CN1177005A - Method for gene clone of inhibitory factor for hyperplasia of inner blood vessel cells in human body's real tumor, and using same to anti tumer blood vessel regeneration - Google Patents
Method for gene clone of inhibitory factor for hyperplasia of inner blood vessel cells in human body's real tumor, and using same to anti tumer blood vessel regeneration Download PDFInfo
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- CN1177005A CN1177005A CN 97107112 CN97107112A CN1177005A CN 1177005 A CN1177005 A CN 1177005A CN 97107112 CN97107112 CN 97107112 CN 97107112 A CN97107112 A CN 97107112A CN 1177005 A CN1177005 A CN 1177005A
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- 210000004204 blood vessel Anatomy 0.000 title abstract 4
- 206010020718 hyperplasia Diseases 0.000 title 1
- 230000002401 inhibitory effect Effects 0.000 title 1
- 239000002299 complementary DNA Substances 0.000 claims abstract description 10
- 101500026378 Homo sapiens Endostatin Proteins 0.000 claims abstract description 9
- 210000004185 liver Anatomy 0.000 claims abstract description 7
- 230000000694 effects Effects 0.000 claims abstract description 6
- 230000035755 proliferation Effects 0.000 claims abstract description 3
- 108010079505 Endostatins Proteins 0.000 claims description 21
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- 239000012634 fragment Substances 0.000 claims description 15
- 210000001043 capillary endothelial cell Anatomy 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
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- 230000004663 cell proliferation Effects 0.000 claims description 7
- 210000003556 vascular endothelial cell Anatomy 0.000 claims description 7
- 102100031162 Collagen alpha-1(XVIII) chain Human genes 0.000 claims description 6
- 241000588724 Escherichia coli Species 0.000 claims description 6
- 108091008146 restriction endonucleases Proteins 0.000 claims description 5
- 230000000118 anti-neoplastic effect Effects 0.000 claims description 4
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- 108010037936 CCCGGG-specific type II deoxyribonucleases Proteins 0.000 claims description 3
- 108010035532 Collagen Proteins 0.000 claims description 3
- 102000008186 Collagen Human genes 0.000 claims description 3
- 108010001463 Collagen Type XVIII Proteins 0.000 claims description 3
- 102000047200 Collagen Type XVIII Human genes 0.000 claims description 3
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims description 3
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 3
- 230000001919 adrenal effect Effects 0.000 claims description 3
- 210000004899 c-terminal region Anatomy 0.000 claims description 3
- 229920001436 collagen Polymers 0.000 claims description 3
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- 230000037396 body weight Effects 0.000 claims description 2
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention can inhibit the proliferation of blood vessel endothelium and resist the regeneration activity of tumour blood vessel, so that it can be used as a biological prepn. in the treatment of tumour. The characterisitc lies in that: (a). human endostain gene is obtd. from human collagen-albumen 18gene 1503-2055 cDNA active section and selected from human liver cDNA pool through PCR; (b) two kinds of gene section are obtd. from human endostain gene by endonuclease; (c). human endostatin gene and two kinds of gene section obtd. in term (b) are expressed in colon bacillus through the gene clone method.
Description
The present invention is human collagen 18 (human type XVIII collagen) gene 1503 to 2055cDNA active fragments (Genomics 1994:19,494-499 sees document 1) screen from people's liver cDNA storehouse by PCR method and obtain a kind of people's noumenal tumour vascular endothelial cell proliferation supressor (human endostatin)-be the human endostatin.This kind factor is that single composition can be used as biological products and is used among the anti entity tumour revascularization treatment.
Noumenal tumour clinical treatment method is varied at present, but be to adopt diverse ways mostly at different tumours, and the overwhelming majority is to treat at tumour cell, blocks or to suppress the therapy of noumenal tumour revascularization and blood supply aspect less with inhibitor or blocker.Carrying out the antineoplastic vascular regenerative therapy with recombinant human endostatin albumen does not at home and abroad appear in the newspapers as yet.
The object of the present invention is to provide a species specificity supressor, suppress different noumenal tumour capillary endothelial cell propagation and revascularizations, blood supply and source of nutrition with blocking-up or single-minded inhibition tumour make the noumenal tumour atrophy, the apoptosis of tumor cells necrosis.
