CN101092452A - Preparation method for both of micromolecule polypeptide of tumor chalone for anti angiogenesis, and fusion protein - Google Patents
Preparation method for both of micromolecule polypeptide of tumor chalone for anti angiogenesis, and fusion protein Download PDFInfo
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- CN101092452A CN101092452A CNA2007100722510A CN200710072251A CN101092452A CN 101092452 A CN101092452 A CN 101092452A CN A2007100722510 A CNA2007100722510 A CN A2007100722510A CN 200710072251 A CN200710072251 A CN 200710072251A CN 101092452 A CN101092452 A CN 101092452A
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Abstract
This invention discloses a method for preparing tumstatin anti-angiogenic small molecular polypeptide, and fusion protein prepared from the small molecular polypeptide. This invention is aim to resolving the problem of goodpasture syndrome caused by tumor treatment with tumstatin. The amino acid sequence of tumstatin anti-angiogenic small molecular polypeptide is MPFLFCNVNDVCNFASRNDYS, and that of the fusion protein is MPFLFCNVNDVCNFASRGDYSGGASPFLECHGRGTCNYYSNS. The sense strand and anti-sense strand coding the tumstatin anti-angiogenic small molecular polypeptide and the fusion protein are synthesized in a nucleotide automatic synthesis apparatus. After recombinant conversion, the sense strand and anti-sense strand can be used for mass production of tumstatin anti-angiogenic small molecular polypeptide.
Description
Technical field
The fusion rotein and the preparation method for both that the present invention relates to a kind of micromolecule polypeptide and prepare by its.
Background technology
Tumor chalone has by suppressing the new vessel generation, breaks off tumor tissues blood supply and the direct dual anti-tumor activity that suppresses growth of tumour cell, transfer.But existing tumor chalone can cause lung nephrorrhagia syndrome as autoantigen, can not be used for the clinical treatment of tumour, and the anti-angiogenesis activity of tumor chalone and antitumor cell growth, transfer activity lay respectively on the small segment of tumor chalone different zones.
Summary of the invention
Purpose of the present invention can cause the syndromic problem of lung nephrorrhagia in order to solve tumor chalone when treating tumour, and proposes micromolecule polypeptide of tumor chalone for anti angiogenesis, fusion rotein and preparation method for both first.The aminoacid sequence of micromolecule polypeptide of tumor chalone for anti angiogenesis is:
MetProPheLeuPheCysAsnValAsnAspValCysAsnPheAlaSerArgAsnAspTyrSer。The nucleotides sequence of micromolecule polypeptide of tumor chalone for anti angiogenesis is classified as:
TATGCCGTTCTTATTCTGCAATGTTAACGATGTATGCAACTTCGCATCTCGTAATGATTACTCC。Micromolecule polypeptide of tumor chalone for anti angiogenesis is realized by the following method: remove the N-terminal Threonine of tumor chalone 25 peptides, with deputy methionine(Met) as aminoterminal, remove tyrosine, methionine(Met) and three amino acid of leucine of carboxyl terminal, transform 25 peptides as 21 peptides, obtain micromolecule polypeptide of tumor chalone for anti angiogenesis.The aminoacid sequence of tumor chalone micromolecule polypeptide fusion rotein is:
MetProPheLeuPheCysAsnValAsnAspValCysAsnPheAlaSerArgGlyAspTyrSerGlyGlyAlaSerProPheLeuGluCysHisGlyArgGlyThrCysAsnTyrTyrSerAsnSer。The nucleotides sequence of tumor chalone micromolecule polypeptide fusion rotein is classified as:
