CN102443586A - Protein for treating tumor - Google Patents
Protein for treating tumor Download PDFInfo
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- CN102443586A CN102443586A CN2011103196335A CN201110319633A CN102443586A CN 102443586 A CN102443586 A CN 102443586A CN 2011103196335 A CN2011103196335 A CN 2011103196335A CN 201110319633 A CN201110319633 A CN 201110319633A CN 102443586 A CN102443586 A CN 102443586A
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Abstract
The invention relates to a nucleotide sequence and an amino acid sequence of a protein for treating tumor. The sequence is an artificially synthesized DNA sequence, the concrete nucleotide sequence of which is the sequence under item 1 in a sequence list (400) and the concrete amino acid sequence of which is the sequence under item 2 in the sequence list (400). The artificially synthesized DNA sequence is recombined into Pichia genome and a recombinant protein is expressed in Pichia. The purified protein can effectively inhibit proliferation of human vascular endothelial cells and can effectively treat tumor. The invention creates a necessary condition for the research, development and application of the production of a specific drug for tumor treatment, and is of important theoretical and practical significance.
Description
Technical field
The present invention relates to a kind of protein, be specifically related to a kind of protein that is used to treat tumour.
Background technology
Tumour is one of main reason that threatens human health.The M & M of tumour all is annual in the world wide number disease with high that causes death, and does not still have the specific treat-ment so far.The method of multiple treatment tumour is arranged at present, and perform the operation, radiation and chemotherapy is the most frequently used method.Yet postoperative tumour usually recurs; And radiation and chemotherapy killing tumor cell not only also kills and wounds the normal cell of body simultaneously, and spinoff is very big.The cancer therapy drug of seeking low property and high curative effect is a difficult task always.The development need blood vessel of solid tumor and be that tumour provides nutrition.If suppress the angiogenesis of tumour, can die of hunger tumour so.Angiostatin is one type of protein that suppresses vasculogenesis, and the anticancer clinical test results of Angiostatin shows that Angiostatin is slight to human non-toxic property or toxicity.Studying Angiostatin anticancer is the focus of current anticancer research.
Summary of the invention
The purpose of this invention is to provide a kind of protein of treating tumour, this protein has the function of angiogenesis inhibiting, has the characteristic of Angiostatin.Be through the synthetic section of DNA sequence of dna sequence dna compound method; This segment DNA sequence is reconstituted in the pichia spp expresses; And expression product carried out purifying, the expression product activation analysis proves that institute's synthetic gene encoded protein has angiogenesis inhibiting and the BA that suppresses tumor development.Described in its nucleotide sequence and its aminoacid sequence such as 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.
ATGCACGGAGACTCAGAATACAACATCATGTTTGGTCCCGACATCTGTGGCCCTGGCACCAAGAAGGTTCATGTCATCTTCAACTACAAGGGCAAGAACGTGCTGATCAACAAGGACATCCGTTGCAAGGATGATGAGTTTACACACCTTACACTGATTGTGCGGCCAGACAACCATCATCATCATCATCATTAG
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 test of chick chorioallantoic membrane vasculogenesis and treatment experiment mice melanoma model trial.The result is that Scl albumen suppresses the generation of chicken embryo fine hair allantois new vessel; Its restraining effect to the chick chorioallantoic membrane vasculogenesis is better than Angiostatin (accompanying drawing 3); Suppress the experiment mice tumour significantly at the intravital development of machine (accompanying drawing 4), have the effect of treatment tumour.Utilizing above-mentioned protein to prepare medicine, can be used for treating tumour, is a kind of medicine of treating tumour.
Advantage of the present invention:
The artificial synthetic scl gene of the present invention, the Scl albumen with the scl recombination is expressed acquisition in the pichia spp genome can suppress the chick chorioallantoic membrane angiogenesis effectively with the Scl albumen that obtains behind the expression product purifying, has the function of treatment tumour.Angiostatin has important purposes treating through the angiogenesis that suppresses tumour in the tumour.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.Treat the research and development application of the medicine of tumour for the production specific and created necessary condition, have important theory and practice significance.
