CN1060521C - 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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- CN1060521C CN1060521C CN 97107112 CN97107112A CN1060521C CN 1060521 C CN1060521 C CN 1060521C CN 97107112 CN97107112 CN 97107112 CN 97107112 A CN97107112 A CN 97107112A CN 1060521 C CN1060521 C CN 1060521C
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Abstract
The present invention relates to a method for gene clone of an inhibitory factor for the hyperplasia of inner blood vessel cells in real tumors of a human body and application thereof in the treatment of resisting tumor blood vessel regeneration. The present invention is characterized in that a, human endostatin genes are sieved from a human liver cDNA library via PCR from human collagen 18 gene 1503 to a 2055cDNA active segment; b, the human endostatin genes are in enzyme cutting via incision enzyme to obtain two kinds of gene segments; c, the human endostatin genes and the two gene segments in the b are expressed in colibacillus via a gene cloning method, and have the functions of inhibiting the hyperplasia of inner blood vessel cells and the regenerating activity of tumor blood vessels to be used as biological products for treating real tumors.
Description
The present invention is human collagen 18 (human type X VIII collagen) gene 1503 to 2055cDNA active fragments (Genomics 1994:19,494-499, see document 1) screen from people's liver cDNA storehouse by PCR method that to 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 endostatin gene is to screen by PCR method from human collagen 18 (human type X VIII 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 Nde I and two kinds of restriction endonucleases of BamH I
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 Pst I restriction endonuclease to cut 162 Nucleotide or adopt the Pst I and Sma I 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.
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 Nde I and BamH I 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 Pst I restriction endonuclease to cut 162 Nucleotide from people endostatin gene N-terminal, or adopts Pst I and Sma I 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 for suppressing people's entity tumor revascularization and is better than in theory recombinating with mouse Endostatin albumen, this is to be because adopt people's endostatin gene outcome to be used for the treatment of people's entity tumor Belong to homology, do not repelled by body. Mouse endostatin albumen and recombinant human endostatin wherein recombinate The amino acid sequence of albumen only has 85.33% homology (seeing attached list). Recombinant human endostatin albumen selectivity Act on the capillary endothelial cell of propagation, inoperative to many other cells of body, and entity tumor is thin Born of the same parents breed expansion and must follow intratumoral vasculature constantly to regenerate, and the capillary endothelium of regeneration is constantly bred in the tumour, Existing capillary endothelial cell is also constantly bred in the tumour, so recombinant human endostatin albumen selectivity Suppress these constantly endothelial cells of propagation, make tumor vessel can not regenerate 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, from And entity tumor produces atrophy, apoptosis of tumor cells necrosis. Another characteristics of the present invention have provided the treatment tumour Another means, then can reach from the hemotrophic nutrition supply that suppresses entity tumor the purpose for the treatment of and this Therapy also can be carried out in conjunction with existing many therapies for tumour cell (such as radiotherapy, chemotherapy etc.) combination, from And treat simultaneously from suppressing entity tumor revascularization and indirect, direct two kinds of therapies of kill tumor cell, Can greatly improve its curative effect like this. The 3rd characteristics of the present invention are that people endostatin gene is in Escherichia coli Expression and purification obtains people endostatin albumen, and technology is relatively simple, and cost is relatively cheap, is beneficial to and applies. Activated two the recombinant human endostatin protein fragments molecular weight of tool are little, are easy to enter in the body. Especially Little a kind of recombinant human endostatin protein fragments is 42 amino acid only, are convenient to from now on artificial synthetic and extensive Produce.
Subordinate list: a kind of people's noumenal tumour vascular endothelial cell proliferation supressor (human endostatin)
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 (1)
1. the gene clone method of people's noumenal tumour vascular endothelial cell proliferation supressor, it is characterized in that: a: the gene clone method of people endostatin gene is to screen by PCR method from the human collagen shown in subordinate list 18 (human type X VIII 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 Nde I and two kinds of restriction endonucleases of BamH I
2With PET15b, PET9c genophore, then at expression in escherichia coli, purifying obtains activated recombinant human endostatin protein; B. adopt Pst I restriction endonuclease to cut the gene fragment clone that obtains behind 162 Nucleotide and go into PMAL-c from people endostatin gene N-terminal
2In the genophore; Adopt Pst I and Sma I restriction endonuclease respectively after people endostatin gene N-terminal and C-terminal cut 162 and 264 Nucleotide resulting gene fragment clone go into PET15b and the PET9c genophore, respectively at expression in escherichia coli, purifying obtains activated two kinds of recombinant human endostatin protein fragments then.
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 CN1177005A (en) | 1998-03-25 |
| CN1060521C true CN1060521C (en) | 2001-01-10 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| 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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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3840262B2 (en) | 1995-10-23 | 2006-11-01 | ザ チルドレンズ メディカル センター コーポレイション | Therapeutic anti-angiogenic compositions and methods |
| US6346510B1 (en) * | 1995-10-23 | 2002-02-12 | 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 |
| 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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1997
- 1997-09-10 CN CN 97107112 patent/CN1060521C/en not_active Expired - Lifetime
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