeuTyrSerGlyPheSerPhe
354045
LeuPheValGlnGlyAsnGlnArgAlaHisGlyGlnAspLeuGlyThr
505560
LeuGlySerCysLeuGlnArgPheThrThrMetProPheLeuPheCys
65707580
AsnValAsnAspValCysAsnPheAlaSerArgAsnAspTyrSerTyr
859095
TrpLeuSerThrProAlaLeuMetProMetAsnMetAlaProIleThr
100105110
GlyArgAlaLeuGluProTyrIleSerArgCysThrValCysGluGly
115120125
ProAlaIleAlaIleAlaValHisSerGlnThrThrAspIleProPro
130135140
CysProHisGlyTrpIleSerLeuTrpLysGlyPheSerPheIleMet
145150155160
PheThrSerAlaGlySerGluGlyThrGlyGlnAlaLeuAlaSerPro
165170175
GlySerCysLeuGluGluPheArgAlaSerProPheLeuGluCysHis
180185190
GlyArgGlyThrCysAsnTyrTyrSerAsnSerTyrSerPheTrpLeu
195200205
AlaSerLeuAsnProGluArgMetPheArgLysProIleProSerThr
210215220
ValLysAlaGlyGluLeuGluLysIleIleSerArgCysGlnValCys
225230235240
MetLysLysArgHisAspAsnGluLysLeuArgLysProLysHisLys
245250255
LysLeuLysGlnProAlaAspGly
260
<210>8
<211>246
<212>PRT
<213>liver targeting peptides and Canstatin fusion protein amino acid sequence 1
<400>8
AspAsnGluLysLeuArgLysProLysHisLysLysLeuLysGlnPro
151015
AlaAspGlyValSerIleGlyTyrLeuLeuValLysHisSerGlnThr
202530
AspGlnGluProMetCysProValGlyMetAsnLysLeuTrpSerGly
354045
TyrSerLeuLeuTyrPheGluGlyGlnGluLysAlaHisAsnGlnAsp
505560
LeuGlyLeuAlaGlySerCysLeuAlaArgPheSerThrMetProPhe
65707580
LeuTyrCysAsnProGlyAspValCysTyrTyrAlaSerArgAsnAsp
859095
LysSerTyrTrpLeuSerThrThrAlaProLeuProMetMetProVal
100105110
AlaGluAspGluIleLysProTyrIleSerArgCysSerValCysGlu
115120125
AlaProAlaIleAlaIleAlaValHisSerGlnAspValSerIlePro
130135140
HisCysProAlaGlyTrpArgSerLeuTrpIleGlyTyrSerPheLeu
145150155160
MetHisThrAlaAlaGlyAspGluGlyGlyGlyGlnSerLeuValSer
165170175
ProGlySerCysLeuGluAspPheArgAlaThrProPheIleGluCys
180185190
AsnGlyGlyArgGlyThrCysHisTyrTyrAlaAsnLysTyrSerPhe
195200205
TrpLeuThrThrIleProGluGlnSerPheGlnGlySerProSerAla
210215220
AspThrLeuLysAlaGlyLeuIleArgThrHisIleSerArgCysGln
225230235240
ValCysMetLysAsnLeu
245
<210>9
<211>246
<212>PRT
<213>liver targeting peptides and Canstatin fusion protein amino acid sequence 2
<400>9
ValSerIleGlyTyrLeuLeuValLysHisSerGlnThrAspGlnGlu
151015
ProMetCysProValGlyMetAsnLysLeuTrpSerGlyTyrSerLeu
202530
LeuTyrPheGluGlyGlnGluLysAlaHisAsnGlnAspLeuGlyLeu
354045
AlaGlySerCysLeuAlaArgPheSerThrMetProPheLeuTyrCys
505560
AsnProGlyAspValCysTyrTyrAlaSerArgAsnAspLysSerTyr
65707580
TrpLeuSerThrThrAlaProLeuProMetMetProValAlaGluAsp
859095
GluIleLysProTyrIleSerArgCysSerValCysGluAlaProAla
100105110
IleAlaIleAlaValHisSerGlnAspValSerIleProHisCysPro
115120125
AlaGlyTrpArgSerLeuTrpIleGlyTyrSerPheLeuMetHisThr
130135140
AlaAlaGlyAspGluGlyGlyGlyGlnSerLeuValSerProGlySer
145150155160
CysLeuGluAspPheArgAlaThrProPheIleGluCysAsnGlyGly
165170175
ArgGlyThrCysHisTyrTyrAlaAsnLysTyrSerPheTrpLeuThr
180185190
ThrIleProGluGlnSerPheGlnGlySerProSerAlaAspThrLeu
195200205
LysAlaGlyLeuIleArgThrHisIleSerArgCysGlnValCysMet
210215220
LysAsnLeuAspAsnGluLysLeuArgLysProLysHisLysLysLeu
225230235240
LysGlnProAlaAspGly
245
<210>10
<211>567
<212>PRT
<213>liver targeting peptides and thrombospondin-1 fusion amino acid sequence 1
<400>10
AspAsnGluLysLeuArgLysProLysHisLysLysLeuLysGlnPro
151015
AlaAspGlyMetGlyLeuAlaTrpGlyLeuGlyValLeuPheLeuMet
