CN101306192A - Use of neuritis auxin in preparing antineoplastic medicine - Google Patents
Use of neuritis auxin in preparing antineoplastic medicine Download PDFInfo
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- CN101306192A CN101306192A CNA2008100374432A CN200810037443A CN101306192A CN 101306192 A CN101306192 A CN 101306192A CN A2008100374432 A CNA2008100374432 A CN A2008100374432A CN 200810037443 A CN200810037443 A CN 200810037443A CN 101306192 A CN101306192 A CN 101306192A
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Abstract
The invention belongs to the field of a biological medicine. The invention provides the new use of human protein neurite growth substance, namely, the application of the human protein neurite growth substance in preparing antineoplastic drugs. The human protein neurite growth substance shows obvious up-regulated expression in such malignant tumors as lung cancer, liver cancer, breast cancer, alimentary tract adenocarcinoma and so on, and can promote the tumor formation of mice. Accordingly, reducing the expression of neurite growth substance means inhibiting the growth of tumor. The human protein neurite growth substance provided by the invention can be used for early diagnosis of tumors, and can be used as a medicine for medicinal target screening and inhibiting the growth of tumor. The monoclonal antibody of the protein neurite growth substance can be used for preventing tumor blood vessels from growing, thus inhibiting the development of tumors. The invention further provides a kit and a biochip used for screening antineoplastic drugs.
Description
Technical field
The present invention relates to genetic engineering and field of medicaments, particularly, the present invention relates to the application of people's albumen neurite-outgrowth element in the preparation antitumor drug.
Background technology
(Neuritin NRN1) is a neurotrophic factor of finding in 1997, total length 142aa, molecular weight 15.3KD to the neurite-outgrowth element.Mainly be expressed in the satellite cell of brain, liver, skeletal muscle, heart and lung also have a small amount of expression.With phosphinositides anchored site (GPI), be anchored on the cell membrane, can pass through the effect of phospholipase C (PLC), be distributed in the body fluid from the film disengaging and with soluble form.Work as a kind of cytokine.The neurite-outgrowth element has very strong promotion neurite-outgrowth, synapse connects and sophisticated effect, stimulate former foster hippocampal neuron and the brain cortex cell of being commissioned to train with recombinant expressed neurite-outgrowth fibroin, can cause that neural axon and dendron showed increased prolong, in the research of Xenopus laevis and mammal optic element enation, the neurite-outgrowth element has also shown identical effect.Confirmed equally that in our early-stage Study the neurite-outgrowth element can promote the growth of PC12 cell process.The neurite-outgrowth element is an albumen very conservative in the evolution, protein sequence 100% homology of people and rat, and also its express spectra also is not limited to nervous system, proves that it is the very important albumen of biological function.Particularly nearly 2 years, the effect of neurite-outgrowth element outside nervous system was familiar with by people gradually.The neurite-outgrowth element is high expressed in the closely-related satellite cell regenerating with skeletal muscle, helps to repair after the damage of skeletal muscle, proves that the neurite-outgrowth element can regulate the motion of cytoskeleton, also has guiding function outside nervous system.
Tumor disease has now risen to No. the 2nd, the world " killer ", and its death toll is only second to the cardiovascular diseases.In the past few years, the foreign medical science bound pair has had new understanding again in the pathogeny of tumor disease on cell base.Based on the further understanding to tumor invasion mechanism, people utilize various approach to develop and develop can be special, effectively killing tumor cell and to the avirulent medicine of normal cell.At present, for treatment for cancer is first-selection with chemotherapy and radiotherapy still, though both have obtained suitable curative effect to tumor treatment, but since lack to the specificity of tumor cell thus have bigger toxic and side effects and some tumor cell to chemotherapy and radiation handle insensitive, therefore limited their application in clinical to a great extent.Tumor vascular being formed in tumor growth and the evolution process is vital.When many tumors begin to form, there is no angiogenic activity, its growth generally also is no more than 2～3mm3; Only after opening the tumor-blood-vessel growth phenotype, tumor just is able to malignancy and transfer.Tumor growth and transfer are the one of the main reasons of anticancer therapy failure.The authoritative scholar Folkman in angiogenesis field once proposed should regard tumor as vascular endothelial cell group and tumor cell group mutually promote the process of fellowship tumor development and transfer in the process of treatment and research tumor.Although the both is the cell mass of fast breeding, tumor cell genetic background is complicated and changeable, and gene expression profile varies, and the cell physiological habit is rather unstable also, and endotheliocyte is much all relatively stable from every side.By contrast, can haggle over the difference on the various details in the tumor cell, and directly act on the tumor vessel of stable in properties at the anti-angiogenic medicaments of endotheliocyte.Therefore, he think will be to the oncotherapy new hope of taking with being improved based on the combination of the treatment means of angiogenesis and traditional treatment means based on tumor cell.
