CN102409056A - Associated gene sequence for inhibiting angiogenesis - Google Patents
Associated gene sequence for inhibiting angiogenesis Download PDFInfo
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- CN102409056A CN102409056A CN2011102706415A CN201110270641A CN102409056A CN 102409056 A CN102409056 A CN 102409056A CN 2011102706415 A CN2011102706415 A CN 2011102706415A CN 201110270641 A CN201110270641 A CN 201110270641A CN 102409056 A CN102409056 A CN 102409056A
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Abstract
The invention discloses a gene sequence for inhibiting a blood vessel from generating activity associated protein. The gene sequence is a DNA (deoxyribonucleic acid) sequence which is artificially synthesized by an overlap PCR (polymerase chain reaction) method. According to the invention, the gene can be subjected to recombinant expression to produce proteins, and the purified proteins can be used as a medicament for treating angiodysplasia.
Description
Technical field
The present invention relates to the gene field in the molecular biology, is about suppressing the relevant gene order of angiogenic activity.
Background technology
The propagation of normal adult human vascular endothelial remains static, and does not have new vasculogenesis.But under some pathologic condition, the propagation of vascular endothelial cell is in the immobilized state and is broken vascular endothelial cell proliferation then, new vasculogenesis.
So far the chemicals that does not still have effective angiogenesis inhibiting.Angiostatin is the medicine that one type of specificity suppresses vascular endothelial cell proliferation, does not develop immunity to drugs and toxicity.Research Angiostatin treatment angiogenesis is current research focus.
Summary of the invention
The purpose of this invention is to provide a kind of new gene order that suppresses vasculogenesis.Be through the synthetic section of DNA sequence of dna sequence dna compound method, this segment DNA sequence be reconstituted in the pichia spp express that expression product activation analysis proof has and suppresses vascular endothelial cell proliferation and the activity that suppresses new vasculogenesis.SEQ ID NO.1 is said in its nucleotide sequence such as the sequence table; SEQ ID NO.2 is said in its aminoacid sequence such as the sequence table.
The object of the invention is realized through following technical measures:
1.DNA sequence is synthetic.
Design following dna sequence dna, and carry out its sequence with the intussusception PCR method and synthesize.With its sequence called after scl.
ATGCACGGAGACTCAGAATACAACATCATGTTTGGTCCCGACATCTGTGGCCCTGGCAC?CAAGAAGGTTCATGTCATCTTCAACTACAAGGGCAAGAACGTGCTGATCAACAAGGACA?TCCGTTGCAAGGATGATGAGTTTACACACCTTACACTGATTGTGCGGCCAGACAACCAT?CATCATCATCATCATTAG
2.DNA the proof of sequence encoding protein function
(1) dna sequence dna is reconstituted in the yeast and expresses
Through the effect of DNA restriction endonuclease and T4DNA ligase enzyme, with the MCS of above synthetic dna fragmentation insertion yeast expression vector pPIC9K, with its expression vector called after pPIC9K-scl (accompanying drawing 1).Electricity consumption is changeed method pPIC9K-scl is transformed pichia spp GS115, obtains engineering bacteria GS115 (pPIC9K-scl).With BMGY-BMMY GS115 (pPICP9K-scl) the abduction delivering Scl albumen that ferments.
(2) expression product activation analysis
Fermented liquid supernatant is crossed Ni-Agarose His label protein purification column and is carried out purifying (accompanying drawing 2), behind the liquid process suction filtration desalination that contains target protein of results, suppresses human umbilical vein endothelial cell propagation and suppresses the test of chick chorioallantoic membrane vasculogenesis.The result is that Scl albumen brings out human vascular endothelial apoptosis (accompanying drawing 3), suppresses chicken embryo fine hair allantois new vessel and generates, and its restraining effect to the chick chorioallantoic membrane vasculogenesis is better than Angiostatin (accompanying drawing 4).These results show that the albumen of scl genes encoding has the activity that suppresses vascular endothelial cell proliferation and the generation of inhibition new vessel, and scl suppresses vasculogenesis genes involved sequence.