202530
HisValCysGlyThrAsnArgIleProGluSerGlyGlyAspAsnSer
354045
ValPheAspIlePheGluLeuThrGlyAlaAlaArgLysGlySerGly
505560
ArgArgLeuValLysGlyProAspProSerSerProAlaPheArgIle
65707580
GluAspAlaAsnLeuIleProProValProAspAspLysPheGlnAsp
859095
LeuValAspAlaValArgAlaGluLysGlyPheLeuLeuLeuAlaSer
100105110
LeuArgGlnMetLysLysThrArgGlyThrLeuLeuAlaLeuGluArg
115120125
LysAspHisSerGlyGlnValPheSerValValSerAsnGlyLysAla
130135140
GlyThrLeuAspLeuSerLeuThrValGlnGlyLysGlnHisValVal
145150155160
SerValGluGluAlaLeuLeuAlaThrGlyGlnTrpLysSerIleThr
165170175
LeuPheValGlnGluAspArgAlaGlnLeuTyrIleAspCysGluLys
180185190
MetGluAsnAlaGluLeuAspValProIleGlnSerValPheThrArg
195200205
AspLeuAlaSerIleAlaArgLeuArgIleAlaLysGlyGlyValAsn
210215220
AspAsnPheGlnGlyValLeuGlnAsnValArgPheValPheGlyThr
225230235240
ThrProGluAspIleLeuArgAsnLysGlyCysSerSerSerThrSer
245250255
ValLeuLeuThrLeuAspAsnAsnValValAsnGlySerSerProAla
260265270
IleArgThrAsnTyrIleGlyHisLysThrLysAspLeuGlnAlaIle
275280285
CysGlyIleSerCysAspGluLeuSerSerMetValLeuGluLeuArg
290295300
GlyLeuArgThrIleValThrThrLeuGlnAspSerIleArgLysVal
305310315320
ThrGluGluAsnLysGluLeuAlaAsnGluLeuArgArgProProLeu
325330335
CysTyrHisAsnGlyValGlnTyrArgAsnAsnGluGluTrpThrVal
340345350
AspSerCysThrGluCysHisCysGlnAsnSerValThrIleCysLys
355360365
LysValSerCysProIleMetProCysSerAsnAlaThrValProAsp
370375380
GlyGluCysCysProArgCysTrpProSerAspSerAlaAspAspGly
385390395400
TrpSerProTrpSerGluTrpThrSerCysSerThrSerCysGlyAsn
405410415
GlyIleGlnGlnArgGlyArgSerCysAspSerLeuAsnAsnArgCys
420425430
GluGlySerSerValGlnThrArgThrCysHisIleGlnGluCysAsp
435440445
LysArgPheLysGlnAspGlyGlyTrpSerHisTrpSerProTrpSer
450455460
SerCysSerValThrCysGlyAspGlyValIleThrArgIleArgLeu
465470475480
CysAsnSerProSerProGlnMetAsnGlyLysProCysGluGlyGlu
485490495
AlaArgGluThrLysAlaCysLysLysAspAlaCysProIleAsnGly
500505510
GlyTrpGlyProTrpSerProTrpAspIleCysSerValThrCysGly
515520525
GlyGlyValGlnLysArgSerArgLeuCysAsnAsnProThrProGln
530535540
PheGlyGlyLysAspCysValGlyAspValThrGluAsnGlnIleCys
545550555560
AsnLysGlnAspCysProIle
565
<210>11
<211>567
<212>PRT
<213>liver targeting peptides and thrombospondin-1 fusion amino acid sequence 2
<400>11
MetGlyLeuAlaTrpGlyLeuGlyValLeuPheLeuMetHisValCys
151015
GlyThrAsnArgIleProGluSerGlyGlyAspAsnSerValPheAsp
202530
IlePheGluLeuThrGlyAlaAlaArgLysGlySerGlyArgArgLeu
354045
ValLysGlyProAspProSerSerProAlaPheArgIleGluAspAla
505560
AsnLeuIleProProValProAspAspLysPheGlnAspLeuValAsp
65707580
AlaValArgAlaGluLysGlyPheLeuLeuLeuAlaSerLeuArgGln
859095
MetLysLysThrArgGlyThrLeuLeuAlaLeuGluArgLysAspHis
100105110
SerGlyGlnValPheSerValValSerAsnGlyLysAlaGlyThrLeu
115120125
AspLeuSerLeuThrValGlnGlyLysGlnHisValValSerValGlu
130135140
GluAlaLeuLeuAlaThrGlyGlnTrpLysSerIleThrLeuPheVal
145150155160
GlnGluAspArgAlaGlnLeuTyrIleAspCysGluLysMetGluAsn
165170175
AlaGluLeuAspValProIleGlnSerValPheThrArgAspLeuAla
180185190
SerIleAlaArgLeuArgIleAlaLysGlyGlyValAsnAspAsnPhe
195200205
GlnGlyValLeuGlnAsnValArgPheValPheGlyThrThrProGlu
210215220
AspIleLeuArgAsnLysGlyCysSerSerSerThrSerValLeuLeu
225230235240
ThrLeuAspAsnAsnValValAsnGlySerSerProAlaIleArgThr
245250255
AsnTyrIleGlyHisLysThrLysAspLeuGlnAlaIleCysGlyIle