Description of drawings
Accompanying drawing 2. purification of Recombinant Scl albumen
M. albumen Marker; 1. purified recombinant Scl albumen.
Accompanying drawing 3.Scl albumen suppresses the chick chorioallantoic membrane neovascularity and generates test
A.PBS; B.Scl albumen (20 μ g); C.Angiostatin albumen (20 μ g)
Accompanying drawing 4.Scl protein for treatment test mice tumour
A.PBS; B.Scl albumen
Embodiment
1.DNA sequence is synthetic
(1) designs following dna sequence dna.
ATGCACGGAGACTCAGAATACAACATCATGTTTGGTCCCGACATCTGTGGCCCTGGCACCAAGAAGGTTCATGTCATCTTCAACTACAAGGGCAAGAACGTGCTGATCAACAAGGACATCCGTTGCAAGGATGATGAGTTTACACACCTTACACTGATTGTGCGGCCAGACAACCATCATCATCATCATCATTAG
(2) synthetic following primer:
p1.
5’ATGCACGGAGACTCAGAATACAACATCATGTTTGGTCCCGACATCTGTGGCCCTGGCAC3’
p2.
5’TTCTTGCCCTTGTAGTTGAAGATGACATGAACCTTCTTGGTGCCAGGGCCACAGATGTC3’
p3.
5’TTCAACTACAAGGGCAAGAACGTGCTGATCAACAAGGACATCCGTTGCAAGGATGATGA3’
p4.
5’TGGTTGTCTGGCCGCACAATCAGTGTAAGGTGTGTAAACTCATCATCCTTGCAACGGAT3’
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:
ATGCACGGAGACTCAGAATACAACATCATGTTTGGTCCCGACATCTGTGGCCCTGGCACCAAGAAGGTTCATGTCATCTTCAACTACAAGGGCAAGAACGTGCTGATCAACAAGGACATCCGTTGCAAGGATGATGAGTTTACACACCTTACACTGATTGTGCGGCCAGACAACCATCATCATCATCATCATTAG
2. the proof of nucleotide 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 T4DNA ligase enzyme, 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) Scl albumen angiogenesis inhibiting activation analysis
With above results contain the proteic liquid of Scl through the suction filtration desalination after, through suppressing the test of chicken embryo fine hair vasculogenesis.The Scl albumen of proof purifying has the activity (accompanying drawing 3) that suppresses the new vessel generation.
(3) Scl protein for treatment tumour test
1) practical example one
In the back of 7 all male C57BL6/J mouse inoculation 3 * 10
6Every mouse of B16 MC/0.2ml/.Inoculate 20 mouse altogether.According to formula: gross tumor volume V=0.52 * length * wide calculating gross tumor volume.When tumour reaches average 100mm
3The time, animal is divided into two groups of A, B, 10 every group.A group injection PBS solution, B group are injected Scl albumen (5mg/kg/d) every day, medication every day once, continuous use 12d.Write down tumor size every day.A group and the gross tumor volume result of variations of B group during medication are recorded in table 1.Two groups tumor size is significant difference (P＜0.01).
The MV that A group and B group tumor size change during table 1. medication
| Sequence number in the group | A | |
| 1 | 100mm3 | 100mm3 |
| 2 | 289mm3 | 95mm3 |
| 3 | 397mm3 | 87mm3 |
| 4 | 876mm3 | 85mm3 |
| 5 | 1067mm3 | 68mm3 |
| 6 | 1260mm3 | 57mm3 |
| 7 | 1403mm3 | 43mm3 |
| 8 | 1851mm3 | 38mm3 |
| 9 | 2283mm3 | 32mm3 |
| 10 | 2693mm3 | 27mm3 |
| 11 | 3170mm3 | 26mm3 |
| 12 | 3575mm3 | 25mm3 |
2) practical example two
In the back of 7 all male C57BL6/J mouse inoculation 3 * 10
6Every mouse of B16 MC/0.2ml/.According to formula: gross tumor volume V=0.52 * length * wide calculating gross tumor volume.When tumour reaches average 100mm
3The time, animal is divided into two groups of A, B, 10 every group.A group injection PBS solution, B organizes and injects Scl albumen (5mg/kg/d) every day.Medication 12d always.