Advantage of the present invention:
Angiostatin has important purposes in the treatment vascular proliferative disease.Though it is multiple that the Angiostatin of having found has, Angiostatin gene order of the present invention is little, and the medicine that molecular weight is little helps in the intravital transmission of machine, and the effect of Scl albumen inhibition vasculogenesis is stronger.
Description of drawings
Accompanying drawing 2. purification of Recombinant Scl albumen
M. albumen Marker; 2. purified recombinant Scl albumen.
Accompanying drawing 3.Scl albumen brings out the apoptosis of vascular endothelial cell test
a.PBS b.Scl
Accompanying drawing 4.Scl albumen suppresses the chick chorioallantoic membrane neovascularity and generates test
a.PBS；b.Scl；c.Angiostatin
Embodiment
1.DNA sequence is synthetic.
(1) designs following dna sequence dna.
ATGCACGGAGACTCAGAATACAACATCATGTTTGGTCCCGACATCTGTGGCCCTGGCAC?CAAGAAGGTTCATGTCATCTTCAACTACAAGGGCAAGAACGTGCTGATCAACAAGGACA?TCCGTTGCAAGGATGATGAGTTTACACACCTTACACTGATTGTGCGGCCAGACAACCAT?CATCATCATCATCATTAG
(2) synthetic following primer:
p1.
5’ATGCACGGAGACTCAGAATACAACATCATGTTTGGTCCCGACATCTGTGGCCCTGGCA
C3’
p2.
5’TTCTTGCCCTTGTAGTTGAAGATGACATGAACCTTCTTGGTGCCAGGGCCACAGATGT
C3’
p3.
5’TTCAACTACAAGGGCAAGAACGTGCTGATCAACAAGGACATCCGTTGCAAGGATGAT
GA3’
p4.
5’TGGTTGTCTGGCCGCACAATCAGTGTAAGGTGTGTAAACTCATCATCCTTGCAACGGA
T3’
p5.CTAATGATGATGATGATGATGGTTGTCTGGCCGCACAA3’
(3) carry out the synthetic of above sequence with following intussusception PCR method, the steps include:
1. Using P 1 is carried out PCR with the p2 primer, obtains product 1.
2. carry out pcr amplification with P3 and p4 primer, obtain product 2.
The mixture of 3. above product that obtains 1 and product 2 is a substrate, increases with primer P1 and p5, obtains product 3.
(4) above PCR product 3 is connected in the T carrier, carries out the consistent of dna sequence dna that dna sequence analysis proof obtained and design:
ATGCACGGAGACTCAGAATACAACATCATGTTTGGTCCCGACATCTGTGGCCCTGGCAC?CAAGAAGGTTCATGTCATCTTCAACTACAAGGGCAAGAACGTGCTGATCAACAAGGACA?TCCGTTGCAAGGATGATGAGTTTACACACCTTACACTGATTGTGCGGCCAGACAACCAT?CATCATCATCATCATTAG
2. the proof of sequence encoding protein function
(1) dna sequence dna is reconstituted in the yeast and expresses and the purifying of expression product
Through the effect of DNA restriction endonuclease and T4 dna ligase, with the MCS of above synthetic dna fragmentation insertion yeast expression vector pPIC9K, with its expression vector called after pPPIC9K-scl (accompanying drawing 1).Electricity consumption is changeed method pPIC9K-scl is transformed pichia spp GS115, obtains engineering bacteria GS115 (pPIC9K-scl).With BMGY-BMMY substratum fermentation GS115 (pIPIC9K-scl) abduction delivering Scl albumen.Fermented liquid supernatant is crossed Ni-Agarose His label protein purification column, obtain the target protein (accompanying drawing 2) of conformance with standard molecular weight.
(2) expression product activation analysis
Behind the liquid process suction filtration desalination that contains target protein with above results, suppress human umbilical vein's cell proliferation test and suppress the test of chicken embryo fine hair vasculogenesis.Results expression Scl albumen brings out human vascular endothelial apoptosis (accompanying drawing 3), suppresses chicken embryo fine hair allantois new vessel and generates, and its restraining effect to the chick chorioallantoic membrane vasculogenesis is better than Angiostatin (accompanying drawing 4).These results show that the albumen of scl genes encoding has the activity that suppresses vascular endothelial cell proliferation and the generation of inhibition new vessel, and scl suppresses vasculogenesis genes involved sequence.