260265270
SerCysAspGluLeuSerSerMetValLeuGluLeuArgGlyLeuArg
275280285
ThrIleValThrThrLeuGlnAspSerIleArgLysValThrGluGlu
290295300
AsnLysGluLeuAlaAsnGluLeuArgArgProProLeuCysTyrHis
305310315320
AsnGlyValGlnTyrArgAsnAsnGluGluTrpThrValAspSerCys
325330335
ThrGluCysHisCysGlnAsnSerValThrIleCysLysLysValSer
340345350
CysProIleMetProCysSerAsnAlaThrValProAspGlyGluCys
355360365
CysProArgCysTrpProSerAspSerAlaAspAspGlyTrpSerPro
370375380
TrpSerGluTrpThrSerCysSerThrSerCysGlyAsnGlyIleGln
385390395400
GlnArgGlyArgSerCysAspSerLeuAsnAsnArgCysGluGlySer
405410415
SerValGlnThrArgThrCysHisIleGlnGluCysAspLysArgPhe
420425430
LysGlnAspGlyGlyTrpSerHisTrpSerProTrpSerSerCysSer
435440445
ValThrCysGlyAspGlyValIleThrArgIleArgLeuCysAsnSer
450455460
ProSerProGlnMetAsnGlyLysProCysGluGlyGluAlaArgGlu465
470475480
ThrLysAlaCysLysLysAspAlaCysProIleAsnGlyGlyTrpGly
485490495
ProTrpSerProTrpAspIleCysSerValThrCysGlyGlyGlyVal
500505510
GlnLysArgSerArgLeuCysAsnAsnProThrProGlnPheGlyGly
515520525
LysAspCysValGlyAspValThrGluAsnGlnIleCysAsnLysGln
530535540
AspCysProIleAspAsnGluLysLeuArgLysProLysHisLysLys
545550555560
LeuLysGlnProAlaAspGly
565
<210>12
<211>636
<212>DNA
<213>liver targeting peptides and recombinant human endothelial inhibin fusion corresponding nucleotide sequence
<400>12
atggggggttctcatcaccatcaccatcacagccaccgcgacttccagccggtgctccac60
ctggttgcgctcaacagccccctgtcaggcggcatgcggggcatccgcggggccgacttc120
cagtgcttccagcaggcgcgggccgtggggctggcgggcaccttccgcgccttcctgtcc180
tcgcgcctgcaggacctgtacagcatcgtgcgccgtgccgaccgcgcagccgtgcccatc240
gtcaacctcaaggacgagctgctgtttcccagctgggaggctctgttctcaggctctgag300
ggtccgctgaagcccggggcacgcatcttctcctttgacggcaaggacgtcctgaggcac360
cccacctggccccagaagagcgtgtggcatggctcggaccccaacgggcgcaggctgacc420
gagagctactgtgagacgtggcggacggaggctccctcggccacgggccaggcctcctcg480
ctgctggggggcaggctcctggggcagagtgccgcgagctgccatcacgcctacatcgtg540
ctctgcattgagaacagcttcatgactgcctccaaggacaacgagaaattaaggaaacca600
aaacataaaaaattaaagcaaccagcggatggttaa636
<210>13
<211>1242
<212>DNA
<213>liver targeting peptides and angiostatin fusion corresponding nucleotide sequence 1
<400>13
gacaacgagaaattaaggaaaccaaaacataaaaaattaaagcaaccagcggatggtatg60
gctgaaaacaggaagtcctccataatcattaggatgagagatgtagttttatttgaaaag120
aaagtgtatctctcagagtgcaagactgggaatggaaagaactacagagggacgatgtcc180
aaaacaaaaaatggcatcacctgtcaaaaatggagttccacttctccccacagacctaga240
ttctcacctgctacacacccctcagagggactggaggagaactactgcaggaatccagac300
aacgatccgcaggggccctggtgctatactactgatccagaaaagagatatgactactgc360
gacattcttgagtgtgaagaggaatgtatgcattgcagtggagaaaactatgacggcaaa420
atttccaagaccatgtctggactggaatgccaggcctgggactctcagagcccacacgct480
catggatacattccttccaaatttccaaacaagaacctgaagaagaattactgtcgtaac540
cccgatagggagctgcggccttggtgtttcaccaccgaccccaacaagcgctgggaactt600
tgtgacatcccccgctgcacaacacctccaccatcttctggtcccacctaccagtgtctg660
aagggaacaggtgaaaactatcgcgggaatgtggctgttaccgtgtccgggcacacctgt720
cagcactggagtgcacagacccctcacacacataacaggacaccagaaaacttcccctgc780
aaaaatttggatgaaaactactgccgcaatcctgacggaaaaagggccccatggtgccat840
acaaccaacagccaagtgcggtgggagtactgtaagataccgtcctgtgactcctcccca900
gtatccacggaacaattggctcccacagcaccacctgagctaacccctgtggtccaggac960