TATGCCGTTCTTATTCTGCAATGTTAACGATGTATGCAACTTCGCATCTCGTGGAGATTACTCCGGAGGAGCTAGCCCTTTCCTAGAATGTCATGGAAGAGGAACGTGCAACTACTACTCAAACTCC。The preparation method of tumor chalone micromolecule polypeptide fusion rotein makes by following steps: 1, remove the N-terminal Threonine of tumor chalone 25 peptides, with deputy methionine(Met) as aminoterminal, remove tyrosine, methionine(Met) and three amino acid of leucine of carboxyl terminal, transform 25 peptides as 21 peptides; 2, the Asn with the 18th of 21 peptide changes Gly into, forms the RGD sequence with the amino-acid residue on both sides then, obtains introducing 21 peptides of RGD sequence; 3, will introduce and add 2 Gly between 21 peptides of RGD sequence and 19 peptides and connect, obtain tumor chalone micromolecule polypeptide fusion rotein.Antisense strand 3 ' the end of above-mentioned tumor chalone micromolecule polypeptide and fusion rotein DNA of encoding lacks and two bases of sense strand 5 ' end TA paired, after antisense strand and the pairing of sense strand separately form two strands, sense strand 5 ' end has more two bases of TA, form cohesive end, can be connected with the cohesive end that Nde I enzyme is cut; Sense strand 3 ' is a flush end, can be connected with the flush end that Sma I enzyme is cut.The nucleotide sequence of codes for tumor chalone micromolecule polypeptide fusion rotein of the present invention is according to the preferences design of intestinal bacteria to password, recombinate with the pTYB2 carrier through Nde I and Sma I double digestion in the sense strand of codes for tumor chalone micromolecule polypeptide fusion rotein and antisense strand phosphorylation annealing back, again transformed into escherichia coli BL21 (DE3).Genetic engineering bacterium BL21 (DE3) induces through IPTG, just can extract purifying tumor chalone micromolecule polypeptide fusion rotein with the chitin affinity column.The codes for tumor chalone angiogenesis inhibitor micromolecule polypeptide of the present invention's design and the sense strand of fusion rotein and antisense strand are synthetic by the Nucleotide automatic DNA synthesizer DNA, available biological engineering method scale operation micromolecule polypeptide of tumor chalone for anti angiogenesis and tumor chalone micromolecule polypeptide fusion rotein after recombinant conversion.Micromolecule polypeptide of tumor chalone for anti angiogenesis among the present invention and tumor chalone micromolecule polypeptide fusion protein molecule amount are little, be fit to vein, muscle and subcutaneous injection, be easy to absorb, drug safety, can not cause lung nephrorrhagia syndrome, the clinical treatment that can be used for tumour fully, find with experimentation on animals contrast back: 19 peptide tumour inhibiting rates are 40～42.41%, 21 peptide tumour inhibiting rates are 40～40.08%, the 21 peptide tumour inhibiting rates of introducing RGD are 40～46.88%, more than the inhibitory rate to 57.59% of 21 peptides+1 9 peptide fusion proteins of the introducing RGD among the present invention.
Description of drawings
Fig. 1 is the recombinant plasmid sequencer map of coding 19 peptides, and Fig. 2 is coding 21 peptide recombinant plasmid sequencer maps, the antisense strand sequence of presentation code 21 peptides; Fig. 3 is the 21 peptide recombinant plasmid sequencer maps that coding is introduced the RGD sequence, represents 21 peptide-coding sequences; Fig. 4 introduces RGD sequence 21 peptides+19 peptide fusion protein recombinant plasmid sequencer maps, shows the fusion rotein encoding sequence; Fig. 5 is the transmission electron microscope picture that does not add the control group of tumor chalone micromolecule polypeptide, Fig. 6 to Fig. 8 is the transmission electron microscope picture that adds ovarian cancer cell behind the tumor chalone micromolecule polypeptide in the nutrient solution, wherein Fig. 6 is the transmission electron microscope picture that adds 21 peptide groups of 21 peptides, Fig. 7 is the transmission electron microscope picture that adds 19 peptide groups of 19 peptides, and Fig. 8 is the transmission electron microscope that adds the fusion rotein group of fusion rotein. figure; Fig. 9 is the control group that does not add the tumor chalone micromolecule polypeptide, Figure 10 to Figure 12 is the transmission electron microscope picture that adds huve cell behind the tumor chalone micromolecule polypeptide in the nutrient solution, wherein Figure 10 is the transmission electron microscope picture that adds 21 