Summary of the invention
The new purposes that the purpose of this invention is to provide people's albumen neurite-outgrowth element (be people Neuritin, the NCBI accession number is NP_057672).
The invention provides the application of people's albumen neurite-outgrowth element in the preparation antitumor drug.
Described tumor comprises the optimum or malignant tumor at positions such as liver, lung, digestive tract or mammary gland.
The invention provides the application of people's albumen neurite-outgrowth element in the preparation anti-tumor angiogenesis drug.People's albumen neurite-outgrowth element of the present invention can promote migration of vascular endothelial cells, adhesion external; Utilize the recombinant expressed neurite-outgrowth fibroin of mice cornea angiogenesis model discovery can induce new vessels.The neurite-outgrowth fibroin can be subjected to hypoxia inducible, and presents obvious up-regulated expression in malignant tumor such as pulmonary carcinoma, hepatocarcinoma, Kaposi's sarcoma, digestive tract adenoma.
As seen, thereby the neurite-outgrowth element can promote tumor-blood-vessel growth to promote tumor growth, and therefore, the expression that suppresses the neurite-outgrowth element just means the inhibition tumor growth.
People's albumen neurite-outgrowth element of the present invention can be used as the medicine target in screening and the preparation antitumor drug.
Among the present invention, term " neurite-outgrowth fibroin (NRN1) " refer to promote tumor growth, sequence and (the NCBI number of landing
NP_057672) shown in the NRN1 polypeptide and the variant form thereof of peptide sequence at least 70% homology.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) aminoacid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar aminoacid of performance.Again such as, add one or several aminoacid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragment of people NRN1 and reactive derivative.
The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people NRN1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum of anti-people NRN1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people NRN1 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people NRN1 polypeptide.Usually, this fragment have people NRN1 peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
The present invention also provides the analog of people NRN1 albumen or polypeptide.The difference of these analog and natural human NRN1 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic agent and produce random mutagenesis, also can pass through direct mutagenesis method or the biological technology of other known moleculars.Analog also comprises having the analog that is different from the amino acid whose residue of natural L-(as D-aminoacid), and has non-natural analog that exist or synthetic aminoacid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representative polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylation or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as phosphotyrosine, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-Proteolytic enzyme performance or optimized solubility property by modifying.
NRN1 albumen of the present invention can adopt the preparation method preparation of various routines.As gene engineering method or artificial synthesis etc.For example, can be that albumen gets with NRN1 gene expression.
NRN1 gene of the present invention can adopt the preparation method preparation of various routines.NRN1 gene order of the present invention can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be according to NRN1 nucleotide sequence of the present invention, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of conventional method well known by persons skilled in the art as template, amplification and must relevant sequence.
In the present invention, term " NRN1 gene " refer to the encode nucleotide sequence of polypeptide with NRN1 protein active is as the serial number of nucleotide sequence and the degenerate sequence thereof for (the NCBI number of landing NM_016588).This degenerate sequence is meant in this nucleotide sequence open reading frame, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, so be low to moderate also can encode out the sequence of neuron differentiation associated gene NRN1 of about 70% degenerate sequence with this nucleotide sequence open reading frame sequence homology.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition, with the nucleotide sequence of NM_016588 open reading frame sequence hybridization.This term also comprises the homology at least 70% with the NM_016588 open reading frame sequence, preferably at least 80%, and at least 90% nucleotide sequence more preferably.