tgctaccatggtgatggacagagctaccgaggcacatcctccaccaccaccacaggaaag1020
aagtgtcagtcttggtcatctatgacaccacaccggcaccagaagaccccagaaaactac1080
ccaaatgctggcctgacaatgaactactgcaggaatccagatgccgataaaggcccctgg1140
tgttttaccacagaccccagcgtcaggtgggagtactgcaacctgaaaaaatgctcagga1200
acagaagcgagtgttgtagcacctccgcctgttgtcctgtaa1242
<210>14
<211>1242
<212>DNA
<213>liver targeting peptides and angiostatin fusion corresponding nucleotide sequence 2
<400>14
atggctgaaaacaggaagtcctccataatcattaggatgagagatgtagttttatttgaa60
aagaaagtgtatctctcagagtgcaagactgggaatggaaagaactacagagggacgatg120
tccaaaacaaaaaatggcatcacctgtcaaaaatggagttccacttctccccacagacct180
agattctcacctgctacacacccctcagagggactggaggagaactactgcaggaatcca240
gacaacgatccgcaggggccctggtgctatactactgatccagaaaagagatatgactac300
tgcgacattcttgagtgtgaagaggaatgtatgcattgcagtggagaaaactatgacggc360
aaaatttccaagaccatgtctggactggaatgccaggcctgggactctcagagcccacac420
gctcatggatacattccttccaaatttccaaacaagaacctgaagaagaattactgtcgt480
aaccccgatagggagctgcggccttggtgtttcaccaccgaccccaacaagcgctgggaa540
ctttgtgacatcccccgctgcacaacacctccaccatcttctggtcccacctaccagtgt600
ctgaagggaacaggtgaaaactatcgcgggaatgtggctgttaccgtgtccgggcacacc660
tgtcagcactggagtgcacagacccctcacacacataacaggacaccagaaaacttcccc720
tgcaaaaatttggatgaaaactactgccgcaatcctgacggaaaaagggccccatggtgc780
catacaaccaacagccaagtgcggtgggagtactgtaagataccgtcctgtgactcctcc840
ccagtatccacggaacaattggctcccacagcaccacctgagctaacccctgtggtccag900
gactgctaccatggtgatggacagagctaccgaggcacatcctccaccaccaccacagga960
aagaagtgtcagtcttggtcatctatgacaccacaccggcaccagaagaccccagaaaac1020
tacccaaatgctggcctgacaatgaactactgcaggaatccagatgccgataaaggcccc1080
tggtgttttaccacagaccccagcgtcaggtgggagtactgcaacctgaaaaaatgctca1140
ggaacagaagcgagtgttgtagcacctccgcctgttgtcctggacaacgagaaattaagg1200
aaaccaaaacataaaaaattaaagcaaccagcggatggttaa1242
<210>15
<211>306
<212>DNA
<213>liver targeting peptides and human plasminogen kringles5 fusion corresponding nucleotide sequence 1
<400>15
gacaacgagaaattaaggaaaccaaaacataaaaaattaaagcaaccagcggatggtatg60
tttgggaatgggaaaggataccgaggcaagagggcgaccactgttactgggacgccatgc120
caggactgggctgcccaggagccccatagacacagcattttcactccagagacaaatcca180
cgggcgggtctggaaaaaaattactgccgtaaccctgatggtgatgtaggtggtccctgg240
tgctacacgacaaatccaagaaaactttacgactactgtgatgtccctcagtgtgcggcc300
ccttaa306
<210>16
<211>306
<212>DNA
<213>liver targeting peptides and human plasminogen kringles5 fusion corresponding nucleotide sequence 2
<400>16
atgtttgggaatgggaaaggataccgaggcaagagggcgaccactgttactgggacgcca60
tgccaggactgggctgcccaggagccccatagacacagcattttcactccagagacaaat120
ccacgggcgggtctggaaaaaaattactgccgtaaccctgatggtgatgtaggtggtccc180
tggtgctacacgacaaatccaagaaaactttacgactactgtgatgtccctcagtgtgcg240
gcccctgacaacgagaaattaaggaaaccaaaacataaaaaattaaagcaaccagcggat300
ggttaa306
<210>17
<211>795
<212>DNA
<213>liver targeting peptides and tumor chalone fusion corresponding nucleotide sequence 1
<400>17
gacaacgagaaattaaggaaaccaaaacataaaaaattaaagcaaccagcggatggtcca60
ggtttgaaaggaaaacgtggagacagtggatcacctgcaacctggacaacgagaggcttt120
gtcttcacccgacacagtcaaaccacagcaattccttcatgtccagaggggacagtgcca180