peptide groups of 21 peptides, Figure 11 is the transmission electron microscope picture that adds 19 peptide groups of 19 peptides, and Figure 12 is the transmission electron microscope picture that adds the fusion rotein group of fusion rotein; Figure 13 to Figure 16 is that the TUNEL method detects cervical cancer cell apoptosis figure as a result, wherein Figure 13 is that the control group TUNEL method that does not add the tumor chalone micromolecule polypeptide detects cervical cancer cell apoptosis figure as a result, Figure 14 is that the 21 peptide group TUNEL methods that add 21 peptides detect cervical cancer cell apoptosis figure as a result, Figure 15 is that the 19 peptide group TUNEL methods that add 19 peptides detect cervical cancer cell apoptosis figure as a result, and Figure 16 is that the fusion rotein group TUNEL method that adds fusion rotein detects cervical cancer cell apoptosis figure as a result; Figure 17 to Figure 20 is that the TUNEL method detects huve cell apoptosis figure as a result, wherein Figure 17 is that the control group TUNEL method that does not add the tumor chalone micromolecule polypeptide detects huve cell apoptosis figure as a result, Figure 18 is that the 21 peptide group TUNEL methods that add 21 peptides detect huve cell apoptosis figure as a result, Figure 19 is that the 19 peptide group TUNEL methods that add 19 peptides detect huve cell apoptosis figure as a result, and Figure 20 is that the fusion rotein group TUNEL method that adds fusion rotein detects huve cell apoptosis figure as a result; Figure 21 to Figure 25 is that flow cytometer detects huve cell cell cycle and apoptosis figure as a result, wherein Figure 21 is the control group that does not add the tumor chalone micromolecule polypeptide, Figure 22 is that the 19 peptide group flow cytometers that add 19 peptides detect huve cell cell cycle and apoptosis figure as a result, Figure 23 is that the 21 peptide group flow cytometers that add 21 peptides detect huve cell cell cycle and apoptosis figure as a result, Figure 24 is that the fusion rotein group flow cytometer that adds fusion rotein detects huve cell cell cycle and apoptosis figure as a result, and Figure 25 is that the RGD21 peptide group flow cytometer that adds the RGD21 peptide detects huve cell cell cycle and apoptosis figure as a result; Figure 26 to Figure 30 presses down the knurl experimental result picture in the mouse body, wherein Figure 26 is the control group that does not add the tumor chalone micromolecule polypeptide, Figure 27 is the interior knurl experimental result picture that presses down of 21 peptide group mouse bodies that adds 21 peptides, Figure 28 is the interior knurl experimental result picture that presses down of RGD21 peptide group mouse body that adds the RGD21 peptide, Figure 29 is the interior knurl experimental result picture that presses down of 19 peptide group mouse bodies that adds 19 peptides, and Figure 30 is the interior knurl experimental result picture that presses down of fusion rotein group mouse body that adds fusion rotein.
Embodiment
Embodiment one: the aminoacid sequence of micromolecule polypeptide of tumor chalone for anti angiogenesis is in the present embodiment:
MetProPheLeuPheCysAsnValAsnAspValCysAsnPheAlaSerArgAsnAspTyrSer。
Embodiment two: the nucleotides sequence of micromolecule polypeptide of tumor chalone for anti angiogenesis is classified as in the present embodiment:
TATGCCGTTCTTATTCTGCAATGTTAACGATGTATGCAACTTCGCATCTCGTAATGATTACTCC。
Embodiment three: micromolecule polypeptide of tumor chalone for anti angiogenesis is realized by the following method in the present embodiment: the tumor chalone anti-angiogenic peptides is 25 peptides, remove the N-terminal Threonine of tumor chalone 25 peptides, with deputy methionine(Met) as aminoterminal, remove tyrosine, methionine(Met) and three amino acid of leucine of carboxyl terminal, transform 25 peptides as 21 peptides, obtain micromolecule polypeptide of tumor chalone for anti angiogenesis.