Purpose of the present invention can reach by following measure:
A kind of people's noumenal tumour vascular endothelial cell proliferation supressor (human endostatin)-be human endostatin's gene clone method and as the application of biological products among the treatment noumenal tumour is characterized in that:
A. the gene clone method of people endostat in gene is to screen by PCR method from human collagen 18 (human typeXVIII collagen) gene 1503 to 2055cDNA active fragmentss to obtain from people's liver cDNA storehouse, at first prepare two kinds of primers and be respectively ATTCATATGCACAGCCACCGCGACTTCCAGCCG and GCCGGATCCCTACTTGGAGGCAGTCATGAAGCT, with people's liver cDNA storehouse is template, with the PCR condition be 94 ℃ 1 minute, 55 ℃ of 1 minute and 72 ℃ amounted to 32 in 1 minute and circulate and obtain people endostatin gene (gene nucleotide series is seen attached list), were cloned into PMAL-c by NdeI and two kinds of restriction endonucleases of BamHI
2(NewEngland Biolabs Catalog sees document 2) and PET19b, PET9c (Novagen.Inc Catalog sees document 3) genophore, then at expression in escherichia coli, purifying obtains activated recombinant human endostatin protein;
B. adopt the PstI restriction endonuclease to cut 162 Nucleotide or adopt PstI and the SmaI restriction endonuclease cuts gained gene fragment behind 162 and 264 Nucleotide and is cloned into PMAL-c from people endostatin gene N-terminal and C-terminal respectively from people endostatin gene N-terminal
2With PET15b, PET9c genophore, then at expression in escherichia coli, purifying obtains activated recombinant human endostatin protein fragments;
C. described 2 the recombinant protein fragments of recombinant human endostat in protein that purifying is obtained and b item add and contain in the bovine adrenal capillary endothelial cell nutrient solution of bFGF, cultivate after 3 days by cell counting, measure their capillary endothelial cell inhibited proliferation activity intensity.
People endostatin gene of the present invention can obtain by gene clone methods such as PCR.Recombinant human endostatin albumen can obtain by the engineered protein purification process.
Embodiment:
1, synthetic two kinds of primers are respectively ATTCATATGCACAGCCACCGCGACTTCCAGCCG and GCCGGATCCCTACTTGGAGGCAGTCATGAAGCT.
2, be template with 1 microlitre people liver cDNA, add each 2 microlitre (concentration is every microlitre 0.1 microgram) of 1 described two kinds of primer, add 5 microlitre 2.5mMdNTP, 5 microlitres, 10 PCR damping fluids and 1 microlitre Taq or PwoDNA polysaccharase, on the PCR instrument 94 ℃ 1 minute, 55 ℃ of 1 minute and 72 ℃ amounted to 32 circulations in 1 minute, obtaining people endostatin gene with the separation of 1.5% agarose gel electrophoresis then.
3, adopt NdeI and BamHI endonuclease digestion from 2 people endostatin genes that obtain, be cloned into PMAL-c
2Among PET15b, PET9c genophore, express recombinant people endostatin albumen in escherichia coli respectively then, adopt amylose resin beads according to producer's handbook (NewEngland Biolabs Catalog sees document 2) purification of recombinant human endostatin albumen and routine biochemistry means of purification purification of recombinant human endostatin albumen (gal4 amino acid is seen attached list in proper order) such as employing ultrafiltration, high speed centrifugation, gel-filtration respectively.
4, people endostatin gene adopts the PstI restriction endonuclease to cut 162 Nucleotide from people endostatin gene N-terminal, or adopts PstI and SmaI restriction endonuclease to cut two kinds of gene fragments of gained behind 162 and 264 Nucleotide respectively and be cloned into PMAL-c from the N-terminal of people endostatin gene and C-terminal
2Among PET15b, PET9C genophore, obtain recombinant human endostatin activated protein fragment by 3 described method purifying then.
5, adopt bovine adrenal capillary endothelial cell (BCE) to be cultured on 24 well culture plates, cell concn is every milliliter of 25000 cells, every milliliter of recombinant human endostatin protein fragments and two kinds of different recombinant human endostatin protein fragments that add 1 nanogram bFGF and 0.008 microgram to 1 microgram different concns are cultivated after 3 days and are measured the activity that suppresses capillary endothelial cell propagation by cell counting.
6, the mouse hypodermic inoculation lung carcinoma cell makes tumour grow to 100-200mm
3The time, mouse is divided into three groups at random, one group compares, distinguish subcutaneous injection recombinant human endostatin albumen and a kind of people endostatin protein fragments for two groups in addition, every day, the per kilogram of body weight injection was 20 milligrams, injection is 20 days continuously, to observe the effect that suppresses the noumenal tumour revascularization in animal body.