ctctacagtgggttttcttttctttttgtacaaggaaatcaacgagcccacggacaagac240
cttggaactcttggcagctgcctgcagcgatttaccacaatgccattcttattctgcaat300
gtcaatgatgtatgtaattttgcatctcgaaatgattattcatactggctgtcaacacca360
gctctgatgccaatgaacatggctcccattactggcagagcccttgagccttatataagc420
agatgcactgtttgtgaaggtcctgcgatcgccatagccgttcacagccaaaccactgac480
attcctccatgtcctcacggctggatttctctctggaaaggattttcattcatcatgttc540
acaagtgcaggttctgagggcaccgggcaagcactggcctcccctggctcctgcctggaa600
gaattccgagccagcccatttctagaatgtcatggaagaggaacgtgcaactactattca660
aattcctacagtttctggctggcttcattaaacccagaaagaatgttcagaaagcctatt720
ccatcaactgtgaaagctggggaattagaaaaaataataagtcgctgtcaggtgtgcatg780
aagaaaagacactga795
<210>18
<211>795
<212>DNA
<213>liver targeting peptides and tumor chalone fusion corresponding nucleotide sequence 2
<400>18
ccaggtttgaaaggaaaacgtggagacagtggatcacctgcaacctggacaacgagaggc60
tttgtcttcacccgacacagtcaaaccacagcaattccttcatgtccagaggggacagtg120
ccactctacagtgggttttcttttctttttgtacaaggaaatcaacgagcccacggacaa180
gaccttggaactcttggcagctgcctgcagcgatttaccacaatgccattcttattctgc240
aatgtcaatgatgtatgtaattttgcatctcgaaatgattattcatactggctgtcaaca300
ccagctctgatgccaatgaacatggctcccattactggcagagcccttgagccttatata360
agcagatgcactgtttgtgaaggtcctgcgatcgccatagccgttcacagccaaaccact420
gacattcctccatgtcctcacggctggatttctctctggaaaggattttcattcatcatg480
ttcacaagtgcaggttctgagggcaccgggcaagcactggcctcccctggctcctgcctg540
gaagaattccgagccagcccatttctagaatgtcatggaagaggaacgtgcaactactat600
tcaaattcctacagtttctggctggcttcattaaacccagaaagaatgttcagaaagcct660
attccatcaactgtgaaagctggggaattagaaaaaataataagtcgctgtcaggtgtgc720
atgaagaaaagacacgacaacgagaaattaaggaaaccaaaacataaaaaattaaagcaa780
ccagcggatggttga795
<210>19
<211>741
<212>DNA
<213>liver targeting peptides and Canstatin fusion protein corresponding nucleotide sequence 1
<400>19
gacaacgagaaattaaggaaaccaaaacataaaaaattaaagcaaccagcggatggtgtc60
agcatcggctacctcctggtgaagcacagccagacggaccaggagcccatgtgcccggtg120
ggcatgaacaaactctggagtggatacagcctgctgtacttcgagggccaggagaaggcg180
cacaaccaggacctggggctggcgggctcctgcctggcgcggttcagcaccatgcccttc240
ctgtactgcaaccctggtgatgtctgctactatgccagccggaacgacaagtcctactgg300
ctctctaccactgcgccgctgcccatgatgcccgtggccgaggacgagatcaagccctac360
atcagccgctgttctgtgtgtgaggccccggccatcgccatcgcggtccacagtcaggat420
gtctccatcccacactgcccagctgggtggcggagtttgtggatcggatattccttcctc480
atgcacacggcggcgggagacgaaggcggtggccaatcactggtgtcaccgggcagctgt540
ctagaggacttccgcgccacaccattcatcgaatgcaatggaggccgcggcacctgccac600
tactacgccaacaagtacagcttctggctgaccaccattcccgagcagagcttccagggc660
tcgccctccgccgacacgctcaaggccggcctcatccgcacacacatcagccgctgccag720
gtgtgcatgaagaacctgtga741
<210>20
<211>741
<212>DNA
<213>liver targeting peptides and Canstatin fusion protein corresponding nucleotide sequence 2
<400>20
gtcagcatcggctacctcctggtgaagcacagccagacggaccaggagcccatgtgcccg60
gtgggcatgaacaaactctggagtggatacagcctgctgtacttcgagggccaggagaag120
gcgcacaaccaggacctggggctggcgggctcctgcctggcgcggttcagcaccatgccc180
ttcctgtactgcaaccctggtgatgtctgctactatgccagccggaacgacaagtcctac240
tggctctctaccactgcgccgctgcccatgatgcccgtggccgaggacgagatcaagccc300
tacatcagccgctgttctgtgtgtgaggccccggccatcgccatcgcggtccacagtcag360