This term also comprises and can coded sequence number be the nucleotide sequence variation form of NP_057672 aminoacid sequence.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) nucleotide.
After having obtained the NRN1 gene order, just the NRN1 gene order can be inserted suitable expression, to obtain recombinant expression plasmid.In case obtained to contain the recombinant expression plasmid of NRN1 gene order, just it can be transformed into and carry out protein expression among the corresponding host.
In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, select commercially available carrier for use, the nucleotide sequence with code book invention polypeptide operationally is connected in expression regulation sequence then, can form protein expression vector.
As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion targeting sequencing) DNA operationally is connected in polypeptid DNA so; If transcribing of promoter control sequence, it is operationally to be connected in coded sequence so; When if ribosome binding site is placed in the position that can make its translation, it is operationally to be connected in coded sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion targeting sequencing.
In the present invention, term " host cell " comprises prokaryotic cell and eukaryotic cell.The example of prokaryotic host cell commonly used comprises escherichia coli, bacillus subtilis etc.Eukaryotic host cell commonly used comprises yeast cells, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.
The DNA sequence of at present, can be fully coming code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This DNA sequence can be introduced then in the various dna moleculars (as carrier) and cell in this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The proteic fragment of the present invention is except available recombination method produces, and also available solid phase technique is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., SanFrancisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (FosterCity CA) synthesizes peptide automatically with the 431A type peptide synthesizer of Applied Biosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical method then.
The proteic coded sequence of the present invention also can be used for gene mapping.For example,, the cDNA clone is hybridized with the chromosome of metaphase of cell division, can carry out chromosome mapping exactly by fluorescence in situ hybridization technique (FISH).This technology can be used the cDNA that is as short as about 500bp; Also can use and grow to about 2000bp or longer cDNA.For this technology, can be referring to people such as Verma, Human Chromosomes:A Manual of BasicTechniques, Pergamon Press, New York (1988).
In case sequence is located in certain exact position on the chromosome, the physical location of sequence on chromosome can be associated with the genetic map data.These genetic map data can obtain, for example by Mendel (Mendelian) people genetic database (can obtain on the net by Johns Hopkins University Welch Medical Library).Then, come identified gene by linkage analysis and be positioned dependency between the disease of same chromosomal region.
Then, be necessary to determine the cDNA between diseased individuals and the healthy individual or the difference of genome sequence aspect.Be not present in normal individual if a certain sudden change is present in part or all of diseased individuals, this sudden change may be exactly the paathogenic factor of this disease so.
Utilize albumen of the present invention,, can filter out with NRN1 interactional material takes place, as receptor, inhibitor or antagonist etc. by various conventional screening techniques.
As target except NRN1 albumen, can also be the expression product of mRNA, NRN1 of NRN1 gene, NRN1 and aforementioned three's specificity segment thereof.Described " specificity segment " is meant the segment that can distinguish NRN1 of the present invention and other gene.
Among the present invention, described antitumor drug is the pharmaceutical composition that NRN1 albumen inhibitor and carrier pharmaceutically or excipient are formed.
Among the present invention, described medicine is tablet, injection, powder, oral liquid or injection.
Among the present invention, in the described medicine consumption of NRN1 albumen inhibitor can be every day 1 microgram/kilogram to 10 mg/kg body weight.
Albumen of the present invention, inhibitor, antagonist or receptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can be with being changed to some extent by preparation Substance Properties and disease to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, Intradermal or topical.
With people NRN1 albumen of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains protein and the pharmaceutically acceptable carrier or the excipient for the treatment of effective dose.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.People NRN1 albumen of the present invention can be made into the injection form, for example is prepared by conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.Pharmaceutical composition such as tablet and capsule can be prepared by conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of active component is the treatment effective dose, for example every day about 1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When people NRN1 protein polypeptide inhibitor of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to mammal, wherein should treat effective dose usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The present invention also provides a kind of test kit that is used to screen antitumor drug, comprises NRN1 albumen in this test kit.This test kit also can contain operation instruction, molecular marker, buffer etc.
Test kit of the present invention can will suppress NRN1 expressing quantity or active candidate compound as the antineoplastic drug candidate in use.