Embodiment four: the aminoacid sequence of tumor chalone micromolecule polypeptide fusion rotein is in the present embodiment:
MetProPheLeuPheCysAsnValAsnAspValCysAsnPheAlaSerArgGlyAspTyrSerGlyGlyAlaSerProPheLeuGluCysHisGlyArgGlyThrCysAsnTyrTyrSerAsnSer。
Embodiment five: the nucleotides sequence of tumor chalone micromolecule polypeptide fusion rotein is classified as in the present embodiment:
TATGCCGTTCTTATTCTGCAATGTTAACGATGTATGCAACTTCGCATCTCGTGGAGATTACTCCGGAGGAGCTAGCCCTTTCCTAGAATGTCATGGAAGAGGAACGTGCAACTACTACTCAAACTCC。
Embodiment six: the preparation method of tumor chalone micromolecule polypeptide fusion rotein makes by following steps in the present embodiment: 1, remove the N-terminal Threonine of tumor chalone 25 peptides, with deputy methionine(Met) as aminoterminal, remove tyrosine, methionine(Met) and three amino acid of leucine of carboxyl terminal, transform 25 peptides as 21 peptides; 2, the Asn with the 18th of 21 peptide changes Gly into, forms RGD (arginine-glycine-l-asparagine) sequence with the amino-acid residue on both sides then, obtains introducing 21 peptides of RGD sequence; 3, will introduce and add 2 Gly between 21 peptides of RGD sequence and 19 peptides and connect, obtain tumor chalone micromolecule polypeptide fusion rotein.
The nucleotide sequence of codes for tumor chalone angiogenesis inhibitor micromolecule polypeptide and fusion rotein thereof is according to the preferences design of intestinal bacteria to password in the present embodiment, recombinate with the pTYB2 carrier through Nde I and Sma I double digestion in the sense strand of codes for tumor chalone angiogenesis inhibitor micromolecule polypeptide and fusion rotein thereof and antisense strand phosphorylation annealing back, again transformed into escherichia coli BL21 (DE3).Genetic engineering bacterium BL21 (DE3) induces through IPTG, just can extract purifying micromolecule polypeptide of tumor chalone for anti angiogenesis and fusion rotein thereof with the chitin affinity column.The codes for tumor chalone angiogenesis inhibitor micromolecule polypeptide of the present invention's design and the sense strand of fusion rotein thereof and antisense strand are synthetic by the Nucleotide automatic DNA synthesizer DNA, available biological engineering method scale operation tumor chalone micromolecule polypeptide fusion rotein after recombinant conversion.
The aminoacid sequence of 19 peptides is in the present embodiment:
AlaSerProPheLeuGluCysHisGlyArgGlyThrCysAsnTyrTyrSerAsnSe r, the sense strand sequence of the DNA of 19 peptides of encoding is: TATGGCTAGCCCTTTCCTAGAATGTCATGGAAGAGGAACGTGCAACTACTACTCAA ACTCC.Improved 21 peptide ammino acid sequences are:
MetProPheLeuPheCysAsnValAsnAspValCysAsnPheAlaSerArgAsnAs pTyrSer, the DNA sense strand nucleotides sequence of 21 peptides of encoding is classified as:
TATGCCGTTCTTATTCTGCAATGTTAACGATGTATGCAACTTCGCATCTCGTAATGATTACTCC。The 21 peptide ammino acid sequences of introducing the RGD sequence are:
MetProPheLeuPheCysAsnValAsnAspValCysAsnPheAlaSerArgGlyAs pTyrSer, coding is introduced the DNA sense strand nucleotides sequence of 21 peptides of RGD sequence and is classified as:
TATGCCGTTCTTATTCTGCAATGTTAACGATGTATGCAACTTCGCATCTCGTGGAGATTACTCC。Present embodiment is introduced RGD21 peptide fusion protein recombinant plasmid to 19 peptides, 21 peptides, 21 peptides of introducing the RGD sequence and 19+ and has been carried out dna sequencing figure; As shown in Figure 1, Figure 2, Figure 3 and Figure 4: can confirm that by figure the aminoacid sequence of several micromolecule polypeptides in the present embodiment reaches to its coded DNA sequence is real, and present embodiment is fully feasible.