gatgtctccatcccacactgcccagctgggtggcggagtttgtggatcggatattccttc420
ctcatgcacacggcggcgggagacgaaggcggtggccaatcactggtgtcaccgggcagc480
tgtctagaggacttccgcgccacaccattcatcgaatgcaatggaggccgcggcacctgc540
cactactacgccaacaagtacagcttctggctgaccaccattcccgagcagagcttccag600
ggctcgccctccgccgacacgctcaaggccggcctcatccgcacacacatcagccgctgc660
caggtgtgcatgaagaacctggacaacgagaaattaaggaaaccaaaacataaaaaatta720
aagcaaccagcggatggttga741
<210>21
<211>1701
<212>DNA
<213>liver targeting peptides and thrombospondin-1 fusion corresponding nucleotide sequence 1
<400>21
gacaacgagaaattaaggaaaccaaaacataaaaaattaaagcaaccagcggatggtatg60
gggctggcctggggactaggcgtcctgttcctgatgcatgtgtgtggcaccaaccgcatt120
ccagagtctggcggagacaacagcgtgtttgacatctttgaactcaccggggccgcccgc180
aaggggtctgggcgccgactggtgaagggccccgacccttccagcccagctttccgcatc240
gaggatgccaacctgatcccccctgtgcctgatgacaagttccaagacctggtggatgct300
gtgcgggcagaaaagggtttcctccttctggcatccctgaggcagatgaagaagacccgg360
ggcacgctgctggccctggagcggaaagaccactctggccaggtcttcagcgtggtgtcc420
aatggcaaggcgggcaccctggacctcagcctgaccgtccaaggaaagcagcacgtggtg480
tctgtggaagaagctctcctggcaaccggccagtggaagagcatcaccctgtttgtgcag540
gaagacagggcccagctgtacatcgactgtgaaaagatggagaatgctgagttggacgtc600
cccatccaaagcgtcttcaccagagacctggccagcatcgccagactccgcatcgcaaag660
gggggcgtcaatgacaatttccagggggtgctgcagaatgtgaggtttgtctttggaacc720
acaccagaagacatcctcaggaacaaaggctgctccagctctaccagtgtcctcctcacc780
cttgacaacaacgtggtgaatggttccagccctgccatccgcactaactacattggccac840
aagacaaaggacttgcaagccatctgcggcatctcctgtgatgagctgtccagcatggtc900
ctggaactcaggggcctgcgcaccattgtgaccacgctgcaggacagcatccgcaaagtg960
actgaagagaacaaagagttggccaatgagctgaggcggcctcccctatgctatcacaac1020
ggagttcagtacagaaataacgaggaatggactgttgatagctgcactgagtgtcactgt1080
cagaactcagttaccatctgcaaaaaggtgtcctgccccatcatgccctgctccaatgcc1140
acagttcctgatggagaatgctgtcctcgctgttggcccagcgactctgcggacgatggc1200
tggtctccatggtccgagtggacctcctgttctacgagctgtggcaatggaattcagcag1260
cgcggccgctcctgcgatagcctcaacaaccgatgtgagggctcctcggtccagacacgg1320
acctgccacattcaggagtgtgacaagagatttaaacaggatggtggctggagccactgg1380
tccccgtggtcatcttgttctgtgacatgtggtgatggtgtgatcacaaggatccggctc1440
tgcaactctcccagcccccagatgaacgggaaaccctgtgaaggcgaagcgcgggagacc1500
aaagcctgcaagaaagacgcctgccccatcaatggaggctggggtccttggtcaccatgg1560
gacatctgttctgtcacctgtggaggaggggtacagaaacgtagtcgtctctgcaacaac1620
cccacaccccagtttggaggcaaggactgcgttggtgatgtaacagaaaaccagatctgc1680
aacaagcaggactgtccaatt1701
<210>22
<211>1701
<212>DNA
<213>liver targeting peptides and thrombospondin-1 fusion corresponding nucleotide sequence 2
<400>22
atggggctggcctggggactaggcgtcctgttcctgatgcatgtgtgtggcaccaaccgc60
attccagagtctggcggagacaacagcgtgtttgacatctttgaactcaccggggccgcc120
cgcaaggggtctgggcgccgactggtgaagggccccgacccttccagcccagctttccgc180
atcgaggatgccaacctgatcccccctgtgcctgatgacaagttccaagacctggtggat240
gctgtgcgggcagaaaagggtttcctccttctggcatccctgaggcagatgaagaagacc300
cggggcacgctgctggccctggagcggaaagaccactctggccaggtcttcagcgtggtg360
tccaatggcaaggcgggcaccctggacctcagcctgaccgtccaaggaaagcagcacgtg420
gtgtctgtggaagaagctctcctggcaaccggccagtggaagagcatcaccctgtttgtg480
caggaagacagggcccagctgtacatcgactgtgaaaagatggagaatgctgagttggac540