The present invention also provides a kind of biochip, contains NRN1 albumen on the carrier of this biochip.Can will suppress proteic expression of NRN1 or active candidate compound promptly as the antineoplastic drug candidate.This biochip can be with the direct point sample of NRN1 albumen on suitable biochip carrier in preparation, for example nitrocellulose membrane.Then, detect candidate compound and whether can and influence the proteic activity of NRN1 with the NRN1 interactions between protein.
NRN1 of the present invention can be used as target, and screening suppresses the material or the method for NRN1 vigor.Screening technique may further comprise the steps: A selects candidate compound; B provides NRN1 expression or the active system of detecting; The detection architecture of C NRN1 is selected candidate compound; D determines NRN1 expression or active change under the influence of candidate compound.
Among the present invention, the computer simulation three dimensional structure of candidate compound can be combined with the proteic computer simulation three dimensional structure of NRN1, the computer simulation three dimensional structure of candidate compound and the proteic active site of NRN1 (N end) are combined closely, and this candidate compound is the active component of antitumor drug.
Among the present invention, the candidate compound that obtains is added the influence that detects NRN1 protein expression in this chemical compound pair cell in the cell culture fluid.Perhaps the candidate compound that obtains is added and contain in the proteic solution of NRN1, then, it is active with combining of its receptor to detect NRN1 albumen.The proteic expression of NRN1 or with candidate compound that receptor-binding activity descends be the active component of antitumor drug.
Among the present invention, after the combination of computer simulation three dimensional structure, the candidate compound that obtains can be changed in the tumor, the candidate compound that causes tumor growth to slow down is the active component of antitumor drug.
Described candidate compound can be nucleic acid, aminoacid or micromolecular compound.Wherein, nucleic acid comprises DNA, RNA etc., can be siRNA, antisense oligonucleotide, ribozyme or with complementary other nucleic acid molecules of NRN1 nucleotide sequence; Aminoacid comprises peptide, antibody, interact protein etc.; Micromolecular compound then comprises natural and artificial synthetic.
The invention provides the new purposes of people's albumen neurite-outgrowth element, i.e. its application in the preparation antitumor drug.People's albumen neurite-outgrowth element of the present invention presents obvious up-regulated expression in malignant tumor such as pulmonary carcinoma, hepatocarcinoma, Kaposi's sarcoma, digestive tract adenocarcinoma, can promote mice to become tumor.Therefore, the activity of reduction neurite-outgrowth element just means the inhibition tumor growth.People's albumen neurite-outgrowth element of the present invention can be used for the early diagnosis of tumor, can be as the medicine of medicine target screening treatment and inhibition tumor growth.The monoclonal antibody of albumen neurite-outgrowth element of the present invention can be used to block tumor vascular growth to suppress tumor development.
Description of drawings
Fig. 1 is excretory NRN1 detection figure in the various tumor cell strains.Various tumor cell strains can secretory nerve projection element, Huh-7 wherein, QGY, SK-Hep1, HepG
2Deng the cell strain secretory volume than higher, Focus, cell strain secretory volumes such as YY are lower.QGY, YY, Focus, the Huh-7 cell strain can be buied from Chinese Academy of Sciences's cell.SK-Hep1, HepG
2All buy from ATCC, its article No. is respectively HTB-52 and HB-8065.
Fig. 2 is the proteic expression and purification figure of NRN1.Get NRN1-HIS behind the left figure arrow points purification, right figure arrow points determines that with the plain specific antibody of neurite-outgrowth it is NRN1 albumen really that our purification gets gained albumen.
Fig. 3 is that the nervous process element is to vascular endothelial cell proliferation capability result figure.Wherein, Blank is a blank, and PBS is a phosphate buffer, and FT is albumen when dialysis liquid (filtered solution) by dialyzer, is retained in to be nervous process fibroin (NRN1-his) on the dialyzer, and FBS is a hyclone, as positive control.Scheme as seen thus, the neurite-outgrowth element is to not effect of vascular endothelial cell proliferation.
