CN1238052C - Obtainment of cell growth inhibiting polypeptide and its usage - Google Patents
Obtainment of cell growth inhibiting polypeptide and its usage Download PDFInfo
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Abstract
The present invention discloses a polypeptide composed of 25 amino acids, a corresponding cDNA coding sequence, a preparation method thereof, and specific purposes of the polypeptide, the perylene derivative and the corresponding cDNA in tumor therapy and other fertile diseases. The present invention is characterized in that the key function that the polypeptide of the 25 amino acids on the NH2 terminal of the p55PIK albumins (the regulatory subunit of a phosphatidylinositol-3-kinase) is determined, and phosphorylation of Rb is intercepted in cells to achieve the biological effect of eliminating cell dissociation. The polypeptide and the perylene derivative or the transfection of the corresponding cDNA is used for expressing the polypeptide in cells, and thereby, the function of effectively eliminating cell growth and tumor growth can be achieved, and the present invention has a unique use value in the preparation of clinical therapy, tumor drugs and diagnostic reagents for tumor and relevant diseases of cell dissociation. The present invention also discloses a new method for intercepting the phosphorylation process of Rb and also provides a way for designing a new medicine for curing tumors.
Description
One, technical field:
The present invention relates to a kind of application of polypeptide in the treatment of tumor and other proliferative disease that constitutes by 25 aminoacid (methionine-aspartic acid-arginine-aspartic acid-aspartic acid-alanine-aspartic acid-tryptophan-arginine-glutamic acid-valine-methionine-methionine-proline-tyrosine-serine-threonine-glutamic acid-leucine-isoleucine-phenylalanine-tyrosine-isoleucine-glutamic acid-methionine), belong to the bioengineering field.
Two, background technology:
Tumor is a kind of disease of serious harm human health, and its mortality rate has become second the deadly cause of disease that is only second to cardiovascular and cerebrovascular disease at home at present.Main treatment with chemotherapy, radiotherapy and operative treatment in clinical, these methods have formed the treatment system of comparative maturity, have obtained effect preferably.Yet, because these method selectivitys are low, be difficult to avoid that toxic and side effects is strong, the little shortcoming of treatment window between normal cell and the tumor, further improve curative effect and even reach that to cure the purpose development potentiality limited, therefore demand researching and developing new effective Therapeutic Method urgently.
Recently the fast development of cellular biology of tumor, provide real probability for satisfying this social need, wherein, along with illustrating of the signal transduction pathway (Signaling transduction pathways) of cell, found more and more to can be used as the medicine target for the treatment of tumor.There have some successfully to be applied to be clinical, obtained extraordinary effect.Such as ErbB2 in many breast cancer cells (a kind of epithelial growth factor receptor that can promote protein phosphorylation) overexpression, and its protein kinase activity also strengthens, use a species specificity and can effectively suppress the ErbB2 protein kinase activity, block this protein mediated signal transduction at this protein monoclonal antibody.This antibody has obtained the FDA approval at present and has entered clinical use.
The signal of cell growth is to be participated in being delivered to nucleus through a series of albumen by the factor that promotes growth inside and outside the cell, causes many adjusting protein structures of cell cycle and changes of function and causes cell division.Regulate in the albumen of cell growth cycle at these, optic nerve blastoma albumen (Retinoblastoma Protein, Rb) taking on an extremely important role, Rb is the nucleoprotein with strong inhibition cell growth, and it suppresses fissional ability is by the degree decision of its phosphorylation.When the signal of stimulating growth inside and outside the cell passes to nucleus, the protein kinase (such as CDK4 or 6) of some energy phosphorylation modification Rb is activated, make tens serines and threonine phosphorylation among the Rb, this result causes Rb to suppress the afunction of cell cycle, and therefore cell enters division cycle.Therefore, the phosphorylation of blocking-up Rb is the approach of a up-and-coming exploitation treatment tumor new drug.Up to now, the enzyme and the protein of a plurality of participation Rb phosphorylations are found, but because Rb phosphorylation approach is a very complicated process that has a plurality of albumen to participate in, its mechanism is not clear, do not have effective method or chemical compound can directly block the phosphorylation of Rb at present, reach the cell growth inhibiting effect.