gtccccatccaaagcgtcttcaccagagacctggccagcatcgccagactccgcatcgca600
aaggggggcgtcaatgacaatttccagggggtgctgcagaatgtgaggtttgtctttgga660
accacaccagaagacatcctcaggaacaaaggctgctccagctctaccagtgtcctcctc720
acccttgacaacaacgtggtgaatggttccagccctgccatccgcactaactacattggc780
cacaagacaaaggacttgcaagccatctgcggcatctcctgtgatgagctgtccagcatg840
gtcctggaactcaggggcctgcgcaccattgtgaccacgctgcaggacagcatccgcaaa900
gtgactgaagagaacaaagagttggccaatgagctgaggcggcctcccctatgctatcac960
aacggagttcagtacagaaataacgaggaatggactgttgatagctgcactgagtgtcac1020
tgtcagaactcagttaccatctgcaaaaaggtgtcctgccccatcatgccctgctccaat1080
gccacagttcctgatggagaatgctgtcctcgctgttggcccagcgactctgcggacgat1140
ggctggtctccatggtccgagtggacctcctgttctacgagctgtggcaatggaattcag1200
cagcgcggccgctcctgcgatagcctcaacaaccgatgtgagggctcctcggtccagaca1260
cggacctgccacattcaggagtgtgacaagagatttaaacaggatggtggctggagccac1320
tggtccccgtggtcatcttgttctgtgacatgtggtgatggtgtgatcacaaggatccgg1380
ctctgcaactctcccagcccccagatgaacgggaaaccctgtgaaggcgaagcgcgggag1440
accaaagcctgcaagaaagacgcctgccccatcaatggaggctggggtccttggtcacca1500
tgggacatctgttctgtcacctgtggaggaggggtacagaaacgtagtcgtctctgcaac1560
aaccccacaccccagtttggaggcaaggactgcgttggtgatgtaacagaaaaccagatc1620
tgcaacaagcaggactgtccaattgacaacgagaaattaaggaaaccaaaacataaaaaa1680
ttaaagcaaccagcggatggt1701
<210>23
<211>57
<212>DNA
<213>liver targeting peptides CSPI-plus
<400>23
gacaacgagaaattaaggaaaccaaaacataaaaaattaaagcaaccagcggatggt57
<210>24
<211>27
<212>DNA
<213>nucleotide sequence that human endostatin aminoterminal adds
<400>24
atggggggttctcatcaccatcaccat27
<210>25
<211>20
<212>DNA
<213>primer P1
<400>25
ccgcaccacagctcctacgt20
<210>26
<211>20
<212>DNA
<213>primer P2
<400>26
tactgcaccctgcctgaccc20
<210>27
<211>55
<212>DNA
<213>primer P3
<400>27
ggaattccatatggggggttctcatcaccatcaccatcacagccaccgcgacttc55
<210>28
<211>55
<212>DNA
<213>primer P4
<400>28
ttttatgttttggtttccttaatttctcgttgtccttggaggcagtcatgaagct55
<210>29
<211>55
<212>DNA
<213>primer P5
<400>29
ggccgctcgagttaaccatccgctggttgctttaattttttatgttttggtttcc55
Claims (8)
1. liver targeting peptides and an Angiostatin fusion, is characterized in that, its amino acid sequence is by a kind of bloodPipe generates inhibiting factor and CSPI-plus sequence composition; The system of described liver targeting peptides and Angiostatin fusionPreparation Method, comprises the following steps:
S1. antigen-4 fusion protein gene builds: utilize SOE-PCR method to add at the c-terminus of people's Angiostatin geneCSPI-plus gene order;
S2. above-mentioned antigen-4 fusion protein gene is built to recombinant expression carrier;
S3. by recombinant expression carrier transformed host cell, obtain the engineering bacteria of expressed fusion protein, fermented and cultured;
S4. purifies and separates obtains liver targeting peptides and Angiostatin fusion;
Described Angiostatin be endostatin, angiostatin, human plasminogen kringles5, tumor chalone,Canstatin albumen or thrombospondin-1; The amino acid of described liver targeting peptides and Angiostatin fusionSequence is as shown in SEQIDNO:1 ~ 11, and its corresponding nucleotide sequence is as shown in SEQIDNO:12 ~ 22.