Fig. 4 is the plain migration data figure that promotes endotheliocyte of nervous process.For detecting of the influence of nervous process element, in the nervous process fibroin adding ECV cell culture medium with purification, detect the variation of migration of vascular endothelial cells ability by scratch experiment to the migration of vascular endothelial cells ability.Wherein FBS (hyclone) is as positive control, and FT (filtered solution) is as negative control.23h behind the cut, the FBS group is all healings almost, and the NRN1 group is taken second place, and still there is very big white space in the FT group.Prompting nervous process element can promote the migration of endotheliocyte.
Fig. 5 is above-mentioned experiments experiment data analysis figure.As shown in the figure, the NRN1 protein groups of 100nm is more very fast than FT group migration rate, behind cut 4 hours, significant difference occurred, and along with the increase along with the time, the gap of the two migration area increases gradually.23h behind the cut, the FBS group is all healings almost, and the NRN1 group is taken second place, and still there is very big white space in the FT group.Prompting nervous process element can promote the migration of endotheliocyte.
Fig. 6 is a NRN1 albumen migration experiment block diagram.Wherein, (fibroblast growth factor 100ng) is positive control to bFGF.The scratch experiment result of nervous process fibroin dose gradient shows, (500ng/ml), the area of migration of vascular endothelial cells increases gradually for 100ng/ml, 250ng/ml, and migration rate improves gradually along with the increase of nervous process fibroin concentration.
Fig. 7 is the datagram that influences of nervous process fibroin pair cell adhesive attraction.
Fig. 8 is the expression figure of NRN1 in QGY, Fucos, L-02, Huh-7, HepG2, Hep3B, YY, SK-Hep1 various cancers cell strain, and visible neurite-outgrowth element is all a large amount expression in multiple cancer cell strain.
Fig. 9 utilizes the NRN1 expression in the DFO hypoxia inducible tumor to increase figure as a result.
Figure 10 utilizes CoCl
2NRN1 expression in the hypoxia inducible tumor increases figure as a result.
Figure 11 is that NRN1 promotes mice cornea angiogenesis block diagram as a result.Wherein PBS is as positive control, and bFGF is as positive control.As seen, NRN1 can obviously promote mice cornea angiogenesis.
Figure 12 detects the expression figure of nervous process element in the digestive tract adenocarcinoma tissue for immunohistochemical experiment.The bronzing place of marking is NRN1.The result shows that NRN1 low scale in above-mentioned normal structure reaches, and the superelevation scale reaches in cancerous tissue.
Figure 13 is the interior angiogenesis function figure as a result that urgees of body that the experiment of mice cornea detects NRN1.Wherein PBS is as positive control, and bFGF is as positive control.As seen, NRN1 can obviously promote mice cornea angiogenesis.
Figure 14 is the expression figure of NRN1 in various normal structures and tumor tissues.Wherein, A is a normal lung tissue, and B is a cancerous lung tissue, and C is a normal liver tissue, and D is a liver cancer tissue, and E is a normal galactophore tissue, and F is a breast cancer tissue, and G is a normal skin tissue, and H is the Kaposi's sarcoma tissue.The bronzing place of marking is NRN1.The result shows that NRN1 expresses hardly or low scale reaches in above-mentioned normal structure, and a large amount is expressed in cancerous tissue.
The specific embodiment
1) get the cell that is cultured to exponential phase, trypsinization is collected in the centrifuge tube and counting, and is centrifugal.Get requirement cell kind in the 60mm culture dish.
2) after 24 hours, change serum-free medium
3) serum-free medium is collected in 24 backs, and 3, the centrifugal 10min of 000rpm collects supernatant
4) get the loading buffer that 200 microlitre cells and supernatant add 2X, 98 ℃ of degeneration 5 minutes, sample is gone up the sample electrophoresis detection with the cell sample after the degeneration.
The result shows, various tumor cell strains can secretory nerve projection element, Huh-7 wherein, QGY, SK-Hep1, HepG
2Deng the cell strain secretory volume than higher, Focus, cell strain secretory volumes such as YY are lower.
The expression of embodiment 2.NRN1 full-length proteins in Pichia sp.
2.1 transform
1) the PIC9K plasmid of NRN-HIS is equipped with in a large amount of extractings, with Sac I single endonuclease digestion plasmid, reclaims purification.
2) YPD inoculation of medium GS115,30 degree incubator shaken cultivation are to OD600 value 1.3-1.5.