Three, summary of the invention:
Under study for action, we have found the interaction of a subunit of Rb and phosphatidylinositol-3-kinase (PI3K).PI3K forms by regulating subunit and catalytic subunit, the serine on some lipid of catalytic subunit catalysis and the protein and the activity of threonine phosphorylation are the proteic core effect of PI3K ingredients, yet, the activity of catalytic subunit is regulated the regulation and control of subunit, and regulation and control realize by following two mechanism: 1. the activity of catalytic subunit itself is regulated subunit influences; 2. regulate subunit by catalytic subunit being positioned target protein with other proteic interactions.We discover that the interaction of Rb and PI3K is that (sequence is: methionine-aspartic acid-arginine-aspartic acid-aspartic acid-alanine-aspartic acid-tryptophan-arginine-glutamic acid-valine-methionine-methionine-proline-tyrosine-serine-threonine-glutamic acid-leucine-isoleucine-phenylalanine-tyrosine-isoleucine-glutamic acid-methionine by aminoterminal 1-25 aminoacid in the adjusting subunit (p55PIK) of 55 kilodaltons among the PI3K; See " aminoacid and nucleotides sequence tabulation ") participate in, this polypeptide fragment is called as Rb combined function district (Rb bindingdomain), it interconnects PI3K and Rb, and the protein kinase activity of PI3K has caused the phosphorylation of Rb, thereby has caused cell division.According to this model, we imagine, if we only just express Rb combined function district among the p55PIK in cell, this polypeptide can be incorporated into Rb effectively, the combination of PI3K and Rb in the competitive inhibition cell is not because this polypeptide has any phosphorylated protein kinase activity, so polypeptide will suppress the phosphorylation process of Rb in conjunction with Rb, keep the cytostatic function of Rb, thereby reach the purpose that stops the tumour cell division process.
The crucial part that proves this model is synthetic this polypeptide and in this polypeptide importing somatoblast, observes the splitted influence of this polypeptide pair cell again.For reaching this purpose, we adopt molecular biological means, make a DNA construct, this construct coding is by the fusion rotein of this Rb combined function district and green fluorescent protein (GFP), again this construct is changed in the cultured cell, because it is quite ripe to detect the technology of GFP, we separate the cell (containing the DNA construct that changes over to) that produces the GFP signal separately, analyze its cell division situation.Its result and the cell (transfection contrasts DNA construct accordingly) of only expressing GFP compare, to confirm whether the Rb combined function district polypeptide among the single expression p55PIK can cell growth inhibiting.
Experimental result shows, in several different cultured cell systems, express this 25 amino acid whose polypeptide with growth splitting ability, the DNA of these cells observation index synthetic and other several proof cell growths all significantly reduce, and have proved that this polypeptide has cytostatic ability.
Compare with existing cytostatic method, adopt the present invention to reach following effect (accompanying drawing 1 is seen in explanation):
1. the Rb combined function district of single expression p55PIK can be incorporated into Rb in the cell, the phosphorylation process that has suppressed Rb, thereby effectively improve the ability of the inhibition cell cycle of Rb, design and screening for treatment cell growth abnormity disease new drug provide a kind of new method and approach.
2. the sequence of this polypeptide is present in the albumen of cell, and toxic and side effects is low, and a little less than the antigenicity, experimental result is also found the not significantly influence of this polypeptide pair cell apoptosis, so this polypeptide does not have tangible lethal effect to normal cell.
3. this polypeptide and corresponding cDNA can suppress cell proliferation effectively in cell.
4. this polypeptide molecular weight is little can chemosynthesis, is convenient to directly apply on a large scale clinical, and simultaneously also available other method (such as with plasmid and viral vector) is put into this polypeptide in the cell, to reach the purpose of treatment tumor.
Four, description of drawings
Accompanying drawing 1:N25 (p55PIK Rb combined function district) blocks cell division.Phosphatidylinositol-3-kinase in cell (PI3K) interacts by p55PIK and Rb, and this interaction has activated a series of protein kinases that promote the Rb phosphorylation, causes the phosphorylation of Rb.The phosphorylation of Rb makes the cytostatic afunction of Rb, and cell enters growth cycle.Rb combined function district N25 polypeptide has the ability in conjunction with Rb, but this combination can not activate the protein kinase of the phosphorylation that causes Rb, thereby has kept low phosphorylation and its cytostatic ability of Rb, has blocked the division of cell.