The present invention has following advantage compared with prior art:
Recombinant human endostatin albumen is used to suppress people's noumenal tumour revascularization and is better than the albumen with mouse reorganization endostatin in theory, this is because adopting people's endostatin gene product to be used for the treatment of people's noumenal tumour is to belong to homology, is not repelled by body.Wherein recombinate mouse endostatin albumen and the proteic aminoacid sequence of recombinant human endostatin only has 85.33% homology (seeing attached list).Recombinant human endostatin albumen specificity acts on the capillary endothelial cell of propagation, inoperative to many other cells of body, and the expansion of solid tumor cell propagation must follow intratumoral vasculature constantly to regenerate, the regenerated capillary endothelium is constantly bred in the tumour, existing capillary endothelial cell is also constantly bred in the tumour, so recombinant human endostatin albumen specificity suppresses these constantly endotheliocytes of propagation, tumor vessel can not be regenerated or suppress revascularization, tumor tissues can not get enough nutrition supplies, also can not get the stimulation of tumour cell that vascular endothelial cell proliferation brings, thereby noumenal tumour produces atrophy, apoptosis of tumor cells necrosis.Another characteristics of the present invention have provided another means of treatment tumour, then can reach the purpose of treatment from the hemotrophic nutrition supply that suppresses noumenal tumour, and this therapy also can be in conjunction with existing many therapies at tumour cell (as radiotherapy, chemotherapy etc.) in conjunction with carrying out, thereby treat simultaneously with indirect, direct two kinds of therapies of kill tumor cell from suppressing the noumenal tumour revascularization, can improve its curative effect greatly like this.The 3rd characteristics of the present invention are that people endostatin gene obtains people endostatin albumen at the expression in escherichia coli purifying, and technology is simple relatively, and cost is cheap relatively, is beneficial to and applies.It is little to have active two recombinant human endostatin protein fragments molecular weight, is easy to enter in the body.Especially Zui Xiao a kind of recombinant human endostatin protein fragments 42 amino acid only are convenient to from now on synthetic and scale operation.Subordinate list: a kind of people's noumenal tumour vascular endothelial cell proliferation supressor (human endostatin)
The cDNA nucleotide sequence
The?nucleotide?sequence?of?human?endostatin?cDNA
CATATGCACAGCCACCGCGACTTCCAGCCGGTGCTCCACCTGGTTGCGCTCAACAGCCCCCTGTCAGGCGGCATGHuman H S H R D F Q P V L H L V A L N S P L S G G MMouse T Q T
CGGGGCATCCGCGGGGCCGACTTCCAGTGCTTCCAGCAGGCGCGGGCCGTGGGGCTGGCGGGCACCTTCCGCGCCHuman R G I R G A D F Q C F Q Q A R A V G L A G T F R AMouse S
TTCCTGTCCTCGCGCCTGCAGGACCTGTACAGCATCGTGCGCCGTGCCGACCGCGCAGCCGTGCCCATCGTCAACHuman F L S S R L Q D L Y S I V R R A D R A A V P I V NMouse G S
CTCAAGGACGAGCTGCTGTTTCCCAGCTGGGAGGCTCTGTTCTCAGGCTCTGAGGGTCCGCTGAAGCCCGGGGCAHuman L K D E L L F P S W E A L F S G S E G P L K P G AMouse V S D S Q Q V Q
CGCATCTTCTCCTTTGACGGCAAGGACGTCCTGAGGCACCCCACCTGGCCCCAGAAGAGCGTGTGGCATGGCTCGHuman R I F S F D G K D V L R H P T W P Q K S V W H G SMouse R A
GACCCCAACGGGCGCAGGCTGACCGAGAGCTACTGTGAGACGTGGCGGACGGAGGCTCCCTCGGCCACGGGCCAGHuman D P N G R R L T E S Y C E T W R T E A P S A T G QMouse S M T T G
GCCTCCTCGCTGCTGGGGGGCAGGCTCCTGGGGCAGAGTGCCGCGAGCTGCCATCACGCCTACATCGTGCTCTGCHuman A S S L L G G R L L G Q S A A S C H H A Y I V L CMouse S E K N S
ATTGAGAACAGCTTCATGACTGCCTCCAAGTGAHuman I E N S F M T A S KMouse S (F) * Human (people), Mouse (mouse)
Claims (2)
1, a kind of people's noumenal tumour vascular endothelial cell proliferation supressor (human endostatin)-be human endostatin's gene clone method is characterized in that:
A. the gene clone method of people endostatin gene is to screen by PCR method from human collagen 18 (human typeXVIII collagen) gene 1503 to 2055cDNA active fragmentss to obtain from people's liver cDNA storehouse, at first prepare two kinds of primers and be respectively ATTCATATGCACAGCCACCGCGACTTCCAGCCG and GCCGGATCCCTACTTGGAGGCAGTCATGAAGCT, with people's liver cDNA storehouse is template, with the PCR condition be 94 ℃ 1 minute, 55 ℃ of 1 minute and 72 ℃ amounted to 32 in 1 minute and circulate and obtain people endostatin gene, were cloned into PMAL-c by NdeI and two kinds of restriction endonucleases of BamHI
2With PET19b, PET9c genophore, then at expression in escherichia coli, purifying obtains activated recombinant human endostatin protein;
B. adopt the PstI restriction endonuclease to cut 162 Nucleotide or adopt PstI and the SmaI restriction endonuclease cuts gained gene fragment behind 162 and 264 Nucleotide and is cloned into PMAL-c from people endostatin gene N-terminal and C-terminal respectively from people endostatin gene N-terminal
2With PET15b, PET9c genophore, then at expression in escherichia coli, purifying obtains activated recombinant human endostatin protein fragments;
C. described 2 the recombinant protein fragments of recombinant human endostatin protein that purifying is obtained and b item add and contain in the bovine adrenal capillary endothelial cell nutrient solution of bFGF, cultivate after 3 days by cell counting, measure their capillary endothelial cell inhibited proliferation activity intensity.