3) 1, the centrifugal 5min of 500rpm abandons supernatant, and the water that adds the pre-cooling of 30 milliliters of ice is resuspended,
4) 1, the centrifugal 5min of 500rpm abandons supernatant, and the water that adds the pre-cooling of 10 milliliters of ice is resuspended
5) 1, the centrifugal 5min of 500rpm abandons supernatant, and it is resuspended to add 2.5 milliliters of ice pre-cooling sorbitol
6) 1, the centrifugal 5min of 500rpm abandons supernatant, and the sorbitol that adds the pre-cooling of 0.5 milliliter of ice is resuspended, and this moment, the OD600 value was 50-60
7) get 80 microlitre yeast competent cells, add 5-20 microgram linearisation DNA (being dissolved among the 5-10 microlitre TE)
8) after the mixing, be transferred in the electric revolving cup ice bath 30min.
9) select Pichia sp. electricity carryover preface in the electroporation, electric shock cell.
10) in electric revolving cup, add 1 milliliter of sorbitol of icing pre-cooling at once
11) draw 300 microlitre bacterium liquid and be coated on the MD plate, 30 degree are cultured to bacterium colony and occur.
2.2 multicopy in-vitro screening
1) get 96 orifice plates, add 200 microlitre YPD/ holes, choose single bacterium colony to every hole, 30 degree were cultivated two days,
2) get 96 new orifice plates, add 190 microlitre YPD/ holes, draw the bacterium liquid of cultivating in first 96 orifice plate of 10 microlitres and join in the new plate, corresponding one by one, 30 degree overnight incubation,
3) get 96 new orifice plates, add 190 microlitre YPD/ holes, draw the bacterium liquid of cultivating in second batch 96 orifice plate of 10 microlitres and join in the new plate, corresponding one by one, 30 degree overnight incubation,
4) with liquid-transfering gun with bacterium liquid piping and druming in the 3rd batch of 96 orifice plates evenly, each is drawn 10 microlitre culture fluid and is added to and contains 0.25,0.5, on 1.0,1.5,2.0,3.0, the 4.0 mg/ml G418YPD plates, and numbering.
Whether 5) 30 degree are cultivated, observed bacterium colony and occur in 2 or 3 days.
2.3 albumen is expressed in a small amount
1) on the picking G4I8 plate in bacterium colony to the 10 milliliter BMGY culture medium, 30 degree are cultured to OD600 value 2-6.
2) 5000rpm, centrifugal 5min removes supernatant, and adding BMMY culture medium is resuspended, makes the OD600 value equal 1.
3) sampling 100 microlitres, all the other 30 degree inducing culture. every 24 hours, add methanol, making its final concentration is 0.5%6h, 12h, 24h, 36h, 48h, 60h and 72h 100 microlitres of respectively taking a sample.
4) will collect sample 5000rpm, centrifugal 5min collects respectively and goes up cleer and peaceful precipitation.
2.4 supernatant is handled
1) in supernatant, adds ammonium sulfate, to final concentration 55%.
2) place 2h on ice, albumen is fully precipitated
3) 10000rpm, centrifugal 30min removes supernatant, adds the SDS-PAGE sample-loading buffer, and 98 degree boil 8min
4) 12%SDS-PAGE electrophoresis
2.5 albumen great expression.
1) on the picking G4I8 plate in bacterium colony to the 50 milliliter BMGY culture medium, 30 degree are cultured to OD600 value 2-6.
2) 5000rpm, centrifugal 5min removes supernatant, and adding BMMY culture medium is resuspended, makes the OD600 value equal 1.
3) 30 degree inducing culture. every 24 hours, add methanol, making its final concentration is to receive bacterium behind the 0.5%36h.
2.6 protein purification
1) 5000rpm, centrifugal 5min gets supernatant.
2) in supernatant, add ammonium sulfate, to final concentration 55%.