21 peptides of 19 peptides in the present embodiment, 21 peptides and introducing RGD sequence also all can be used for tumor treatment, but effect is compared with the tumor chalone micromolecule polypeptide fusion rotein in the present embodiment, effect but has than big-difference, find with experimentation on animals contrast back: 19 peptide tumour inhibiting rates are 42.41%, 21 peptide tumour inhibiting rates are 40.08%, the 21 peptide tumour inhibiting rates of introducing RGD are 46.88%, and the 19+ in the present embodiment introduces the inhibitory rate to 57.59% of RGD21 peptide fusion protein.
Tumor chalone micromolecule polypeptide in the present embodiment and the anti-tumor activity of micromolecule polypeptide fusion rotein and anti-angiogenic regeneration tests:
1, get the good Proliferation of Human Ovarian Cell of cultivation conditions, cell is divided into four groups and experimentizes, difference called after 19 peptide groups, 21 peptide groups, fusion rotein group and control group, with final concentration is 19 peptides of 44 μ g/mL, 21 peptides and fusion rotein are put into experimental group separately respectively, control group then adds the PBS of equal volume, cultivate 24h, the trysinization of each experimental group cell 0.25%, the PBS washing, 4 ℃ of glutaraldehyde are 24h fixedly, carry out transmission electron microscope observing, extremely shown in Figure 8 as Fig. 5: we find by the contrast experiment: behind the dosing 24h, the a large amount of cells of 19 peptide groups begin to occur the features of apoptosis form and change, and caryorrhexis or the complete disintegration of cell appear in some cells, but membrane structure is intact.The organoid form that small amounts of cells just appears in 21 peptide groups changes, and individual cells has the morphological feature of early apoptosis.During the fusion rotein group then occurs, non-viable apoptotic cell and a large amount of downright bad cells, membrane structure is destroyed, illustrate that the tumor killing effect of the 19+ introducing RGD21 peptide fusion protein in the present embodiment is better than other tumor chalone micromolecule polypeptide.
2, get the good Human umbilical vein endothelial cells of cultivation conditions, cell is divided into four groups and experimentizes, difference called after 19 peptide groups, 21 peptide groups, the fusion rotein group, with final concentration is 19 peptides of 44 μ g/mL, 21 peptides and fusion rotein are put into experimental group separately respectively, control group then adds the PBS of equal volume, cultivate 24h, the trysinization of each experimental group cell 0.25%, the PBS washing, 4 ℃ of glutaraldehyde are 24h fixedly, carries out transmission electron microscope observing, as Fig. 9 to shown in Figure 12: 19 peptides and 21 peptides are more or less the same to the influence of endotheliocyte: cell volume diminishes, the apoptosis form change in early stage appears in 1/3 cytoplasmic membrane swelling nearly.The apoptotic cell of fusion rotein group is more, and coming off and the change of organoid of apoptotic body arranged, and it is many that the tumor killing effect that the 19+ introducing RGD21 peptide fusion protein in the present embodiment be described is better than other tumor chalone small molecules. peptide.
3, get the good human cervical carcinoma cell of growth conditions, with ordinary method digestion, preparation cell suspension, counting, adjusting cell concn is 5 * 10
4Individual/mL.The cover glass of handling well is put into each hole of 6 orifice plates, the 1mL cell suspension is accurately inoculated in each hole, after cultivation 24h treats cell attachment, every kind of cell is divided into four groups and experimentizes, difference called after 19 peptide groups, 21 peptide groups, fusion rotein group, with final concentration is that 19 peptides, 21 peptides and the fusion rotein of 44 μ g/mL put into experimental group separately respectively, and control group adds the PBS of equal volume.Continue to cultivate 24h, take out cover glass, through dry air, paraformaldehyde solution is fixed, endogenous peroxydase blocking-up and cell are penetrating, mark, after signal transforms and analyzes, under fluorescent microscope and under the light microscopic, observe the quantity of fluorescence respectively, intensity and cellular form, change in color, the result as Figure 13 to scheming shown in the .16: a little less than light microscopic shows that down 21 peptides are to human cervical carcinoma cell effect, apoptotic cells is less, accidentally catch brown cell, the visible more staining cell of 19 peptide groups and some have the apoptotic cell that outstanding cell surface has apoptotic body, the effect of fusion rotein group is stronger than 19 peptides, illustrates that the knurl effect of killing of the 19+ introducing RGD21 peptide fusion protein in the present embodiment is better than other tumor chalone micromolecule polypeptide.