Accompanying drawing 2: in cultured cells, detect GFP, the proteic expression of N25-GFP and N25-GFP.With pEGFP-C1 (expressing GFP albumen), the COS7 cell that pEGFP-N25 (expressing the N25-GFP fusion rotein) and each the 10 microgram transfection of pEGFP-C25 (expressing the GFP-N25 fusion rotein) plasmid are cultivated.After 48 hours, lysis.After albumen separates with the 12%SDS polyacrylamide gel electrophoresis in the lysate, transfer to nylon membrane.This film is used for the Western blotting and detects GFP, the proteic expression of N25-GFP and N25-GFP (GFP antibody is from CLONTECH).Arrow shows the position of N25-GFP and GFP-N25 among the figure.
Accompanying drawing 3:N25 polypeptide has suppressed the phosphorylation of Rb.3T3 cell (available from ATCC) is incubated in 10 centimetres of culture dishs.With pEGFP-C1 (expressing GFP albumen), pEGFP-N25 (expressing the N25-GFP fusion rotein) and each 10 microgram transfectional cell of pEGFP-C25 (expressing the GFP-N25 fusion rotein) plasmid.Two days later, the cell of expressing GFP separates with flow cytometer, after the lysis, separates with sds polyacrylamide gel electrophoresis and transfers on the film from the equal protein of different cells, and film is with the antibody test cell Rb phosphorylation level of identification phosphorylation Rb.PpRb represents phosphorylation Rb among the figure.
Five, the specific embodiment
Example 1: the polypeptide cDNA (this polypeptide is designated hereinafter simply as N25) that in the pEGFP plasmid, introduces N-terminal 25 amino acid residues of coding p55PIK.
PEGFP-N1 and pEGFP-C1 plasmid vector purchase that (catalog number (Cat.No.) is: 6085-1 in U.S. Clontech company, 6084-1), plasmid comprises the cDNA of encoding green fluorescent protein (GFP), (purchase Promega company with EcoRI-BamHI in the U.S., catalog number (Cat.No.) is: R6011, R6021) digested plasmid, the plasmid behind the enzyme action separates in agarose gel, purification, is used for later coupled reaction.The test kit that reclaims dna fragmentation from glue is from German Qiagen company (catalog number: 28704).
The cDNA of encoding murine p55PIK full-length proteins derives from mouse testis tissue cDNA library (U.S. Stratagene company, catalog number (Cat.No.) is: 937308), be cloned into pcDNA3 (purchase the company in Invitrogen, catalog number (Cat.No.) is: V79020), the PCR primer sequence of the N25cDNA that is used to increase is:
Primer 1:5 ' TTTTGAATTCTATGGACCGCGATGACGCAGA
Primer 2: 5 ' TTTTGGATCCATTTCAATATAAAATATCAGT
The PCR condition: 94 ℃ 2 minutes; 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ totally 25 circulations in 2 minutes; 72 ℃ were extended 5 minutes.
(catalog number (Cat.No.) is: M1861), the PCR product is through (test kit used of purification is from German Qiagen company, and catalog number is: 28704), use the EcoRI-BamHI enzyme action behind the purification from U.S. Promega company for the PCR test kit; Reuse agarose gel purification, then with the enzyme action purification after carrier pEGFP-N1 or pEGFP-C1 carry out coupled reaction (be connected test kit from U.S. Promega company, catalog number (Cat.No.) be: M1801).(the competence antibacterial is from U.S. Promega company, and catalog number (Cat.No.) is: L2001), the clone screens with conventional bacterium colony PCR behind the transform bacteria.Select positive colony, after the correctness of its cDNA sequence was confirmed by determining nucleic acid sequence, large scale purification prepares plasmid, and (the large scale purification test kit was from U.S. Promega company, and catalog number (Cat.No.) is: A7270), be used for later experiment.These two kinds of plasmids are expressed the fusion rotein of a N25 and GFP respectively at eukaryotic cell.Among the pEGFP-N1, the N that the N25 of expression is connected GFP does not hold, and this fusion rotein is called N25-GFP; N25 is that the C-that is connected in GFP does not hold in the fusion rotein that pEGFP-C1 expresses, and this albumen is called GFP-N25.