3) place 2h on ice, albumen is fully precipitated
4) 10000rpm, centrifugal 30min removes supernatant,
5) add Binding Buffer soluble protein,
6) 10000rpm, centrifugal 30min go precipitation,
7) supernatant is transferred to nickel post 2000rpm, centrifugal, 2min
8) abandon filtrate, add 600 microlitre Wash Buffer2000rpm, centrifugal, 2min washes twice
9) add 200 microlitre Elution Buffer eluting .2000rpm, centrifugal, 2min
10) the gained protein solution is added the PBS dialysis and remove imidazoles with the dialysis cartridge dialysis, and concentrate, the liquid that wherein is retained on the dialyzer is the destination protein of purification, is filter liquor from the filterable liquid of dialyzer.
11) protein quantification.
The result sees Fig. 2 for details.
Embodiment 3. cell proliferation experiment
Measure the cell proliferation situation by the MTS method.
Plant in 96 holes (3 * 103 cells/well) the resuspended back of cell dissociation, left standstill 5 hours,
After treating cell attachment, remove culture medium, wash twice, add serum-free medium then and cultivated 24 hours with PBS.
In culture hole, add final concentration 1nM respectively, 10nM, the nervous process fibroin solution of 100nM (albumen dilutes with the DMEM serum-free), final every hole culture fluid volume is 100 μ l.Only adding DMEM serum-free culture hole is blank, adds the negative contrast in hole of PBS and filtered solution, contains the positive contrast of 1%FBS.
Cultivate after 48h hour, remove culture medium, add 100 μ l DMEM serum-free mediums.
Every hole adds 20 μ l MTS/PMS mixed liquors, continues to cultivate colour developing in 3 hours.
The result shows that NRN1 has the not influence of vascular endothelial cell proliferation ability.
Embodiment 4. scratch experiments
1) the ECV304 cell is resuspended in the DMEM+10%FBS culture medium, is seeded in then in the 35mm ware, density is 4 * 105 cells/well, leaves standstill 8 hours.
2) treat cell attachment after, wash cell twice with PBS, add the DMEM serum-free medium then.
3) cell is after hungry 24 hours, with the Tip head mark that places a wheal in being paved with the ware of cell, washes twice with PBS then, the removal cell debris.
4) add the culture fluid that contains the different stimulated condition, comprise 1%FBS, nervous process fibroin, filtered solution.
5) 0h, 4h, 8h, 12h, 23h takes the cell migration situation with phase contrast microscope, and each every ware of the moment is chosen 12 zones, tracing study.
6) data analysis: migration area=A0-AT.The area of white space when wherein A0 is 0h, AT are the area of white space constantly of taking pictures.
As Fig. 4-shown in Figure 6, the NRN1 protein groups of 100nm is more very fast than FT group migration rate, behind cut 4 hours, significant difference occurred, and along with the increase of time, the gap of the two migration area increases gradually.23h behind the cut, the FBS group is all healings almost, and the NRN1 group is taken second place, and still there is very big white space in the FT group.Prompting nervous process element can promote the migration of endotheliocyte.
The experiment of embodiment 5. cell adhesions
1) added the protein solution of variable concentrations in first day in 96 new orifice plates, 4 degree bags are spent the night.
2) suction in second day is gone more than protein solution, adds 100 μ l 1%BSA solution, 37 degree sealings 1 hour.
3) the resuspended ECV304 cell of digestion adds 1 * 10 in every hole
5Individual cell, adherent 1 hour.
4) carefully draw upper solution, and wash each hole with PBS, totally twice,
5) following steps are that the MTS method is surveyed the adherent cell number, and concrete steps are seen cell proliferation experiment.
Results suggest, NRN1 can significantly promote vascular endothelial cell to adhere to.
The experiment of embodiment 6. mice corneas
1) laboratory animal is the Kunming white mice in 5 to 6 ages in week
2) hydrochloric acid chlore-ammonia ketone and diazepam are pressed 2: 1 proportional arrangement anesthetis, intraperitoneal injection of anesthesia mice.
3) behind the mouse anesthesia, with transparent adhesive tape with its cervical fixation at desktop, cut beard.
4) clamping eyeball gently with toothed ophthalmology tweezer fixes.Choose to the eyeball axial centre along tangent plane with thin syringe needle, needle slope upwards a little to pressing down, stabs an osculum and reaches half place of corneal stroma.