4, get the good Human umbilical vein endothelial cells of growth conditions, with ordinary method digestion, preparation cell suspension, counting, adjusting cell concn is 5 * 10
4Individual/mL.The cover glass of handling well is put into each hole of 6 orifice plates, the 1mL cell suspension is accurately inoculated in each hole, after cultivation 24h treats cell attachment, every kind of cell is divided into four groups and experimentizes, difference called after 19 peptide groups, 21 peptide groups, fusion rotein group, with final concentration is that 19 peptides, 21 peptides and the fusion rotein of 44 μ g/mL put into experimental group separately respectively, and control group adds the PBS of equal volume.Continue to cultivate 24h, take out cover glass, through dry air, paraformaldehyde solution is fixed, endogenous peroxydase is blocked and cell is penetrating, mark, after signal transforms and analyzes, observe quantity, intensity and cellular form, the change in color of fluorescence under light microscopic, result such as Figure 17 are to shown in Figure 20: visible 19 peptides under the mirror, 21 peptides are not obvious to the effect difference of endotheliocyte, and the endotheliocyte quantity of fusion rotein group apoptosis significantly increases.The knurl effect of killing that 19+ introducing RGD21 peptide fusion protein in the present embodiment is described is better than other tumor chalone micromolecule polypeptide.
5, detect the cell cycle experiment with flow cytometer: 4 groups of the Human umbilical vein endothelial cells in the vegetative period of taking the logarithm, 19 peptides, 21 peptides, RGD21 peptide and the fusion rotein that add final concentration respectively and be 40 μ g/mL are as experimental group, the PBS that another group adds equal volume organizes in contrast, after cultivating 24h, the cell of control group and experimental group is washed 2 times with PBS, remove non-viable non-apoptotic cell and apoptotic cell fragment, the cell of laboring cycle detection.Single cell suspension is made in 0.25% trysinization, and 1, the centrifugal 10min of 000r/min abandons supernatant, and PBS washes 2 times, and every pipe adds the ice ethanol of 1～1.5mL70%, makes the cell concn of each sample be at least 0.5～1.0 * 10
6Individual/mL, mixing makes it to be single cell suspension gently, 4 ℃ of fixing 24h.Carry out the detection of flow cytometer cell cycle with CycleTESTTM PLUS DNA Reagent Kit.The detected result of human endothelial cell is seen Figure 21 to Figure 25, compare with control group, flow cytometer detects the cell cycle and the apoptosis of huve cell, the control group G1 phase is that 47%, 19 peptides are that 64.46%, 21 peptides are 62.79%, the RGD21 peptide is 69.86%, fusion rotein is 64.96%, the apoptosis rate of RGD 21 peptides and fusion rotein group is respectively 6.96% and 3.76%, the fusion rotein that present embodiment is described has very strong short endothelial cell apoptosis activity, can suppress the generation of new vessel effectively, the fusion rotein group also detects apoptosis.
6, the intravital knurl that presses down of animal is tested: it is 3 * 10 that mouse H22 liver cancer cell is adjusted to cell concn with physiological saline
7/ ml.The male mouse of kunming of body weight 18-22g, every mouse is in right side armpit subcutaneous vaccination 0.2ml.When treating behind the 2d that mouse can touch subcutaneous nodule respectively by 5mg (kgd)
-1Abdominal injection 19 peptides, 21 peptides, RGD21 peptide and fusion rotein, negative control group is injected the physiological saline of same volume, successive administration 9d.Put to death mouse next day, get knurl under the aseptic condition, claim that knurl is heavy and calculate tumour inhibiting rate, the tumour inhibiting rate calculation formula is: (1-administration group knurl weight/control group knurl is heavy) * 100%.Press down the knurl experimental result in the animal body and see Figure 26 to Figure 30,19 peptide tumour inhibiting rates are that 42.41%, 21 peptide tumour inhibiting rate is 40.08%, and the 21 peptide tumour inhibiting rates of introducing RGD are 46.88%, and the 19+ among the present invention introduces the inhibitory rate to 57.59% of RGD21 peptide fusion protein.It is obvious that the in vitro tests anticancer effect appears in cancer therapy drug regular meeting; the phenomenon that the animal vivo test antitumour activity is then had a greatly reduced quality; and fusion rotein confirms that by pressing down the knurl experimental result in the animal body tumor chalone micromolecule polypeptide fusion rotein has definite high anti-tumor activity in the present embodiment.