12d after medication puts to death mouse, peels tumor tissues and claims its weight (table 2), and the weight of two groups tumour is significant difference (P＜0.01).
The numerical value of root a tree name table 2; According to formula: average tumor inhibiting rate=(the average knurl of the average knurl weight-experimental group of control group is heavy) * average knurl of 100% ÷ control group is heavy, and the average tumor inhibiting rate of its Scl albumen medication group is=(3.36-0.25) * 100% ÷ 3.36=92%.Explain that Scl albumen has stronger restraining effect to tumour.
A group and B group tumor weight behind the table 2. medication 12d
| Sequence number in the | A | B | |
| 1 | 3.2g | 0.27g | |
| 2 | 3.5g | 0.29 |
|
| 3 | 3.5g | 0.27 |
|
| 4 | 3.3g | 0.28g | |
| 5 | 3.4g | 0.28g | |
| 6 | 3.2g | 0.26g | |
| 7 | 3.4g | 0.29g | |
| 8 | 3.5g | 0.30g | |
| 9 | 3.3g | 0.28g | |
| 10 | 3.3g | 0.26g |
Conclusion: Scl albumen has therapeutic action to tumour.
Claims (3)
1. protein of treating tumour is characterized in that the nucleotide sequence of encoding such proteins is:
atgcacggag actcagaata caacatcatg tttggtcccg acatctgtgg ccctggcacc 60
aagaaggttc atgtcatctt caactacaag ggcaagaacg tgctgatcaa caaggacatc 120
cgttgcaagg atgatgagtt tacacacctt acactgattg tgcggccaga caaccatcat 180
catcatcatc attag 195。
2. protein of treating tumour is characterized in that this proteinic aminoacid sequence is:
Met His Gly Asp Ser Glu Tyr Asn Ile Met Phe Gly Pro Asp Ile
1 5 10 15
Cys Gly Pro Gly Thr Lys Lys Val His Val Ile Phe Asn Tyr Lys
20 25 30
Gly Lys Asn Val Leu Ile Asn Lys Asp Ile Arg Cys Lys Asp Asp
35 40 45
Glu Phe Thr His Leu Thr Leu Ile Val Arg Pro Asp Asn His His
50 55 60
His His His His。
3. a kind of proteinic nucleotide sequence and described a kind of proteinic aminoacid sequence of treating tumour of claim 2 of treating tumour according to claim 1 is characterized in that: utilize described nucleotide sequence coded protein preparation to treat the medicine and the pharmaceutical products of tumour.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011103196335A CN102443586A (en) | 2011-10-09 | 2011-10-09 | Protein for treating tumor |
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| CN2011103196335A CN102443586A (en) | 2011-10-09 | 2011-10-09 | Protein for treating tumor |
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| CN102443586A true CN102443586A (en) | 2012-05-09 |
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Cited By (1)
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409056A (en) * | 2011-08-31 | 2012-04-11 | 中国热带农业科学院热带生物技术研究所 | Associated gene sequence for inhibiting angiogenesis |
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2011
- 2011-10-09 CN CN2011103196335A patent/CN102443586A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409056A (en) * | 2011-08-31 | 2012-04-11 | 中国热带农业科学院热带生物技术研究所 | Associated gene sequence for inhibiting angiogenesis |
Non-Patent Citations (1)
| Title |
|---|
| 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 (1)
| 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 |
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Application publication date: 20120509 |