5) toothholder with the ophthalmology tweezer plays opening, and along the tangential enlarged openings of eyeball, needle slope is towards eyeball with thick needle, but the fine rotation syringe needle.The about 1mm of pocket diameter.
6) after breach was opened, the Hydron granule that will have sample with anodontia ophthalmology tweezer was filled in the pocket.
7) all backs are observed under stereomicroscope and are taken pictures.
8) measure new vessels and extend to the extreme length and the hour number (angle of neovascularity continuum, per 30 degree are a hour number) of pocket from edge of cornea, the area computing formula of blood vessel is
Area=0.2 * π * the longest length of vessel * clock number
The result shows that NRN1 can obviously promote mice cornea angiogenesis.
The experiment of embodiment 7. hypoxia inducibles
1) digestion re-suspended cell is with 8 * 10
4Cells/well is inoculated in 24 orifice plates.
2) treat cell attachment after, add and to contain DFO (deferoxamine, Deferoxamine) or the culture medium of cobaltous chloride (CoCl2), concentration is respectively 0 μ M, 20 μ M, 50 μ M, 100 μ M, 200 μ M.
3) after 24 hours, PBS cleans one time, adds the lysis buffer cell lysis, the proteic expression of Western testing goal.
The result shows that anoxia condition can promote the expression of NRN1.
Embodiment 8. organizational groups fractional analysis
1) paraffin section is washed 5min/time 3 times with PBS
2) with confining liquid (confining liquid: 5%BSA, 5%house serum) sealing, room temperature one hour
3) wash 1 time with PBS
4) one anti-be diluted in (12000g * 20min removes impurity) among the 1%BSA, be added in the section, hatched two hours the chamber
5) wash 5min/time 3 times with PBS
6) two anti-be diluted in 1%BSA (12000g * 10min removes impurity), be added in the section incubated at room 1.5 hours
7) wash 5min/time 3 times with PBS, develop the color with ECL
8) with 80% glycerol mounting microscopic examination
The result shows that NRN1 expresses or low the expression hardly, only expresses in cancerous tissue in above-mentioned normal structure.
Claims (10)
1. the application of neurite-outgrowth element in the preparation antitumor drug.
2. application as claimed in claim 1 is characterized in that described antitumor drug is an anti-tumor angiogenesis drug.
3. application as claimed in claim 1 is characterized in that, the neurite-outgrowth element is the medicine target of screening and preparation antitumor drug.
4. application as claimed in claim 1 is characterized in that, described antitumor drug is the pharmaceutical composition that the plain inhibitor of neurite-outgrowth and carrier pharmaceutically or excipient are formed.
5. application as claimed in claim 1 is characterized in that, described tumor is the malignant tumor at liver, lung, digestive tract or mammary gland position.
6. a test kit that is used to screen antitumor drug is characterized in that, comprises the neurite-outgrowth element in this test kit.
7. a biochip is characterized in that, is connected with the neurite-outgrowth element on the carrier of this biochip.
8. application process as claimed in claim 1, it is characterized in that, the computer simulation three dimensional structure of candidate compound is combined with the computer simulation three dimensional structure of neurite-outgrowth fibroin, the computer simulation three dimensional structure of candidate compound and the active site of neurite-outgrowth fibroin are combined closely, and this candidate compound is the active component of antitumor drug.
9. application process as claimed in claim 8, it is characterized in that, the candidate compound adding that obtains is contained in the solution of neurite-outgrowth fibroin, then, detect the expression or the activity of neurite-outgrowth fibroin, the expression of neurite-outgrowth fibroin or the active candidate compound that descends are the active component of antitumor drug.
10. application process as claimed in claim 8 is characterized in that, after the combination of computer simulation three dimensional structure, the candidate compound that obtains is changed in the cancerous cell again, and the candidate compound that causes cancerous cell quantity to reduce is the active component of antitumor drug.
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| CNA2008100374432A CN101306192A (en) | 2008-05-15 | 2008-05-15 | Use of neuritis auxin in preparing antineoplastic medicine |
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| CNA2008100374432A CN101306192A (en) | 2008-05-15 | 2008-05-15 | Use of neuritis auxin in preparing antineoplastic medicine |
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