The detection of example 2:DNA construct expressed fusion protein
Adopt conventional DNA transfection experiment to detect Expression of Fusion Protein.(purchase the ATCC in the U.S., catalog number (Cat.No.) is: CRL-1651) detect Expression of Fusion Protein with the COS7 cell for we.
The transfection reagent box is purchased in American I nvitrogen (catalog number (Cat.No.) is 11668), and transfection experiment is also finished according to the operation instruction that manufacturer provides.The COS7 cell culture is in 10% calf serum DMEM nutritional solution.After the transfection 48 hours, the COS7 cell is washed twice with normal saline (PBS), add the cell pyrolysis liquid cell lysis, behind ultrasound wave destruction DNA, add an amount of 2 mercapto ethanol and bromophenol blue, in boiling water, handled 5 minutes, place on ice and preserve, be splined on 12% SDS-polyacrylamide gel electrophoresis subsequently.Albumen after the separation is transferred on the nylon membrane, and this film detects the generation of fusion rotein with anti-GFP antibody (purchase in American I nvitrogen, catalog number (Cat.No.) is R970), and the result has confirmed GFP-N25 and the N25-GFP expression (seeing accompanying drawing 1) in cell.
The experiment of example 3:N25 polypeptide expression cell growth inhibiting and cell cycle.
With NIH/3T3 (mice becomes the fiber epithelial cell, and available from U.S. ATCC, catalog number (Cat.No.) is: CRL-1658) and MCF-7 (MCF-7, available from U.S. ATCC, catalog number (Cat.No.) is: the HTB-22) influence of detection N25 amino acid polypeptide cell growth.
Cell is at the DMEM culture fluid that contains 10% hyclone (FBS), 37 ℃, 5%CO
2Be incubated at 10 cm cell culture dishs under/95% air condition of culture.
Be used to test plasmid: pEGFP-N25 (expressing the N25-GFP fusion rotein); PEGFP-C25 (expressing the GFP-N25 fusion rotein); PEGFP-N1 or pEGFP-C1 be plasmid in contrast.
Be used for the plasmid of transfection and lipofectamine (available from purchasing in American I nvitrogen, catalog number (Cat.No.) is: after 11668) mixing, room temperature was placed 15 minutes, adding in the cell be incubated at serum-free DMEM nutritional solution (cell density is about 50%) cultivated 5 hours in 37 ℃, add 10% hyclone again, continue to cultivate 48 hours.In fluorescence microscope, express the cell that merges green fluorescent protein or independent fluorescin and be easy to and do not express these proteic cell differentiation come.In addition, adopt flow cytometer, these fluorescin positive cells also are easy to separation and purification and come out.The sign index of several detection cell growths is used to studies confirm that the influence of N25 polypeptide cell growth, and in these experiments, only the cell of express fluorescent protein is organized usefulness in contrast.
At first, we are with above-mentioned plasmid transfection 3T3 and MCF7 cell, two days later, adopt flow cytometer transfection the GFP positive cell of DNA pick out, analyze the cell cycle distribution of these cells.
Experimental result shows: express the not obviously influence of N25 pair cell apoptosis, and N25 can suppress cell cycle and enters the S phase, inducing cell enters the G0/G1 phase, and its effect sees the following form.