Claims (3)
1. one kind is suppressed the white gene order of angiogenesis associated protein, it is characterized in that having following nucleotide sequence:
ATGCACGGAGACTCAGAATACAACATCATGTTTGGTCCCGACATCTGTGGCCCTGGCAC?CAAGAAGGTTCATGTCATCTTCAACTACAAGGGCAAGAACGTGCTGATCAACAAGGACA?TCCGTTGCAAGGATGATGAGTTTACACACCTTACACTGATTGTGCGGCCAGACAACCAT?CATCATCATCATCATTAG
2. one kind is suppressed the white gene order of angiogenesis associated protein, it is characterized in that the coded proteinic aminoacid sequence of its gene order:
Met?His?Gly?Asp?Ser?Glu?Tyr?Asn?Ile?Met?Phe?Gly?Pro?Asp?Ile?Cys?Gly?Pro?Gly?Thr?Lys?Lys?Val?His?Val?Ile?Phe?Asn?Tyr?Lys?Gly?Lys?Asn?Val?Leu?Ile?Asn?Lys?Asp?Ile?Arg?Cys?Lys?Asp?Asp?Glu?Phe?Thr?His?Leu?Thr?Leu?Ile?Val?Arg?Pro?Asp?Asn?His?His?His?His?His?His
3. the coded aminoacid sequence of gene order that the described inhibition angiogenesis associated protein of gene order that inhibition angiogenesis associated protein according to claim 1 is white and claim 2 is white is characterized in that: the pharmaceutical products that utilizes the protein preparation treatment blood vessel hyperplasia of described genes encoding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102706415A CN102409056A (en) | 2011-08-31 | 2011-08-31 | Associated gene sequence for inhibiting angiogenesis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102706415A CN102409056A (en) | 2011-08-31 | 2011-08-31 | Associated gene sequence for inhibiting angiogenesis |
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| CN102409056A true CN102409056A (en) | 2012-04-11 |
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| CN2011102706415A Pending CN102409056A (en) | 2011-08-31 | 2011-08-31 | Associated gene sequence for inhibiting angiogenesis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102363629A (en) * | 2011-10-09 | 2012-02-29 | 中国热带农业科学院热带生物技术研究所 | Protein for treating eye angiogenesis |
| CN102443586A (en) * | 2011-10-09 | 2012-05-09 | 中国热带农业科学院热带生物技术研究所 | Protein for treating tumor |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1524879A (en) * | 2003-09-17 | 2004-09-01 | 中山大学 | Anti endothelial cell element, genes encoding same and pharmaceutical uses thereof |
| WO2008046251A1 (en) * | 2006-10-19 | 2008-04-24 | Sunbio Biotech Pharmaceuticals(Tianjin) Co., Ltd. | Fusion proteins containing n domain of human calreticulin and human papillomavirus type 16 e6 or e7 and uses thereof |
-
2011
- 2011-08-31 CN CN2011102706415A patent/CN102409056A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1524879A (en) * | 2003-09-17 | 2004-09-01 | 中山大学 | Anti endothelial cell element, genes encoding same and pharmaceutical uses thereof |
| WO2008046251A1 (en) * | 2006-10-19 | 2008-04-24 | Sunbio Biotech Pharmaceuticals(Tianjin) Co., Ltd. | Fusion proteins containing n domain of human calreticulin and human papillomavirus type 16 e6 or e7 and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| 屠发志等: "以甘油为碳源发酵Pichia pastoris 组成型表达人血管生成抑制素", 《生物工程学报》 * |
| 潘英文等: "Canstatin-N基因在Pichia pastoris中表达及产物活性分析", 《生物技术》 * |
| 陈宏等: "钙网蛋白122~180片段基因克隆、表达和活性分析", 《生物化学与生物物理进展》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102363629A (en) * | 2011-10-09 | 2012-02-29 | 中国热带农业科学院热带生物技术研究所 | Protein for treating eye angiogenesis |
| CN102443586A (en) * | 2011-10-09 | 2012-05-09 | 中国热带农业科学院热带生物技术研究所 | Protein for treating tumor |
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Application publication date: 20120411 |