Sequence table
＜110〉Harbin Medical University
＜120〉micromolecule polypeptide of tumor chalone for anti angiogenesis, fusion rotein and preparation method for both
<210>1
<211>127
<212>DNA
＜213〉artificial sequence
<220>
＜221〉encoding sequence
<222>(2)...(127)
＜223〉19 peptides+introducing RGD21 peptide fusion protein
<400>1
t?atg?ccg?ttc?tta?ttc?tgc?aat?gtt?aac?gat?gta?tgc?aac?ttc?gca?tct
Met?Pro?Phe?Leu?Phe?Cys?Asn?Val?Asn?Asp?Val?Cys?Asn?Phe?Ala?Ser
1 5 10 15
cgt?gga?gat?tac?tcc?gga?gga?gct?agc?cct?ttc?cta?gaa?tgt?cat?gga
Arg?Gly?Asp?Tyr?Ser?Gly?Gly?Ala?Ser?Pro?Phe?Leu?Glu?Cys?His?Gly
20 25 30
aga?gga?acg?tgc?aac?tac?tac?tca?aac?tcc
Arg?Gly?Thr?Cys?Asn?Tyr?Tyr?Ser?Asn?Ser
35 40
<210>2
<211>42
<212>PRT
＜213〉artificial sequence
<220>
＜223〉19 peptides+introducing RGD21 peptide fusion protein
<400>2
Met?Pro?Phe?Leu?Phe?Cys?Asn?Val?Asn?Asp?Val?Cys?Asn?Phe?Ala?Ser
1 5 10 15
Arg?Gly?Asp?Tyr?Ser?Gly?Gly?Ala?Ser?Pro?Phe?Leu?Glu?Cys?His?Gly
20 25 30
Arg?Gly?Thr?Cys?Asn?Tyr?Tyr?Ser?Asn?Ser
35 40
<210>3
<211>64
<212>DNA
＜213〉artificial sequence
<220>
＜221〉encoding sequence
<222>(2)...(64)
＜223〉tumor chalone angiogenesis inhibitor small molecules 21 peptides
<400>3
t?atg?ccg?ttc?tta?ttc?tgc?aat?gtt?aac?gat?gta?tgg?aac?ttc?gca?tct
Met?Pro?Phe?Leu?Phe?Cys?Asn?Val?Asn?Asp?Val?Cys?Asn?Phe?Ala?Ser
1 5 10 15
cgt?aat?gat?tac?tcc
Arg?Asn?Asp?Tyr?Ser
20
<210>4
<211>21
<212>PRT
＜213〉artificial sequence
<220>
＜223〉tumor chalone angiogenesis inhibitor small molecules 21 peptides
<400>4
Met?Pro?Phe?Leu?Phe?Cys?Asn?Val?Asn?Asp?Val?Cys?Asn?Phe?Ala?Ser
1 5 10 15
Arg?Asn?Asp?Tyr?Ser
20
Claims (6)
1, micromolecule polypeptide of tumor chalone for anti angiogenesis is characterized in that the aminoacid sequence of micromolecule polypeptide of tumor chalone for anti angiogenesis is: MetProPheLeuPheCysAsnValAsnAspValCysAsnPheAlaSerArgAsnAs pTyrSer.