2, a kind of people's noumenal tumour vascular endothelial cell proliferation supressor (human endostatin) is characterized in that as the application of biological products among the treatment noumenal tumour:
A. people endostatin albumen goes into to inoculate the mouse of lung carcinoma cell by 2 milligrams of subcutaneous injections of per kilogram of body weight, to measure its antineoplastic vascular regeneration;
B. recombinant human endostatin albumen and two kinds of fragments thereof are prepared into biological products and are used for the treatment of noumenal tumour according to their antineoplastic vascular regeneration as single agent respectively.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 97107112 CN1060521C (en) | 1997-09-10 | 1997-09-10 | Method for gene clone of inhibitory factor for hyperplasia of inner blood vessel cells in human body's real tumor, and using same to anti tumer blood vessel regeneration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 97107112 CN1060521C (en) | 1997-09-10 | 1997-09-10 | Method for gene clone of inhibitory factor for hyperplasia of inner blood vessel cells in human body's real tumor, and using same to anti tumer blood vessel regeneration |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1177005A true CN1177005A (en) | 1998-03-25 |
| CN1060521C CN1060521C (en) | 2001-01-10 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 97107112 Expired - Lifetime CN1060521C (en) | 1997-09-10 | 1997-09-10 | Method for gene clone of inhibitory factor for hyperplasia of inner blood vessel cells in human body's real tumor, and using same to anti tumer blood vessel regeneration |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000026368A2 (en) * | 1998-10-30 | 2000-05-11 | The Children's Medical Center Corporation | Therapeutic antiangiogenic endostatin compositions |
| AU2004202593B2 (en) * | 1995-10-23 | 2008-01-03 | The Children's Medical Center Corporation | Therapeutic antiangiogenic endostatin compositions |
| US7655458B2 (en) | 1995-10-23 | 2010-02-02 | The Children's Medical Center Corporation | Nucleic acid molecules encoding endostatin protein and peptide fragments thereof |
| CN105646701A (en) * | 2016-03-29 | 2016-06-08 | 徐根兴 | Recombinant human endostatin protein with different amino acid structures, method for preparing recombinant human endostatin protein and application thereof |
-
1997
- 1997-09-10 CN CN 97107112 patent/CN1060521C/en not_active Expired - Lifetime
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2004202593B2 (en) * | 1995-10-23 | 2008-01-03 | The Children's Medical Center Corporation | Therapeutic antiangiogenic endostatin compositions |
| US7655458B2 (en) | 1995-10-23 | 2010-02-02 | The Children's Medical Center Corporation | Nucleic acid molecules encoding endostatin protein and peptide fragments thereof |
| WO2000026368A2 (en) * | 1998-10-30 | 2000-05-11 | The Children's Medical Center Corporation | Therapeutic antiangiogenic endostatin compositions |
| WO2000026368A3 (en) * | 1998-10-30 | 2000-08-10 | Childrens Medical Center | Therapeutic antiangiogenic endostatin compositions |
| CN105646701A (en) * | 2016-03-29 | 2016-06-08 | 徐根兴 | Recombinant human endostatin protein with different amino acid structures, method for preparing recombinant human endostatin protein and application thereof |
| CN105646701B (en) * | 2016-03-29 | 2019-03-05 | 徐根兴 | Recombinant human endostatin albumen of different aminoacids structure and its preparation method and application |
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| Publication number | Publication date |
|---|---|
| CN1060521C (en) | 2001-01-10 |
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