Table 1:N25 suppresses cell cycle, and inducing cell enters the G0/G1 phase and acts on
| The 3T3 cell | The MCF7 cell | |||||
| G0/G1 | S | G2/M | G0/G1 | S | G2/M | |
| The GFP contrast | 30.4％ | 27.9％ | 41.7％ | 72.2％ | 22.6％ | 5.2％ |
| N25-GFP | 42.1％ | 22.1％ | 35.7％ | 87.6％ | 11.1％ | 1.3％ |
| GFP-N25 | 48.9％ | 19.5％ | 31.6％ | 93.1％ | 6.0％ | 0.9％ |
DNA is synthetic to be the sign of cell proliferation.In the next experiment, we detect the synthetic influence of cell DNA of N25.3T3 and MCF7 cell transfection pEGFP-N1 and express N25-GFP and the GFP-N25 plasmid two days later, added the BrdU labeled cell to penetrate in the new synthetic DNA chain 15 hours at cell culture fluid, the reuse immunofluorescence technique the has detected transfection GFP positive cell of plasmid is determined the positive rate (this has represented the cell DNA synthetic ratio) that BrdU infiltrates in these cell DNAs again.The result shows, N25 also strong inhibition cell DNA synthetic.(seeing Table 2)
It is synthetic that table 2:N25 suppresses cell DNA
| The 3T3 cell | The MCF7 cell | |||
| The BrdU positive cell | Suppression ratio | The BrdU positive cell | Suppression ratio | |
| The GFP contrast | 53.8％ | 0％ | 31.2％ | 0％ |
| N25-GFP | 2.5％ | 95.4％ | 2.2％ | 89.5％ |
| GFP-N25 | 1.4％ | 97.4％ | 1.5％ | 95.2％ |
The phosphorylation of example 4:N25 expression inhibiting Rb
The phosphorylation of Rb is closely related with the cytostatic function of Rb.We adopt the Western blotting to detect the phosphorylation level of Rb in the cell of the different plasmid of transfection, with above-mentioned plasmid transfection MCF7 cell two days later, adopt flow cytometer that the separation and purification of GFP positive cell is come out, use the lysate cell lysis, last sample, with SDS-polyacrylamide gel electrophoresis protein isolate, again albumen is transferred on the film, film is used at the antibody of Rb phosphorylation, result's (seeing accompanying drawing 2) shows, Rb mainly exists with phosphorylation state in the cell of expressing GFP, and in the cell of expressing N25, Rb is in the low phosphorylation state.Shown that in these cells, Rb has cytostatic effect.
＜110〉Xia Xianmin
＜120〉acquisition of cytostatic polypeptide and purposes
<160>2
<210>1
<211>25
<212>PRT
＜213〉people (Homo sapiens)
<220>
<400>1
Met Asp Arg Asp Asp Ala Asp Trp Arg Glu Val Met Met Pro Tyr
1 5 10 15
Ser Thr Glu Leu Ile Phe Tyr Ile Glu Met
20 25
<210>2
<211>75
<212>DNA
＜213〉people (Homo sapiens)
<220>
<221>CDS
<222>(1)...(75)
<400>2
ATG GAC CGC GAT GAC GCA GAC TGG AGG GAG 30
Met Asp Arg Asp Asp Ala Asp Trp Arg Glu
1 5 10
GTG ATG ATG CCC TAT TCG ACA GAA CTG ATA 60
Val Met Met Pro Tyr Ser Thr Glu Leu Ile
15 20
TTT TAT ATT GAA ATG 75
Phe Tyr Ile Glu Met
25
Claims (5)
1. a peptide species, be p55PIK Argine Monohydrochloride residue 1-25, this peptide sequence is: methionine-aspartic acid-arginine-aspartic acid-aspartic acid-alanine-aspartic acid-tryptophan-arginine-glutamic acid-valine-methionine-methionine-proline-tyrosine-serine-threonine-glutamic acid-leucine-isoleucine-phenylalanine-tyrosine-isoleucine-glutamic acid-methionine.
The described polypeptide of claim 1 preparation treatment human tumor, and the medicine of other and cell growth abnormity diseases associated in application.
3. the corresponding nucleotide coding sequence of the described polypeptide of claim 1.
The described nucleotide coding sequence of claim 3 preparation treatment human tumor, and the medicine of other and cell growth abnormity diseases associated in application.
5. the application of the described nucleotide coding sequence of claim 3 in the preparation gene therapy vector.
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| CN 03146263 CN1238052C (en) | 2003-07-07 | 2003-07-07 | Obtainment of cell growth inhibiting polypeptide and its usage |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101016340A (en) * | 2007-01-18 | 2007-08-15 | 夏献民 | Fusion polypeptide and use thereof in treatment of tumor and cell growth abnormity correlated disease |
| CN102321158B (en) * | 2011-08-23 | 2013-03-27 | 常州德健生物科技有限公司 | Polypeptide for preventing cell DNA synthesis and inhibiting cell proliferation and application |
| CN106729610B (en) * | 2016-12-16 | 2021-01-29 | 湖北工业大学 | Application of N15 polypeptide in inhibiting staphylococcus aureus inhibitor |
| CN112263672A (en) * | 2020-11-04 | 2021-01-26 | 武汉益承生物科技有限公司 | Application of P55PIK inhibitor in preparation of medicine for treating dry eye |
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