2. liver targeting peptides and Angiostatin fusion according to claim 1, is characterized in that, melts described in S1The gene constructed middle SOE-PCR of the utilization method of hop protein also adds respectively at c-terminus and the aminoterminal of Angiostatin geneEnter restriction enzyme site sequence.
3. according to liver targeting peptides described in claim 1 ~ 2 any one and Angiostatin fusion, it is characterized in that,Described fusion is liver targeting peptides and people's endostatin fusion, its amino acid sequence as shown in SEQIDNO:1, liverThe preparation method of targeting peptides and people's endostatin fusion is as follows:
S1. antigen-4 fusion protein gene build: utilize SOE-PCR method the c-terminus of people's endostatin gene add nucleotides asCSPI-plus gene order shown in SEQIDNO:23 and XhoI restriction enzyme site sequence; Utilize SOE-PCR method also peopleThe aminoterminal of endostatin gene adds genetic fragment and the NdeI restriction enzyme site order of nucleotides as shown in SEQIDNO:24Row;
S2. above-mentioned antigen-4 fusion protein gene is built to recombinant expression carrier;
S3. by recombinant expression carrier transformed host cell, obtain the engineering bacteria of expressed fusion protein, fermented and cultured;
S4. purifies and separates obtains liver targeting peptides and recombinant human endothelial inhibin fusion.
4. liver targeting peptides and Angiostatin fusion according to claim 3, is characterized in that, melts described in S1The gene constructed concrete steps of hop protein are: first amplify people's endostatin gene with primer sets 1, then with primer sets 2 and primerGroup 3 adopts SOE-PCR method to add the CSP of nucleotides as shown in SEQIDNO:23 at the c-terminus of people's endostatin geneI-plus gene order and XhoI restriction enzyme site sequence, add nucleotides as SEQIDNO at endostatin aminopeptidase gene end:Genetic fragment shown in 24 and NdeI restriction enzyme site sequence, primer sets 1 is made up of primer P1 and P2, primer sets 2 by primer P3 andP4 composition, primer sets 3 is made up of primer P3 and P5, the nucleotide sequence of primer P1, P2, P3, P4 and P5 as SEQIDNO:25 ~Shown in 29.
5. a recombinant expression carrier, is characterized in that, by liver targeting peptides described in the MCS insertion claim 1 of carrierGene order with Angiostatin fusion.
6. recombinant expression carrier according to claim 5, is characterized in that, described recombinant expression carrier is recombined pronucleus expressionCarrier.
7. a recombinant cell strain, is characterized in that, contains the recombinant expression carrier described in claim 5 or 6.
8. described in claim 1, liver targeting peptides and Angiostatin fusion suppress liver cancer vascularization medicine in preparationApplication in thing.
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| CN103611151A (en) * | 2013-12-06 | 2014-03-05 | 广东药学院 | Application of circumsporozoite protein polypeptide CSP I-plus of plasmodium in preparing anti-malarial medicine |
| CN109320614B (en) * | 2018-02-02 | 2023-03-24 | 温州医科大学 | FGFR-Fc fusion protein and siRNA |
| CN111875707B (en) * | 2020-05-20 | 2022-05-24 | 华南理工大学 | Fusion polypeptide and preparation method and application thereof |
| CN113735966B (en) * | 2021-09-29 | 2022-11-01 | 陕西巨子生物技术有限公司 | Anti-tumor recombinant collagen and preparation method and application thereof |
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| US20040110671A1 (en) * | 2002-12-05 | 2004-06-10 | Yongzhang Luo | N-terminal modified recombinant human endostatin and its production |
| CN102268093A (en) * | 2011-06-27 | 2011-12-07 | 广东药学院 | Fusion protein of hepatic-targeted peptide and human interferon a2b, and preparation method and application thereof |
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| US20040110671A1 (en) * | 2002-12-05 | 2004-06-10 | Yongzhang Luo | N-terminal modified recombinant human endostatin and its production |
| CN102268093A (en) * | 2011-06-27 | 2011-12-07 | 广东药学院 | Fusion protein of hepatic-targeted peptide and human interferon a2b, and preparation method and application thereof |
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| 内源性血管生成抑制因子的研究进展及在肝癌中的应用前景;邓靖宇等;《中国普外基础与临床杂志》;20060331;第13卷(第2期);184-186 * |
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