2, micromolecule polypeptide of tumor chalone for anti angiogenesis according to claim 1 is characterized in that the nucleotides sequence of micromolecule polypeptide of tumor chalone for anti angiogenesis is classified as: TATGCCGTTCTTATTCTGCAATGTTAACGATGTATGCAACTTCGCATCTCGTAATG ATTACTCC.
3, the preparation method of micromolecule polypeptide of tumor chalone for anti angiogenesis, it is characterized in that micromolecule polypeptide of tumor chalone for anti angiogenesis realizes by the following method: the tumor chalone anti-angiogenic peptides is 25 peptides, remove the N-terminal Threonine of tumor chalone 25 peptides, with deputy methionine(Met) as aminoterminal, remove tyrosine, methionine(Met) and three amino acid of leucine of carboxyl terminal, transform 25 peptides as 21 peptides, obtain micromolecule polypeptide of tumor chalone for anti angiogenesis.
4, tumor chalone micromolecule polypeptide fusion rotein is characterized in that the aminoacid sequence of tumor chalone micromolecule polypeptide fusion rotein is: MetProPheLeuPheCysAsnValAsnAspValCysAsnPheAlaSerArgGlyAs pTyrSerGlyGlyAlaSerProPheLeuGluCysHisGlyArgGlyThrCysAsnT yrTyrSerAsnSer.
5, tumor chalone micromolecule polypeptide fusion rotein according to claim 4 is characterized in that the nucleotides sequence of tumor chalone micromolecule polypeptide fusion rotein is classified as: TATGCCGTTCTTATTCTGCAATGTTAACGATGTATGCACTTCGCATCTCGTGGAGA TTACTCCGGAGGAGCTAGCCCTTTCCTAGAATGTCATGGAAGAGGAACGTGCAACT ACTACTCAAACTCC.
6, the preparation method of tumor chalone micromolecule polypeptide fusion rotein, the preparation method who it is characterized in that tumor chalone micromolecule polypeptide fusion rotein makes by following steps: 1, remove the N-terminal Threonine of tumor chalone 25 peptides, with deputy methionine(Met) as aminoterminal, remove tyrosine, methionine(Met) and three amino acid of leucine of carboxyl terminal, transform 25 peptides as 21 peptides; 2, the Asn with the 18th of 21 peptide changes Gly into, forms the RGD sequence with the amino-acid residue on both sides then, obtains introducing 21 peptides of RGD sequence; 3, will introduce and add 2 Gly between 21 peptides of RGD sequence and 19 peptides and connect, obtain tumor chalone micromolecule polypeptide fusion rotein.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103554266A (en) * | 2013-10-11 | 2014-02-05 | 哈尔滨医科大学 | Tumstatin fusogenic peptide with anti-tumor effect, and preparation method and application thereof |
| CN104593402A (en) * | 2015-01-21 | 2015-05-06 | 吉林农业大学 | Method for producing recombinational anti-tumor 19 peptide by applying escherichia coli as biological reactor |
| CN103755799B (en) * | 2014-01-08 | 2015-11-25 | 哈尔滨医科大学 | A kind of tumor chalone 30 peptide with antitumor action and its preparation method and application |
| CN110105453A (en) * | 2011-08-17 | 2019-08-09 | 科罗拉多大学董事会法人团体 | Transferrin-tumor chalone fused protein and its generation and application method |
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2007
- 2007-05-23 CN CNA2007100722510A patent/CN101092452A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110105453A (en) * | 2011-08-17 | 2019-08-09 | 科罗拉多大学董事会法人团体 | Transferrin-tumor chalone fused protein and its generation and application method |
| CN103554266A (en) * | 2013-10-11 | 2014-02-05 | 哈尔滨医科大学 | Tumstatin fusogenic peptide with anti-tumor effect, and preparation method and application thereof |
| CN103755799B (en) * | 2014-01-08 | 2015-11-25 | 哈尔滨医科大学 | A kind of tumor chalone 30 peptide with antitumor action and its preparation method and application |
| CN104593402A (en) * | 2015-01-21 | 2015-05-06 | 吉林农业大学 | Method for producing recombinational anti-tumor 19 peptide by applying escherichia coli as biological reactor |
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